HANDBOOK
OF MEDIA
STAINS AND
REAGENTS IN
MICROBIOLOGY
- A. M. DESHMUKH
Preface
For conducting microbiology practical’s there is always a problem of getting
composition of media, stains and reagents. We have to search one or other book for
reference. The purpose of "Handbook of Media, Stains and Reagents in Microbiology" is
to collect all necessary information in one book. Author hopes the book will save the
time and efforts of research workers, students; teachers, laboratory technicians,
pathologists, environmental scientists and others.
The author is indebted to Dr. R. K. Trivedy, Dr. P. K. Goel, Shri. S.G. Pathak, Dr.
S.S. Jagdale, Dr. S.C. Kale, Dr. M.B. Gandhi, Dr. S.S. Bajekal, Shri. N.R. Shaikh, and
Shri. T.B. Sawant for graciously offering their suggestions.
The author wishes to express his thanks to -Dr. S.G. Sabnis and Prof. S.H. Shinde
for constant encouragement for writing this book. The author is also thankful to Dr. G.R.
Tathade for reading the manuscript and providing suggestions for improvement.
The author acknowledges his thanks to many known and unknown persons
involved in invaluable help during completion of this work.
- A. M. Deshmukh
Contents
Preface
1. Media for Biochemical Test
4
2. Cultivation Media
35
3. Staining Methods
153
4. Reagents
192
5. The Standard Solutions
206
Appendices
228
References
233
Index
236
1
Media for Biochemical Test
Amino acid Decarboxylation Test
A. Moeller's medium
Composition:
Peptone
Meat extracts
D --- Glucose
Pyridoxal
Bromcresol purple
( 1 in 500 solution)
Cresol red
( 1 in 500 solution)
Distilled water to
PH
5.0 g
5.0 g
0.5 g
0.005 g
5.0 ml
2.5 ml
1000 ml
6.0
Preparation: Dissolve all in gradients except indicators in water and adjust the pH. Add
indicators. Add 1 per cent amino acid (di or mono) Hydrochloride whose decarboxylation
is to be tested. (L-lysine, L-ornithine or. L-arginine hydrochloride). Distribute 1 ml in
each tube.
Add sterile liquid paraffin to provide a layer about 5 mm think above the medium.
Autoclave at 1210 C for 15 min.
Use: To test amino acid decarboxylation ability of bacteria. Inoculate the tubes and
incubate. After incubation positive test is indicated by violet colour of the medium. The
control remains yellow.
B. Falkow’s medium
Composition:
Peptone
5.0 g
Yeast extract
3.0 g
D - Glocose
1.0 g
0.2% bromocresol purple
10 ml
Aminoacid
5.0 g
(L-LysineiL-ornithinek-arginine)
Distilled water to
1000 ml
PH
6.7
Preparation: Dissolve all ingredients in water except bromocresol purple. Adjust the pH.
Add the indicator. Autoclave at 12 1 O C for 15 min.
Use: To test aminoacid decarboxylation ability of bacteria. The medium first becomes
yellow due to acid production. Later violet colour of the medium appears due to
decarboxylation. The control remains yellow.
Amylase Production
Starch agar
Composition:
Starch (soluble)
20.0 g
Peptone
5.0 g
Beef extract
3.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.0
Preparation: Dissolve all ingradients in distilled water. Adjust the pH. Add agar. Steam
sterilize the medium for one hour at 100°C.
Use: To study the starch hydrolysis by microgranisms. After incubation, flood the plate
with Gram's iodine. Amyl lytic colony will be surrounded by clear zone against purple
coloured background.
Casein Hydrolysis
Milk agar
Composition
Nutrient agar (sterile)
875.0 ml
Skimmed milk (sterile)
125.0 ml
Preparation: Sterilize the nutrient agar and milk separately at 121°C for 30 minutes.
Cool the agar at 50°C. Add cooled milk aseptically in the melted nutrient agar.
Use: To set casein hydrolyzing ability of microorganisms. Casein hydrolytic colonies
will develop clear zones around the colony.
Chitin Hydrolyse
Composition:
Chitin
4.0 g
K2HPO4
0.7 g
MgSO4.7H2O
0.5 g
KH2PO4
0.3 g
FeSO4.7H2G
0.01 g
MnCl2.4H2O
0.001 g
ZnSO4.7H2O
0.001 g
Agar
20.0 g
Distillen water to
1000 ml
pH
7.0
0
Preparation: Autoclave the medium 121 C for 20 minutes.
Use: For the isolation of chitin lytic bacteria.
Note: Adjust the pH 8.0 and use the medium for isolation of actinomycetes.
Citrate Utilization Test
A. Citrate broth (Koser's modified)
Composition:
NaCl
5.0 g
MgSO4.7H2O
0.2 g
NH4H2PO4
1.0 g
KH2PO4
1.0 g
Na3C6H5O7.2H2O
5.0 g
(Sodium citrate)
Distillen water to
1000 ml
pH
6.8
Preparation: Dissolve all ingredients in distilled water. Adjust the pH. Distribute in the
tubes and steam sterilize at 12 1 "C for 15 minutes.
Use: To test the ability of an organism to utilize citrate as the sole carbon and energy
source for growth. Positive test is indicated by the presence of turbidity in the broth after
incubation. Observe the turbidity by comparing uninoculated control tube.
B. Citrate agar
(Simmon's citrate medium)
Composition:
Koser's (modified) broth
1000 ml
Bromothymol blue (0.2%)
40.0 ml
Agar
20.0 g
Preparation: Add the indicator in the broth. Dissolve agar. Distribute in the tubes, steam
sterilize at 12 1°C for 15 min. and allow to set as slants.
Use: To test the ability of an organism to utilize citrate as the sole carbon and energy
source for growth. Positive test is indicated by the blue colour of medium after
incubation. Colour of original medium is green.
DNase test
DNase agar
Composition:
Casein (pancreatic digest)
15.0 g
NaCl
5.0 g
Soybean meal (papaic digest)
5.0 g
Deoxyribonucleic acid
2.0 g
Agar
20.0 g
Distilled water to
1000 ml
PH
7.3
Preparation: Add all comporients in distilled water by gentle heating. Steam sterilize at
1180C for 15 min. Pour plates.
Use: Spot inoculate the medium heavily by suspension of test organism. Incubate for 24
hrs. flood the plate with IN HCI. A clear halo around the colony indicate positive test. 1
N HCI precipitates unchanged deoxyribonucleic acid.
Esculin Hydrolysis
Bile esculin medium
Composition :
Peptone
5.0 g
Beef extract
3.0 g
Bile (oxgall)
40.0 g
Esculin
1.0 g
Ferric citrate
15.0 g
Agar
15.0 g
Distilled water to
1000 ml
PH
7.0
Preparation: Dissolve all ingredients in distilled water. Distribute into tubes, sterilize in
autoclave at 12 1 OC for 15 min. and prepare slants.
Use: To set esculine hydrolyzing ability of bacteria. Esculin hydrolysis is indicated by
causing the slant to blacken after incubation.
Note: This test is usually applied for indentifiecation of group D streptococci. Both
enterococcal and nonenterococcal species of group D are able to hydrolyse esculin.
Eijkman Test
MacConkey's broth
Composition: (See Page 79)
Use: This test in used for identification of Escherichia coli. Inaculate MacConkey's broth
with test organism. Incubate at 44* 0.2OC for 24 hrs. E. coli produce gas at this
temperature.
Gelatin hydrolysis (Gelatin liqueefaction test)
Gelation agar
Composition:
Gelatin
4.0 g
Glucose
0.05 g
KH2PO4
0.5 g
K2HPO4
1.5 g
Nutrient agar (melted)
1000 ml
Preparation: Dissolve the phosphates, gelatin and glucose in melted nutrient agar slowly
for even distribution ofthe gelatin. Steam sterilize in a flask at 1210C for 30 min. Prepare
plates.
Use: To test gelatin hydrolyzing ability of microorganisms. Flood the plate containing
growth of the microorganisms with Frazier's solution. Gelatin hydrolysis is indicated by
clear zone around colony against opaque background.
Gluconate Test
Compasition:
Peptone
1.5 g
Yeast extract
1.0 g
K2HPO4
1.0 g
Potassium gluconate
40.0 g
Distilled water to
1000 ml
pH
7.0
Preparation: Dissolve all components in water. Adjust the pH. Distribute in 1 rnl
quantities in each tube and steam sterilize at 121°C for 15 min.
Use: used to test the ability of an organism to oxidize gluconates to the 2 keto-gluconate.
Inoculate the tube with bacterial suspension and incubate at 370C for 48 hours. Then add
1 ml of qualitative benedict's solution and heat the tube for 5-10 minutes. Observe the
change in colours. Benedict's reagents is blue in colour. Positive test is indicated by
development of green to orange coloured precipitate.
Homo and Heterofermentation - Differentiation
Tomato juice-gelatin medium
Composition:
Gelatin
120.0 g
Yeast hydrolysate
2.5 g
Tomato juice
100.0 g
Glucose
50.0 g
Distilled water to
1000 ml
PH
7.0
Preparation: Sterilize glucose separately in 500 ml of water by filtration and remaining
medium at 121°C for 30 minutes. Mix both the solutions aseptically.
Use: For differentiating homofermentative and heterofer-mentative lactic acid bacteria.
Inoculate the tubes with culture. Mix it properly, chill the tubes in cold water to solidity
the gelatin. Then seal with about I inch of melted nutrient agar. Incubate for 2-5 days at
the temperature indicated and examine for gas production.
Hippurate Hydrolysis
Sodium hippurate broth
Composition:
Peptone
5.0 g
Beef extract
3.0 g
Sodium hippurate
10.0 g
Distilled water to
1000 ml
Preparation: Dissolve the ingradients in distilled water. Distribute in tubes. Sterilize in
autoclave at 121°C for min.
Use: To detect hippurate hydrolysis. Incubated broth is centrifuged and 0.8 ml clear
supernate is taken into a kahn tube. 0.4 ml of ferric chloride solution [12g FeC136H2O
dissolved in 100 ml of dilute (5.4 ml concentrated HC1 to 94.6 ml distilled water) HCI] is
added in a kahn tube. Mixture is allowed to stand with occasional shaking for 10 minutes.
Positive test: A heavy cloudy, precipitate persisting after 10 minutes.
Negative test: Clearing of the initial precipitate within 10 minutes.
Hydrogen Sulfide Production
A. SIM agar (Sulfide indole motility agar)
Composition:
Peptone
30.0 g
Beef extract
3.0 g
Ferrous ammonium sulphate
0.2 g
Sodium thiosulphate
0.025 g
Agar
4.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve all components indistilled water. Adjust the pH. Disribute in the
tubes. Sterilize the medium at 121°C for 20 min. in the autoclave.
Use: This medium is used for detection of H,S, indole and motility. H2O producing
organism cause blackening of the medium. H2S reacts with ferrous ammonium sulphate
resulting black insoluble compound. ferrous sulphide. Indole production can be tested by
the method prescribed in the illdole test. Motility is indicated by diffused growth
from the inoculation point.
B. Peptone water 1
Composition:
Peptone
20.0 g
Sodium chloride
5.0 g
Distilled water to
1000 ml
PH
7.2
Preparation: Dissolve all components in warm water. Adjust the pH. Autoclave at 121°C
for 15 min.
Use: Used to test hydrogen sulphide production ability of bacteria. Inoculate the tube of
peptone water by given organism. Dip the filter. paper in saturated solution of lead
acetate. Dry the filter paper, sterilize and keep hanging in the inoculated tube of peptone
water with the help of cotton plug. Incubate the tube.
Positive test: Filter paper turns black.
Negative test: No change in colour of filter paper.
C. Kligler's iron agar I
Composition:
Meat extract
3.0 g
Yeast extract
3.0 g
Peptone
20.0 g
Lactose
10.0 g
Glucose
1.0 g
Sodium chloride
5.0.g
Ferrio citrate
0.3 g
Sodium thiosulphate
0.3 g
Phenol red
0.05 g
Agar
12.0 g
Distilled water to
1000 ml
pH
7.4
Preparation: Dissolve the ingredients in water. Dispense in about 5 min amounts in test
tubes and sterilize them at 121°C for 15 minutes. Prepare slants with a butt.
Use: To detect H,S production and fermentation of lactose and glucose. Interpret the
result like TSI agar.
D. Lysine iron agar
Composition:
Yeast extract
3.0 g
Peptone
5.0 g
L-lysine
10.0 g
Dextrose
1.0 g
Sodium thisulphate
0.04 g
Ferric ammonium citrate
0.5 g
Bromocresol purple
0.02 g
Agar
20.0 g
Distilled water to
1000 ml
Preparation: Dissolve the ingredients by boiling in water. Dispense into tubes. Sterilize
at 121°C for 20 minutes. Prepare slants with deep butts.
Use: To test production of H2O and lysine decarboxylase by Proteus.
(a) The production is detected by blackening of the medium.
(b) The lysine decarboxylase production is detected by development of red colour around
the colony.
Indole Test
Durham's peptonewater (Fermentation basal medium)
Composition: (See peptone water in hydrogen sulphide production test)
Use:
1. This medium is used chiefly as the basic for carbohydrate fermentation media.
2. It is also used fortesting the indole formation ability. Inoculate the tube with
given bacteria. After incubation add few drops of xylene. Shake the tube
vigourously. Add 2 to 3 drops of kovac's reagent along tlie side of the tube.
Note: 1. Use of tryptone water instead of peptone gives good results in indole formation.
2. All commercial peptones do not contain sufficient tryptophan for good indole
production. Therefore the suitable brand of peptone must be selected for this
purpose.
Lysozyme Sensitivity Test
Lysozyme broth
Composition:
A. Glycerol broth
Peptone
5.0 g
Beef extract
3.0 g
Glycerol
70.0 ml
Distilled hater to
1000 ml
Add all components in distilled water. Sterilize at 1210C for 20 minutes.
B. Lysozyme solution
Lysozyme
0.1 g
0.01 N HCI
100.0 ml
Add lysozyme to 0.01N HCI. Mix. Filter sterilize. This solution can be stored at
4°C for 7 days.
Preparation of complete medium
Lysozyme solution
0.5 ml
Glycerol broth
100 ml
Add aseptically and distribute in sterile tubes of 5 ml each.
Use: For differentiation of genera of actinomycetes based on sensitivity to lysozyme
sensitivity actinomycetes do not grou in lysozyme broth. Growth can be compared by
inoculating in glycerol broth.
e.g. Nocardia asteroids -- not sensitive to lysozyme
Streptomyces griseus -- sensitive to Iysozyme.
Lactate Fermentation
Lactate agar
Composition:
Trypticase
20.0 g
Yeast extract
5.0 g
Sodium lactate
12.0 g
Agar
25.0 g
Distilled water to
1000 ml
pH
6.8
Preparation: Dissolve all components in distilled water except agar. Adjust the pH. Add
Agar. Distribute in the tubes. Steam sterilize the medium at 1210C for 20 min.
Use: To test lactate fermentation ability of bacteria. Stab inoculates the tubes. Incubate.
Lactate ferernentation is detected by gas production.
Lecithinase Production
A. Tween phosphate buffered substrate medium
Composition:
Phosphate buffer pH
Tween 80
(Polyoxyethylene sorbitain
monooleate)
Neutral red (1 g/litre)
pH
7.0 40.0 ml
0.2 ml
0.8 ml
6.8 - 7.2
Preparation: Add neutral red solution to phosphate buffer and mix. Add tween 80 and
mix gently. Distribute in 5 ml amounts in tubes and sterilize at 1210C for 20 minutes.
Use: It is used in tween80 hydrolysis test to detect lecithinase production.
B. Egg yolk agar
Composition:
1.
Melted nutrient agar (sterile)
85 ml
2.
Egg yolk suspension
l5 ml
Preparation of egg yolk suspension:
Take fresh egg (less than four days old). Wash with water by brush and a plain alkaline
soap. Rinse in running water for 10 min. Dry the egg by sprinkling methylated spirit and
burning it off. Crack the, tapered end of egg with sterile knife. Separate the egg white
from egg yolk. Collect the egg yolk in sterile container aseptically.
Preparation of complete medium:
Melt tlie agar. cool to 55°C and add tlie egg yolk. Pour plates.
Use: To test lecithinase activity of bacteria. Lecithinase production is shown by wide
zones of opalescence around colonies more intense and larger than the zones caused by
lipolysis.
C. TweenTM 80 hydrolysis
Composition :
Peptone
10.0 g
NaCl
5.0 g
CaCI2
0.1 g
TM
Tween 80
10.0 ml
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
0
Preparation: Autoclave at 121 C for 20 min.
Use: For testing the microorganisms for hydrolyzing TweenTM80 Colonies hydroluzing
TweenTM80 are surrounded by an opaque zone.
Lipolysis Test
A. Fat agar
Composition:
Sterile melted nutrient agar
100 ml
Sterile melted butter (unsalted)
10 ml
Preparation: Mix thoroughly and pour plates.
Use: To study lipolytic activity of bacteria. Inoculate the centre of the solidified fat agar
plate with bacterial suspension. Incubate for 5-7 days at given temperature. Flood the
plates with a saturated aqueous solution of' CuSO4. After 20 minutes pour off the excess
solution. Bright greenish blue insoluble copper soap is formed with the fatty acids around
the colony.
B. Egg yolk agar
composition:
(See lecithinase production)
Use: Used to test lipolytic activity of bacteria. Flood the date with copper sulphate
solution. After 20 minutes pour off the excess solution. Bright greenish blue insoluble
copper soap is formed with the fatty acids around the colony.
C. Tributyrin hydrolysis method
Composition:
Peptone
5.0 g
Yeast extract
3.0 g
Tributyrin
10.0 g
(Glyceryl tributyrate)
Agar
20.0 g
Distilled water to
1000 ml
pH
7.5
Preparation: The medium is prepared such that the tributyrin forms A stable emulsion in
the nutrient agar and adjust the pH
Use: To test hydrolysis of tributyrin. An emulsion of microdroplets of the fat, tributyrin,
in a solid medium makes it opaque. Lipolytic organisms remove the opacity by
converting the fat to water soluble butyric acid.
Litmus Milk Test
Litmus milk medium
Composition:
Preparation of litmus solution
Litmus granules
80.0 g
Ethanol, 40% aq.
300 ml
Grind up the granules, add to a flash containing 150 ml aqueous ethanol. Boil for 1
min. Decant the solution in other flask. Add remainder aqueous ethanol and boil for I
min. Decant and combine both solutions, make the volume 30 ml with 40% aq. ethanol.
Add HCI (IN) drop by drop, shaking continuously until the solution becomes purple.
Preparation of complete medium
Skimmed milk
250 ml
Litmus solution
6.25 ml
Mix both the solutions. Distribute in tubes 5 ml in each. Steam for 20 min. on three
successive days. Colour of medium is blue or purple.
Use: Litmus milk test indicates both saccharolytic and proteolytic properties of bacteria.
Lactose fermenter in litmus milk form acid cause it to become pink or red. Proteolytic
bacteria decompose milk protein and change to a clear dark purple solution.
Melanine Production
Tyrosine agar
Composition:
Glycerol
L-Tyrosine
L-Aspargine
K2HPO4
MgS04.7H2O
NaCl
FeSO4.7H4O
Agar
Distilled water to
Preparation: Steam sterilize the medium at 12I0C for 20 min.
Use: For testing melanine production by actinomycetes.
15.0 g
0.5 g
1.0 g
0.5 g
0.5 g
0.5 g
0.01 g
20.0 g
1000 ml
Malonate Utilization Test.
Composition:
Yeast extract
1.0 g
(NH4)2SO4
2.0 g
K2HPO4
0.6 g
KH2PO4
0.4 g
NaCl
2.0 g
Na – malonate
3.0 g
Bromothymol blue
0.025 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.4
Preparation: Dissolve all components except indicator in water. Adjust the pH. Add the
indicator. Distribute in the tubes. Steam sterilize at 121°C for 15 min.
Use: For testing the ability to utilize malonate by bacteria. Positive results are indicated
by the change in colour of the indicator from green to blue due to the rise in pH (due to
utilization of sodium malonate).
Malonate Utilization and Phenylalanine
Deaminase test
Composition:
(NH4)SO4
2.0 g
K2HPO4
0.6 g
KH2PO4
0.4 g
NaCl
2.0 g
Sodium malonate
3.0 g
DL-Phenylalanine
2.0 g
Yeast extract
1.0 g
Distilled water to
1000 ml
PH
7.4
Preparation: Dissolve all components in distilled water. Steam for 5 minutes and filter
through paper. Add 5ml of a 0.5% solution of bromothymol blue in absolute ethanol.
Distribute Iml in each tube, steam sterilize at 121°C for 20 min.
Use: For following two tests:
1. Malanate utilization test:
Positive test, green to blue colour of medium after inoculation and incubation.
2. Phenylalanine deaminase test:
Add few drops of 0. IN HCI until the colour of the medium becomes yellow. Add few
drops of a 10% aqueous solutions of FeCI3, shake and observe the colour for
phenylalanine deaminase test.
Dark green = Positive test
Yellow buff = Negative test.
Methyl Red Test
MR-VP broth (Glucose phosphate broth)
Composition:
Peptone
5.0 g
K2HPO4
5.0 g
Distilled water to
1000 ml
Glucose 10% solution
50 ml
PH
7.6
Preparation: Dissolve the peptone and phosphate. Adjust the pH Filler. Distribute in 5
ml amounts and steam sterilize and at 121°C for 15 minutes. Sterilize the glucose
solution by filtration and add 0.25 ml to each tube. (final concentration 0.5%).
Use: To perform methyl red and Yoges Proskauer test. Inoculate the tube with given
organism. Incubate. Add about 5 drops of methyl red reagent after incubation. Mix and
observe the colour.
Positive test: Development of bright red colour.
Negative test: No development of bright red colour. Colour remains yellow.
Motility Test
Motility agar
Composition:
Peptone
1.0 g
Sodium chloride
5.0 g
Agar
4.0 g
Distilled water to
1000 ml
PH
7.2
Preparation: Dissolve all the components in water by heating. Adjust the pH. Distribute
in tubes. Steam sterilize at 1210C for 15 minutes.
Use: Motile bacteria forms spreading colony on soft motility agar.
Motility and Nitrate Reduction Test
Motility nitrate medium
Composition:
Beef extract
3.0 g
Peptone
5.0 g
KNO3
1.0 g
Agar
3.0 g
Distilled water to
1000 ml
PH
7.2
Preparation: Dissolve all the ingradients in distilled water by heating. Ajust the pH.
Distribute in tubes. Steam sterilize at 12I0C for 15 min.
Use: For detection of motility and nitrate reduction. Diffuse growth along the line of
inoculation indicates motility. For nitrite detection add few drops of sulphanilic acid
followed by equal volume of alphanaphthylamine. The rapid development of a pink
colour indicates the presence of nitrite.
Nitrification Kest
A. Ammonium medium
Composition:
(NH4)2S04
K2HPO4
NaCl
MgSO4.7H2O
FeSO4
Distilled water to
1.0 g
1.0 g
2.0 g
0.5 g
trace
1000 ml
Preparation: Excess of the sterilized CaCO3 is added to the medium. Sterilisation of the
medium is not necessary if the medium is used freshly.
Use: To test ammonia to nitrite (nitrification) conversion ability of bacteria. This medium
is also used to isolate Nitrosomonas from, soil. Pesence of nitrite can be tested by using
an appropriate colorimetric reagent. (See page 245)
B. Nitrite medium
Composition:
NaNO2
1.0 g
MgSO4.7H2O
0.5 g
FeSO4.7H2O
0.03 g
NaCl
0.3 g
Na2CO3
1.0 g
K2HPO4
1.0 g
Distilled water to
1000 ml
pH
7.3
Preparation: Sterilisation is not necessary if the inoculations are made in the freshly
prepared medium.
Use: To test nitrite to nitrate (nitrification) conversion ability of bacteria. This medium is
also used to isolate Nitrobacter from soil. Fresence of nitrate can be tested by using an
appropriate colorimetric reagent. (See .page 245)
Preparation: If sterilization is desirable for storage, adjust the pH aseptically after
sterilization with sterile 0.1 N HCI.
Nitrate Reduction I
A. Tryptone nitrate broth
Composition:
Trytone
20.0 g
Potassium nitrate
1.0 g
Dissodium phosphate
2.0 g
Dextrose
0.1 g
Agar
1.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve the ingredients in water. Adjust the pH. Distribute in the tubes.
Steam sterilize the medium at 1210C for 20 min.
Use: To test nitrate reduction ability of bacteria. Presence of nitrite can be tested by an
appropriate colorimetric reagent. (See page 245)
B. Nitrate m'edium
Composition:
KNO3
0.2 g
Peptone
5.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve the ingredients in water. Adjust the pH. Distribute in the
tubesfflasks. Steam sterilize the medium at 1210C for 20 min.
Use: To test nitrate reduction ability of bacteria. (Presence of the enzyme nitrate
reductase which causes the reduction of nitrate to nitrite.) Presence of nitrite can be tested
by an appropriate colorimetric reagent. (See page 245)
ONPG Test
ONPG broth
Composition:
Preparation of ONPG solution: I
ONPG (0-nitrophenyl-β -D Galactopyranoside)
1.5 g
Sodium phosphate buffer to (0.01 M, pH 7.5)
250 ml
Preparation of complete medium: I
ONPG solution
250
Peptone water
750 ml
pH
7.2 : 7.4
Filter sterilize ONPG solution. Sterilize peptone water saperately. Adjust the pH of
peptone water 7.2 : 7.4 before sterilization. Sterilize peptone water at 1150C for 20 min.
Aseptically combine the sterile ONPG solution with cooled sterile peptone water. Mix
thoroughly. Distribute in tubes.
Use: For differentiation of late lactose fermenting (having β galactosidase and lacking
permease) and non-lactose fermenting bacteria. (without β galactosidase).
Inoculate ONPG broth and incubate for 24 hrs. If galactosidase is present a yellow
colour (due to o-nitrophenol) is formed.
Oxidation Fermentation Test
Hugh and Leifson medium (of glucose medium)
Composition:
Peptone
NaCl
K2HPO4
Bromothymol blue 3 (1% aq. solution)
Glucose (1 0% solution)
2.0 g
5.0 g
0.3 g
3 ml
100 ml
Agar
3.0 g
Distilled water to
900 ml
pH
7.1
Preparation: Dissolve all the ingredients except the indicator and sugar solution in water.
Adjust the pH. Add the indicator and steam sterilize the medium is a flask at 1210C for
30 min. The sugar solution is sterilized separately by filtration and then added as given in
the composition. The medium is then distributed in the sterile tubes to depth of about 4
cm in aseptic condition.
Use: Used to distinguish between aerobic and anaerobic break down of a sugar.
Duplicate tubes of medium are inoculated by stabbing, 1 tube is promptly covered with a
layer of sterile melted petroleum jelly to a depth of 5-10 mm and tubes are incubated.
After incubation, if acid is produced only at surface of the medium, where conditions are
aerobic, the attack on the sugar is oxidative. If acid is found throughout the tube,
including the lower layers where conditions are anaerobic the break down is fermentative
Pectin Hydrolysis
Hankin's medium
Composition:
Mineral salt solution
500 ml
Yeast extract
1.0 g
Agar
15.0 g
Pectin (citrus or apple)
5.0 g
Distilled water to
500 ml
pH
7.2
Preparation: Dissolve agar power in 400 ml of water with heat. Dissolve pectin and
yeast extract in another 100 ml of water and mix Both solutions. Add mineral salt
solutions. Sterilize by autoclaving at 121°C for 20 min.
Composition of mineral salt solution:
(NH4)2SO4
2.0 g
KH2PO4
4.0 g
Na2HPO4
6.0 g
FeSO4.7H2O
0.2 g
CaCl2
1 mg
H3BO3
10 µg
MnSO4
10 µg
ZnSO4
70 µg
Distilled water to
100 ml
Use: To test degradation of pectin by bacteria. If the plates are flooded with 1 percent
aqueous solution of hexadecyltrirnethyl ammonium bromide, penctinolytic organisms
produce clear zone around colonies. Pectin precipitates in presence of hexadecyltrimethyl
ammonium bromide.
Note: Adjust the pH 5 if fungi are to be tested.
Penicillin Assay
A. Antibiotic assay medium (Muller-Hinton agar)
Composition:
Infusion from beef
300.0 g
Acid hydrolysate of casein
17.5 g
Starch
1.5 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2 – 7.4
0
Preparation: Sterilize the medium at 121 C for 15 minutes.
Use: It is used as medium for antibiotic susceptibility testing by Kirby Bauer disc
sensitivity method.
B. Antibiotic assay medium (Trypticase soy broth)
Composition:
Trypticase peptone
17.0 g
Phytone peptone
3.0 g
Sodium chloride
5.0 g
Dipotassium hydrogen phosphate
2.5 g
Dextrose
2.5 g
Distilled water to
1000 ml
Preparation: Sterilize the medium at 121°C for 15 minutes.
Use: It is used for testing antibiotic sensitivity (Kirby Bauer method.)
C. Penassay seed agar
Composition:
Beef extract
Yeast extract
Casein digest
Peptone
Glucose
Agar
Distilled water to
pH
1.5 g
3.0 g
4.0 g
6.0 g
1.0 g
20.0 g
1000 ml
7.0
Preparation: Steam sterilize the medium at 12 1 "C for 20 min.
Use: Used to test penicillin sensitivity of bacteria.
D. Penassay seed agar
Composition:
Gelatin 6.0 g
Yeast extract 3.0 g
Beef extract 1.5 g
Agar 20.0 g
Distilled water to 1000 ml
pH 7.0
Preparation: Steam sterilize the medium at 121 O C for 20 min.
Use: Used as the antibiotic assay medium.
Phenylalanine Deaminase Test
Composition:
Yeast extract
3.0 g
DL phenylalanine (or L phenylalanine 1g)
2.0 g
Na2HPO4
1.0 g
NaCl
5.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.4
Preparation: Dissolve all the components in water except agar. Adjust the pH. Dissolve
the agar by heating. Distribute in tubes and sterilize by autoclaving at 121°C for 15 min.
Allow to solidify in tubes as slope.
Use: To test phenylalanine deamination ability of an organism. After incubation add few
drops of 10% solution of ferric chloride over the growth on the slant.
Positive test: Development of green colour.
Negative test: No development of green colour.
Phosphatase Production
Phenolphthalein phosphate agar
Composition:
Melted nutrient agar (pH 7.2)
98 ml
Sodium phenolphthalein phosphate solution (0.5%)
2ml
Preparation: A stock solution of sodium phenolphthalein phosphate is prepared,
sterilized by filtration and stored at 40C. Mix the melted sterilized nutrient agar with
sodium phenolphthalein phosphate solution.
Use: To isolate phosphatase producing organism, specifically staphycococci.
Phosphatase liberate free phenolphthalein, colonies become pink when held over an open
bottle of ammonia.
Phosphate Solubilization
A. Katznelson and Bose medium
Composition:
Glucose
1.0 g
K2HPO4 (1 0% solution)
5.0 ml
CaCl2 (1 0% solution)
10.0 ml
Agar
2.0 g
Soil extract
100.0 ml
pH
7.0.
Preparation: Sterilize the soil extract containing glucose and agar at 121°C for 20 min in
autoclave. Sterilize the K2HPO4 and CaCI2 solutions separately. Mix. Adjust the pH with
sterile 0.1N sodium hydroxide. Pour the plates immediately and allow to solidify.
Use: To test phosphate solubilizing activity of micro-organisms. The clearing around the
colonies indicate phosphate solubilization.
B. Pikovskaya's medium
(Modified by Sundara Rao and Sinha)
Composition:
Glucose
10.0 g
Tricalcium phosphate
5.0 g
CaCl2 (10% solution)
0.5 g
KCl
0.2 g
MgS04.7H20
0.1 g
MnSO4
trace
FeSO4
trace
Yeast extract
0.5 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve all the components except tricalcium phosphate in 700 ml water.
Adjust the pH. Sterilize tricalcium phosphate separately in 300 ml water. Sterilize at
121°C for 20 min in autoclave. Cool at about 50°C and then mix both the solutions.
Use: To test phosphate solubilizing activity of microoganisms. Clearing around the
colony indicates phosphate solubilization.
Potassium Cyanide Test
Composition:
A. Basal medium
Peptone
3.0 g
NaCl
5.0 g
KH2PO4
0.23 g
Distilled water to
1000 ml
pH
7.4
Steam sterilize the medium at 12 1 OC for 30 min.
B. Cyanide solution
KCN
0.5 g
Distilled water to (sterile)
100 ml
Preparation of complete medium
Basal medium
1000 ml
Cyanide solution
15 ml
Add the cyanide solution to the cold medium. Distribute insterile tubes, 1 ml in each
tube.
Use: To test the ability of an organism to grow in the presence of cyanide.
Note: The medium can be stored for 4 weeks at 40°C.
Sodium Chloride Tolerance Test
Sodium chloride tolerance broth
Composition:
Heart infusion broth
25.0 g
Sodium chloride
60.0 g
Indicator (1.6g Bromcresol purple in 100 ml 95% ethanol) 1.0 ml
Dextrose
1.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Add all ingredients except. indicator in 1000 ml water. Adjust the pH. Then
add indicator. Distribute in tubes and sterilize in an autoclave for 15 min at 1210C.
Use: This test is used for differentiating the two types of group D streptococci. All
enterococci of group D produce heavy growth in this broth. None of the nonenterococci,
group D grow in this medium. A positive result is indicated by turbidity within 72 hrs. A
colour change of purple to yellow may also be present.
Note: Heart infusion broth (obtainable in dehydrated form) already contain 0.5% sodium
chloride.
Sugar Fermentation
A. Sugar fermentation medium
Composition:
Peptone water
100 ml
Test sugar
0.5 to 1 g
Andrade's indicator (1 %)
0.25 ml
or
Neutral red indicator (1 %)
0.25 ml
or
Phenol red indicator (1 %)
0.18 ml
or
Bromothymol blue (1 %)
0.25 ml
Preparation: Sugar is added in peptone water. Adjust the pH. Distribute in tubes.
Sterilize in autoclave at 121°C for 20 minutes. (steam sterilize at 100°C for 1 hr if the
sugar is thermolabile, like diassaccharide lactose.)
Use: Used to test sugar fermentation ability of bacteria. Durham fermentation tube in
inverted position in the broth is used to detect the gas production. Acid production is
indicated by change in colour I according to type indicator used, e.g., yellow to red when
andrade's indicator is used.
B. Sugar fermentation medium
Composition:
Peptone
5.0 g
Meat extract
3.0 g
Sodium chloride
5.0 g
Distilled water to
1000 ml
Test sugar
1%
pH
7.2
Preparation: Dissolve the ingredients in water. Adjust the pH. Add indicator solution as
given in above medium. Distribute in tubes. Place a Durham's tube in an inverted
manner. Sterilize the tubes at 121°C for 15 min.
Use: To test sugar fermentation ability ~f bacteria, if a more nutritious base is needed.
C. Sugar fermentation (for actinomy'cetes)
Composition:
(NH4)2HPO4
MgS04.7H2O
KCl
Carbohydrate
solution
Bromocresol purple
1.0 g
0.2 g
0.02 g
100.0 ml
(10%)
20.0 ml
Agar
Distilled water to
pH
20.0 g
1000 ml
7.0
Bromocresol purple solution
Bromocresol purple
0.04 g
Ethanol
50.0 ml
Distilled water to
100.0 ml
Preparation: Add all components except carbohydrate solution, bromocresol purple
solution and agar in distilled water. Adjust the pH. Add bromocreasol purple solution and
agar. Dissolve. Steam sterilize at 12 1 "C for 20 minutes. Sterilize carbohydrate solution
by filtration. Mix aseptically.
Use: For identification of actinomycetes to perform carbohydrate fermentation test.
Actinomycetes produce acid from carbohydrate turn the medium yellow.
D. Serum agar fermentation medium
Composition:
Basal medium
Peptone
20.0 g
Sodium chloride
5.0 g
Digest broth pH 7.6
100 ml
Agar
25.0 g
Phenol red solution (0.2 %)
20 ml
Distilled water to
900 ml
pH
7.6
Preparation: Dissolve the peptone and salt in the water by steaming. Adjust to pH 8.4 by
making just alkaline to phenolphtlialein. Steam for 30 min. Filter through a coarse filter
paper. Adjust the pH to 7.6. Add the broth and agar and steam at 100°C for one hr. Bottle
in 100 ml amounts and add 2ml phenol red to each bottle. Autoclave at 100°C for 1 h and
then 110°C for 5 min.
Complete medium
Basal medium
100.0 ml
Sterile serum (guinea pig or rabbit)
5.0 ml
Test sugar (10 % solution, sterile)
10.0 ml
Preparation: Melt the basal medium, cool to 55°C. Add other ingredients with sterile
precautions and distribute in tubes. Prepare slants.
Use: This medium is used for organisms such as meningococci and qnococci that grow
poorly in liquid media. Acid production is indicated by change in colour of the medium
from red to yellow.
E. Sugar fermentation
Serum sugar fermentation medium (broth)
Composition:
Peptone water (PH 7.4)
80.0 ml
Serum
20.0 ml
Phenol red (0.2 %)
5.0 ml
Test sugar
1.0 g
Preparation: Serum medium is sterilized usually by filtration. It may be steamed for 30
min for three successive days.
Use: This medium is suitable for more exacting organisms such as Corynebacterium
diphtheria to test sugar fermentation.
Precaution: Some sample of horse serum may contain saccharolytic enzymes and give
false results.
Sulfatase Test
wayne's sulfatase medium
Composition:
Na2HP04
2.5 g
L-Asparagine
1.0 g
KH2PO4
1.0 g
K2HPO4
1.0 g
Trisodium phenolphthalein
0.65 g
sulfate
Casein (pancreatic digest)
0.5 g
Ferric ammonium citrite
0.05 g
MGS04-7H2O
0.01 g
CaCl2.2H2O
0.5 mg
ZnS04.7H2O
0.1 mg
CuSo4
0.1 mg
Glycerol
10.0 ml
Agar
20.0 g
Distilled water to
1000 ml
pH
7.0
Preparation: First add glycerol 700 ml water. Mix thoroughly. Add other components
and make the volume 1000 ml with distilled water. Gently heat. Distribute in tubes.
Autoclave at 121°C for 20 min.
Use: This test is commonly use for identification of Mycobacterium tuberculosis.
Inoculated tubes with the test organism for 3 to 14 days. Add few drops of amino
solution or .5 to 1 ml of 2N Na2CO3 in the tube. Pink colour indicate positive test.
Mycobacterium
ruberculosis gives negative test.
Snyder Test
Snyder test agar
Composition:
Trytone
20.0 g
Sodium chloride
5.0 g
Dextrose
20.0 g
Bromocresol green
0.02 g
Agar
20.0 g
Distilled water to
1000 ml
pH
4.8
0
Preparation: Steam sterilize at 1 18 C for 30 min.
Use: It is used to measure the rate of acid production from the metabolism of the glucose
by the lactobacilli. Lactobacilli grow on the snyder test agar. They utilize glucose of the
medium converting it to organic acids and these by lowering the pH to 4.4 or lower. At
this pH bromocersol green becomes yellow. Lactobacilli present in mouth (saliva)
responsible for dental caries are tested by referring following table. Caries process begins
at pH 4.4.
Standardized table to determine dental caries susceptibility
.Caries
susceptibility
Marked
Moderate
Slight
Negative
24
Positive
Negative
Negative
Negative
Hours incubation
48
--Positive
Negative
Negative
72
----Positive
Negative
Thiocyanate Utilization Test
Composition:
Basal solution
Solution A
Solution B
Solution C
Basal solution
Na2HPO4
KH2PO4
MgS04.7H2O
100 ml
0.5 ml
2.0 ml
20.0 ml
0.48 g
0.44 g
0.05 g
Distilled water to
100 ml
Steam sterilize at 121°C for 20 min.
Solution A
FeC12.6H2O
1.0 g
CaCl2
0.1 g
Distilled water to
100 ml
Sterilize by filtration.
Solution B
D-Glucose
10.0 g
Distilled water to
100 ml
Sterilize by filtration.
Solution C
Sodium thiocynate
0.5 g
Distilled water to
100 rnl
Sterilize by filtration
Use: Used for testing thiocyanate utilization ability of microoganisms. Thiocyanate is
utilized as a source of nitrogen and sulphur.
TSI Agar Test
Triple sugar-iron agar
Composition:
Peptone
20.0 g
Tryptone
10.0 g
Beef extract
3.0 g
Yeast extract
3.0 g
Lactose
10.0 g
Sucrose
10.0 g
Dextrose
1.0 g
Sodium chloride
5.0 g
Ferrous sulphate
0.2 g
Sodium thiosulphate
0.3 g
Phenol red
0.024 g
Agar
15.0 g
Distilled water to
1000 ml
pH
7.4
Preparation: Dissolve all components in distilled water except phenol red and agar.
Adjust the pH. Add phenol red and agar. Distribute in tubes. Steam sterilize at 1180C for
15 minutes.
Use: To test fermentation of these three sugars and H,S production in one medium.
Key reaction for TSI agar
Reaction in
slants
Orange red
Red
(Alkaline)
Yellow
(Acidic)
Red
(Alkaline)
Reaction in
butts
Orange red
Yellow
(Acidic)
Yellow
(Acidic)
Yellow
Black
Gas
H2S
-
Interpreation
Control
Slight Dextrose fermentation
H2S production.
+
Dextrose and lactose
/sucrose fermentation.
+
+
Dextrose fermentation
And excess H2S
Production.
+ = Positive
- = Negative
Culture characteristics ofmembers of enterobacteriaceae on TSI agar after 18-24
hours at 37°C are as follows.
Organism
Slant
Butt
Gas
H2S
Escherichia Sp.
Y
Y
+
Shigella Sp.
R
Y
S. Typhimurium
R
Y
+
S. typhi
R
Y
+
+
Arizona Sp.
R
Y
+
+
Citrobacter freundil
R
Y
+
+
Klebsiella Sp.
Y
Y
+
+
Entrobacter Sp.
R
Y
Serratia Sp.
Y/R
Y
P. vulgaris
Y/R
Y
+
+
P. mirabilis
R/Y
Y
+
+
P. morganii
R
Y
P. rettgeri
R
Y
Providencia Sp.
R
Y
+/Pasteurella Sp.
Y
Y
+
(48 hrs)
Y = Yellow colour
+ = Positive
R = Red (pink) colour
- + Negative
Urea Hydrolysis
Urea agar (Christensen's medium)
Composition:
Peptone
1.0 g
NaCl
5.0 g
K2HP04
2.0 g
Phenol red (1:500 in aq. sol.)
6.0 ml
Agar
20.0 g
Distilled water to
900.0 ml
Glucose
10% sol.
Urea
20% sol.
pH
6.8 – 7.0
Preparation: Sterilize the glucose and urea solutions by filteration. Prepare the basal
medium without glucose or urea. Adjust the pH and sterilize by autoclaving in a flask at
121°C for 15 min. Cool to above 50°C add the glucose and urea and tube the medium as
slant.
Use: The medium used to detect urease production by the bacteria.Red colour after
incubation indicate urease production.
Note: The medium may be used as broth by omitting the agar.
Voges Proskauer Test
Glucose phosphate broth
Composition: (See methyl red test).
Use: Used to test carbohydrate fermentation ability of bacteria with the production of
acetylmethyl carbinol or 2-3 butylene glycol. This test is usually done in conjuction with
the methyl red test since the production of acety1 methyl carbinol or butylenes glycol
usually results in insuff~cient acid accumulation, giving methyl red test negative.
Method: After incubation add 1 ml of 40 per cent potassium hydroxide and 3 ml of an anaphthol solution. A positive reaction is indicated by the development of pink colour in
2-5 min.
Precaution: a-naphthol is carcinogenic. Avoid body contact.
Xanthine Decomposition
Xanthine agar
Composition:
Medium A
Pancreatic digest of gelatin
5.0 g
Beef extract
3.0 g
Agar
20.0 g
Distilled water to
900 ml
Add components to water gently heat to dissolve the components.
Medium B
Xanthine
Distilled water to Gently heat to dissolve.
4.0 g
100 ml
Complete medium
Medium A
900 ml
Medium B
100 ml
Combine medium at medium by mix thoroughly. Steam sterilize at 121°C for 20
minutes. Poor plates.
Use: Used for testing xanthine decomposition ability of bacteria. Examine the plates
under a low power microscope for disappearance of xanthine crustals. (clearing) around
the bacterial growth.
Catalase Test
Solution required: 3% H,02 solution -1 ml approx.
Procedure: Place a drop of three percent H20, on colonies of nutrient agar.
Result: Prompt effervescence indicates catalase production.
Note: Culture media containing blood are unsuitable for the test asblood contain catalase.
Or
Procedure: Take colony growth on nichrome wire. Insert wire loop in to the H2O 2
solution.
Result: Prompt effervescence indicates catalase production.
Oxidase Negative
Regent Required
a.
Oxidase reagent: One percent solution or tetra methyl-p-phenylene diamine
hydrochloride.
Procedure:
1 . Put a drop of freshly prepared one percent solution of oxidase
reagent on a piece of filter paper.
2.
With a sterile loop pick a test colony and rub it on the paper in the area
impregnated with oxidase reagent.
Result: 1f organikm is oxidase positive, paper will become deep purple blue in colour in
a few seconds.
Procedure:
Pour oxidase reagent over the colonies of the test organism on the culture plate.
Result: The colonies of oxidase positive organisms rapidly develop a deep purple blue
colour.
Bile Solubility Test
Solution required: 10% sodium deoxycholate solution.
Procedure:
1. Add one drop of 10% sodium deoxycholate solution to microbial suspension.
2. Incubate at 37°C.
3: Examine for clearing at 15 min., 20 min. and 1 h.
Result: Clearing should occur within 30 min.
Coagulase Test
1. Slide method
Solution required: Citrated plasma.
Procedure:
1. Place three separate drops of saline on a clean slide.
2. Suspend a loopful of test colony in two of these and a loopful of positive
control organism (Staphylococcus aureus) in the third.
3. With a sterile loop, add a drop of citrated plasma to one test and the positive
suspension.
Result: Occurrence of clumping in test and positive control within. 10 s indicates a
positive result. The saline control should remain evenly suspended. (Negative control)
Note: This test detects the presence of “clumping factor” and is not a true coagulase test.
a. Tube method
Procedure:
1. Prepare 1/10 dilution of plasma in 0.85% saline.
2. Emulsify a few colonies of positive control (Staphylococcus aureus) and the
test isolate into two separate tubes containing 1 a 1/10 dilution of plasma in
0.85% saline.
3. Incubate at 37OC.
4. Examine for coagulation at 1, 3 and 6 h.
Result: Conversion of the plasma into a soft 'or stiff gel, seen on tilling the tube to a
horizontal position indicates a positive, result.
Negler Test I
This test indicates the production of alpha-toxin, an exotoxin by Clostridium perfringens.
Requirement:
Egg yolk agar: Composition-See Lecithinase production.
Use: Divide an egg yolk agar plate into two equal sections. Spread a loopful of
Clostridium perfiingens antitoxin over half the plate and allow to dry. Inoculate the plate
with a loopful of the test culture covering both halves by single streak. Incubate at 37OC
under anaerobic conditions.
Result: Production of an opaque zone of precipitation around the area of growth and
absence of this precipitation in the area with specific alpha-antitoxin indicates positive
test.
Optochin Test
Requirement: Blood agar: See page 78
Use:
1. Divide a blood agar plate into three equal sections.
2. Inocula one with a known Streptococcuspneumoniae, another I with a
Streptococcus viridans and the third with the test isolate.
3. Keep the cultures separately.
4. Place a 5 microgram Optochin disc (ethylene hydrocupreine hydrochloride) in
the centre of the plate.
5. Incubate at 37OC overnight and observe the zone of inhibition.
Hydrolysis of Fats
This test detects the ability of an organism to decompose fats. This causes rancidity in
foods due to the formation of fatty acids.
Use: Prepare nutrient agar plates containing 1% fat (oil/butter/ghee) and neutral red
indicator. Streak the test organism in these plates. Incubate at 37'C for 2 days. Observer
plates for the presence of red globules under the colonies.
Result: Acid production is indicated by change in the colour of indicator from yellow to
red. Maintain a control with uninoculated.
Dehydrogenase Activity
Prepare six tubes containing 1.0 ml. of inoculum. 1.0 of M/10 phosphate buffer of pH 7
and 0.5 ml of resazurin solution.
Add 1.0 ml of water to tube number 1.
Add 1.0 ml of M/10 glucose to tube number 2.
Add l .0 ml of M/10 lactose to tube number 3.
Add 1.0 ml of M/10 sodium lactate to tube number4.
Add 1.0 ml of M/10 sodium succinate to tube number 5.
Add 1.0 ml of M/10 sodium fumarate to tube number 6.
Mix the contents thoroughly.
Incubate at room temperature and observe for colour change from blue to pink every 15
min for one hour. Record the time of colour change.
Use: This test detects the ability of an organism to produce dehydrogenases. This causes
removal of hydrogen from the substrate. The indicator dye reacts with this hydrogen and
changes colour.
2
Cultivation Media
1. Bacteria
Actinomycetes medium
(Bennet’s agar)
Composition:
Glucose
10.0 g
Casein
2.0 g
Yeast extract
1.0 g
Beef extract
1.0 g
Agar
20.0 g
Distilled Water to
1000 ml
pH
7.3
0
Preparation: Autoclave the medium at 121 C for 20 min.
Use: Used for the cultivation of actinomycetes, specially Streptomyces species. This
medium enhances sporulation.
Actinomycetes medium
(Dextrose tryptone agar)
Composition:
Glucose
Tryptone
K2HPO4
NaCl
FeSO4.7H2O
Agar
10.0 g
5.0 g
0.5 g
0.5 g
0.1 g
20.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Steam sterilize the medium at 121°C for 20 min.
Use: Used for isolation of actinomycetes.
Actinomycetes Medium
(Glycerol yeast extract agar)
Composition:
Glycerol
K2HPO4
L-aspargine
Agar
Trace salt solution(see oatmeal agar)
Distilled water to
pH
Preparation: Steam sterilize the medium at 12 1°C for 20 min.
Use: Used for isolation of actinomycetes.
10.0 g
1.0 g
1.0 g
20.0 g
1.0 ml
1000 ml
7.4
Actinomycetes Medium
(Oatmeal agar)
Composition:
Oatmeal
Agar
Trace salt solution
Distilled water to
pH
Pridham and Gottlieb trace salt solution
CuSO4.5H2O
FeSO4.7H2O
20.0 g
20.0 g
1.0 ml
1000 ml
7.2
0.64 g
0.11 g
MnCL2.4H2O
0.79
ZnSO4.7H2O
0.15 g
Distilled water to
1000 ml
Preparation: Cook the oatmenl in 1 lit. water for 20 min. Filter through cloth. Restore
volume of filtrate to 1 litre. Add I ml of trace salt solution. Adjust the pH. Add agar.
Steam sterilize the medium at 121°C for 20 min.
Use: Used for isolation of actinomycetes.
Actinomycetes Medium
(Potato-carrot agar)
Composition:
Diced potato
150.0 g
Diced carrot
30.0 g
Tap water to
1000 ml
pH
6.5
Preparation: Cook the potato and carrot in 1 litre of boiling tap Mater for 30 min. Filter
through muslin and adjust the volume to 1 Litre. Adjust the pH and add agar. Steam
sterilize the medium at 121°C for 20 min.
Use: Used for isolation of actinomycetes.
Actinomycetes Medium
(Carbon utilization medium-Shirling and Gottlieb)
(NH4)2SO4
2.64 g
KH2PO4
2.38 g
K,HP04
5.65 g
MgS04.7H2O
1.0
Trace salt solution (See page No. ....)
1.00 ml
Agar
20.0 g
Carbon source(Sterilize separately by filtration) 10.0 g
Distilled water to
1000 ml
pH
7.2
0
Preparation: Steam sterilize the medium at 121 C for 20 min. Add sterile carbon source
solution after sterilization in the medium.
Use: Used to test carbon utilization ability of actinomycetes, important in identification.
Actinomycetes Medium
(Synthetic agar)
Composition:
K2KPO4
MgSO4.7H2O
KCl
FeSO4.7H2O
NaNO3
Glycerol
Agar
Distilled water to
pH
Preparation: Steam sterilize the medium at 121°C for 20 min.
1.0 g
0.5 g
0.5 g
0.01 g
2.0 g
30.0 g
20.0 g
1000 ml
7.2
Use: Used for isolation of actinomycetes.
Actnomycetes Medium
(Yeast glucose agar)
Composition:
Yeast extract
10.0 g
Glucose
10.0 g
Agar
20.0 g
Distilled water.to
1000 ml
pH
7.2
Preparation: Steam sterilize the medium at 121°C for 20 minutes.
Use: Used for isolation of actinomycetes.
Actinomycetes Medium
(Yeast extract-malt extract agar)
Composition:
Yeast extract
4.0 g
Malt extract
10.0 g
Glucose
4.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.3
0
Preparation: Steam sterilize the medium at 121 C for 20 minutes.
Use: Used for isolation of actinomycetes.
Actinomycetes Medium
Composition:
Glycero
Sodium propionate
Sodium caseinate
K2HPO4
Asparagine
MgS04.7H2O
FeS04.7H2O
Distilled water to
Preparation: Steam sterilize the medium at 121°C for 20 min.
Use: Used for cultivation of Actinomyces species.
Acetobactor Medium
l5.0 g
4.0 g
2.0 g
0.5 g
0.1 g
0.1 g
0.001 g
1000 ml
Composition:
Yeast extract
10.0 g
Glucose
3.0 g
CaCO3
10.0 g
Agar
20.0 g
pH
7.1
0
Preparation: Autoclave at 121 C for 20 min. Mix CaCO3 b: shaking the medium before
pouring.
Use: Used for isolation of Acetobacter and Gluconobacter.
Acetobacter Medium
(Hoyer's medium)
Composition:
(NH4)2SO4
0.1 g
K2HPO4
0.01 g
KH2PO4
0.09 g
MgS04.7H2O
0.025 g
FeCl3.6H2O
0.002 g
Ethanol (15% v/v)
20 ml
Distilled water
80 ml
Preparation: Dissolve the salts in 80 ml of distilled water. Dispense as 4 ml aliquots in to
test tubes and autoclave at 1210C. Ethanol may then be added as sterile filtered solution
at the rate of 1 ml per tube.
Use: Used to cultivate Acetobacter. Acetobacter grow slowly, may take up to 14 days to
develop.
Acetobacter Medium
(Acetobacter broth)
Compostion:
Peptone
Glucose
(NH4)2SO4
Yeast extract
Solution A
Solution B
Distilled water to
Solution A
K2HP04
KH2PO4
3.0 g
18.0 g
1.0 g
2.0 g
5 ml
5 ml
1000 ml
50.0 g
50.0 g
Distilled water to
500 ml
Solution B
MgSO4.7H2O
NaCl
FeSO4
MnSO4
Conc. HCL
Distilled water
Preparation: Steam sterilize at 1210C for 20 minutes.
Use: Used for cultivation of Acetobacter.
20.0 g
1.0 g
1.0 g
1.0 g
1.0 ml
500 ml
Acholeplasma Medium
Composition:
Papaic digest of soyabean meal
PPLO broth without crystal violet
Fresh yeast extract solution
(25.0 g Baker's yeast/100 ml water)
Agar
pH
PPLO broth without crystal violet
Beef heart, infusion from
Peptone
NaCl
Preparation: Autoclave at 1210C for 20 min.
Use: Used for cultivation of Acholeplasma.
10.0 g
900.0 ml
100.0 ml
3.0 g
7.8
225.0 g
9.0 g
4.5 g
Alcaligenes Medium
Composition:
K2HPO4
7.32 g
CaCl2.2H2O
0.014 g
MgSO4.7H2O
0.002 g
FeSO4.7H2O
0.04 g
Ammonium tartarate
4.6 g
KH2PO4
1.09 g
Distilled water to
1000 ml
pH
7.5
0
Preparation: Autoclave the medium at 121 C for 20 min.
Use: Used for cultivation of Achromobacter and Alcaligenes speck.
Agrobacterium Isolation Medium
Composition:
Mannitol
10.0 g
Sodium nitrate
4.0 g
Magnesium chloride
2.0 g
Calcium propionate
1.2 g
Magnesium phosphate.
0.2 g
Magnesium sulphate
0.1 g
Sodium bicarbonate
0.075 g
Magnesium carbonate
0.075 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Autoclave the constituents at 115°C for 20 min. Allow to cool to 50-550C
and aseptically add the following:
Berberine
275 ppm
Sodium selenite
100 ppm
Penicillin G
60 ppm
Streptomycin sulphate
30 ppm
Cyclohexamide (85-100% active)
250 ppm
Tryothicin
1 ppm
Bacitracin (65 units/mg)
100 ppm
Use: Highly specific for the isolation of Agrobacerium tumefaciens,
A. radiobacter group from soil.
Ammonification Medium
Composition:
KH2PO4
3.00 g
KCl
0.20 g
MgSO4.7H2O
0.20 g
NaCl
0.20 g
CaSO4
0.10 g
FeSO4
0.01 g
Peptone
10.00 g
Distilled water to
1000 ml
0
Preparation: Steam sterilize the medium at 121 C for 20 minutes.
Use: Used to isolate ammonifying bacteria. Bacteria are inoculated into tubes containing
5 ml of the above medium. After 10 days of incubation production of ammonias is testd
by means of Nessler’s reagent.
Anaerobic Bacteria Medium
Cooked meat broth (Robertson)
Composition:
Cooked meat filtrate
Fresh bullocks heart
500.0 g
Water
500 ml
Sodium hydroxide (1 N NaOH)
1.5 ml
Mince the heart. Add in alkaline water Simmer for 20 min. Filter through muslin cloth.
Dry minced meat, on filter paper partially.
Complete broth
Cooked meat filtrate
500 ml
Peptone
2.5 g
NaCl
1.25 g
pH
7.8
Preparation: Steam at 100°C for 20 min. Add 1 ml pure HCL and filter. Adjust the pH
8.2. Steam again at 100°C for 30 min. Adjust the reaction to pH 7.8.
Complete medium
Place the meat pieces in tubes to a depth of about 2.5 cm. Cover these pieces with about
10 ml broth. Autoclave at 1210C for 20 min.
Use: Used for isolation of anaerobic bacteria.
Note: Before inoculation boil the tube vigorously in water bath to drive away oxygen.
Cool and inoculate. Put a layer of liquid paraffin if essential.
Anaerobic Broth
Composition:
Casein
20.0 g
Glucose
10.0 g
NaCl
5.0 g
Sodium thioglycollate
2.0 g
Sodium formaldehyde
Sulfoxylate
1.0 g
Methylene blue
2.0 mg
Distilled water to
1000 ml
pH
7.2
Preparation: Distribute the broth in to tubes. Autoclave the medium at 1210C for 20 min.
Use: For cultivation of anaerobic microorganisms especially Clostridium.
Anaerobic Medium
(Thioglycollate medium)
Composition:
Yeast extract (water solube)
5.0 g
Casein hydrolysate (Pancreatic digest)
15.0 g
Glucose
5.5 g
L-cysteine
0.5 g
Agar
0.75 g
Sodium chloride
2.5 g
Sodium thioglycollate
0.5 g
Methylene blue
1.0 ml
Distilled water to
1000 ml
pH
7.1
Preparation: Dissolve the ingredients except thioglycollate and indicator in water by
heating. Add thioglycollate and adjust the pH. Filter. Add indicator solution. Sterilize at
121°C for 15 min. Cool and use.
Use: Used for isolation of anaerobic bacteria.
Note:
1. If the upper third is blue in colour, anaerobic condition should be stored by
boiling again for few minutes and cooling rapidly.
2. Resazurin sodium solution 1 in 1000, may be used replacing methylene blue.
Anaerobic Bacteria Medium
(Lapage et al., 1970)
Composition:
K2HOP4
NH4CL
Na2SO4
MgSl4.7H2O
CaCl2.2H2O
Yeast extract
Ferrous sulphate (see below)
Reducing agent (see below)
Sodium lactate (70%)
Resazuri n (see below)
Distilled water to
pH
Gas N2 : C02
Ferrous sulphate
FeSO4, 7H2O
0.5 g
1.0 g
1.0 g
2.0 g
0.1 g
1.0 g
10.0 ml
10.0 ml
3.5 g
1.0 ml
1000 ml
7.4
80 : 20
0.5 g
Distilled water to
100 ml
Reducing agent
Ascorbic acid
1.0 g
Sodium thioglycollate
1.0 g
Distilled water to
100 ml
Redox indicator (Resazurin)
Resazurin
0.10 g
Distilled water to
100 ml
Use: Used for the cultivation of Desulfomonas, Desulfobacter Desulfotomaculum and
Desuvovibrio.
Anaerobic Bacteria Medium
(Bagnra et at. 1985)
Composition:
K2HPO4
KH2PO4
(NH4)2SO4
MgCl2
CaCl2
FeS04.7H2O
Yeast extract
NaHCO3
Cellulose
Cysteine hydrochloride
Resazurin (See page 51)
Distilled water to
pH
Gas Argon or Nitrogen
Use: Used for cultivation of cellulomonas species.
2.21 g
1.5 g
1.3 g
0.1 g
0.02 g
0.001 g
5.0 g
0.8 g
10.0 g
0.5 g
1.0 ml
1000 ml
7.4
Anaerobic Bacteria Medium
(Hungate, 1950)
Composition:
K2HPO4
KH2PO4
(NH4)2SO4
CaCl,
NaCl
MgSO4
0.5 g
0.2 g
0.5 g
0.05 g
1.0 g
0.05 g
Rumen fluid (see below)
150.0 ml
NaHCO2
5.0 g
Resazurin (see page 51)
1.0 ml
Cysteine hydrochloride
0.5 g
Cellulose
10.0 g
Distilled water to
1000 ml
pH 7.5
7.5
Gas N2 : CO2
80 : 20
Rumen fluid
Take 2000 ml fresh rumen fluid. Allow to settle down coarse particles. Decant supenatant
to 1000 ml. Add 4.0 g yeast extract to it, pass nitrogen and incubate at 370C for
overnight. Then centrifuge at 8000 rpm for 10 min at 6 to 8OC. Collect the supernatant in
screw – capped bottle, pass nitrogen and store in refrigerator or -20°C. Pass nitrogen
on every use. Fermenting slurry of anaerobic digester may be processed in
similar manner and used in place of rumen fluid.
Use: Used for cultivation of clostridiurn, Bacteroides Butyrivibrio
and Bacterium species.
Anaerobic Bacteria Medium
(Sijpesteijn, 1951)
Composition:
K2HPO4
KH2PO4
(NH4)2SO4
NaCl
MgSO4
CaCI2 anhydrous
Yeast extract
NaHCO3
Cellulox
Resazurin (see page 51)
Cysteine hydrochloride
Distilled water to
pH
Gas N2 : CO2
Use: Used for cultivation of Ruminococcus species.
Anaerobic Bacteria Medium
(Sowers et al., 1984)
0.6 g
0.4 g
0.4 g
1.2 g
0.2 g
0.2 g
60.0 g
5.0 g
10.0 g
1.0 ml
1.0 g
1000 ml
7.5
80 : 20
Composition:
K2HPO4
NH4Cl
NaCl
KCI
MgSO4.7H2O
CaCl2.2H2O
Yeast extraat
Trace elements (see p.54)
Trace vitamins (see p.54)
Sodium acetate
Resazurin (see below)
Reducing agent (see below)
Distilled water to
pH
Gas N2 : CO2
Trace elements
Nitrilotriacetic acid
FeC12.4H2O
MnC12.4H2O
CaCl2
CoC12.6H2O
ZnCl2
H3BO3
Sodium molybdate
Distilled water to
Trace vitamins
Biotin
Folic acid
Vitamin B12
Pyridoxine HCl
Thiamine
Riboflavin
Nicotinic acid
DL-Calcium pantothenate
p-aminobenzoic acid
Lipoic acid
Distilled water to
Reducing agent
0.06 g
0.05 g
2.34 g
0.08 g
0.63 g
0.014 g
0.10 g
1.0 ml
1.0 ml
4.1 g
1.0 ml
12.5 ml
1000 ml
6.8
80 : 20
4.5 g
0.4 g
0.1 g
0.02 g
0.17 g
0.1 g
0.019 g
0.01 g
100 ml
2.0 mg
2.0 mg
0.1 mg
10.0 mg
5.0 mg
5.0 mg
5.0 mg
5.0 mg
5.0 mg
5.0 mg
100 ml
Cysteine hydrochloride
2.0 g
Adjust pH
9.0 g
Sodium sulphide 9H2O
2.0 g
Distilled water to
100 ml
Use: Used for cultivation of Methanosarcina, Methanococcoides, Methanlobus and
Methanococcus.
Anaerobic Bacteria Medium
(Corder et al., 1983)
Composition:
K2HPO4
0.3 g
KH2PO4
0.3 g
NH4Cl
2.7 g
(NH4)2SO4
0.3 g
NaCl
0.61 g
MgSO4.7H2O
0.13 g
MgCI2.6H2O
0.053 g
FeSO4.7H2O
0.01 g
Cocl2
0.001 g
Na2MoO4.2H2O
0.0002 g
NiCl2.6H2O
0.0002 g
Nitrilotriacetic acid
0.015 g
Trace vitamins (see page 54)
1.0 ml
Sodium acetate
2.5 g
Resazurin (see page 5 1)
1.0 ml
Reducing agent (see page 54)
12.5 ml
Distilled water to
1000 ml
pH
6.8
Gas H2 : CO2 80 : 20
Use: Used for enrichment and cultivation of methanogenic bacteria specifically
Methanogenium.
Anaerobic Bacteria Medium
(Balch et al., 1979)
Composition:
K2HPO4
NH4Cl
NaCl
KCl
0.14 g
0.25 g
18.0 g
0.35 g
MgSO4.7H2O
3.45 g
MgC12.6H2O
2.75 g
CaCl2.2H2O
0.14 g
Fe(NH4)2(SO4)2
0.002 g
Yeast extract
2.0 g
Trypticase
2.0 g
Trace elements (see below)
1.0 ml
Trace elements (Balch et al.)
Nitrilotriacetic acid
1.5 g
MgSO4.7H2O
3.0 g
MnSO4.2H2O
0.5 g
NaCl
1.0 g
CoCl2
0.1 g
FeSO4.7H2O
0.1 g
CaCL2.2H2O
0.1 g
ZnSO4
0.1 g
CuSO4.5H2O
0.01 g
AIK(SO4)2
0.01 g
H3BO3
0.01 g
Na2MoO4.2H2O
0.01 g
Distilled water to
100 ml
Dissolve nitrilotriacetic acid with KOH to pH 6.5 and then add other salts.
Trace vitamins (see page 54)
1.0 ml
Sodium acetate
1.0 g
Resazurin (see page 5 1)
1.0 ml
Reducing agent (see page 5 1)
12.5 ml
Distilled water to
1000 ml
pH
6.8
Gas H2 : CO2
80 : 20
Use: Used for enrichment and cultivation of Methanobrevibacter spp, Methanococcus,
Methanomicrobium, Methanogenium and Methanoplanus.
Anaerobic Bacteria Medium
(Balch et d, 1979)
Composition:
K2HPO4
KH2PO4
(NH4)2SO4
NaCl
0.3 g
0.3 g
0.3 g
0.6 g
MgSO4.7H2O
0.13 g
CaCl2.2H2O
0.008 g
FeSO4
0.002 g
Yeast extract
2.0 g
Trypticase
2.0 g
Trace elements (see page 54)
1.0 ml
Trace vitamins (see page 54)
1.0 ml
Sodium acetate
2.5 g
Sodium formate
2.5 g
Resazurin (see page 51)
1.0 ml
Reducing agents (see page 54)
12.5 ml
Distilled water to
1000 ml
pH
6.8
Gas N2 : CO2 or H2 : CO2
80 : 20
Use: Used for enrichment of methanogenic bacteria, specifically Methanobacterium,
Methanobrevibacter, Methanogenium and Methanospirillum.
Azotobacter Medium
(Nitrogen free glucose broth)
Composition:
K2HPO4
1.0 g
MgSO4.7H2O
0.2 g
FeSO4.7H2O
0.05 g
CaCl2.2H2O
0.1 g
Na2MoO4.2H2O
0.001 g
Glucose
10.0 g
Distilled water to
1000 ml
Preparation: If this medium is to be used immediately, sterilization is not necessary.
When it must be stored for long time, it should be sterilized.
If it is to be sterilized, the glucose should be sterilized separately in 100 ml of water at
1150C for 15 min. The remainder of the medium is steam sterilized at 121°C for 20 min.
After sterilization, the two solutions are mixed aseptically and dispensed in to
sterilized bottles.
Use: Used for isolation of Azotobacter.
Nitrogen free glucose agar
Add 2.5% agar in above broth. Prepare the medium in similar manner.
Azotobacter Medium
(Ashby's nitrogen free medium-modified)
Composition:
Mannitol
15.0 g
MgSO4.7H2O
0.2 g
K2HPO4
0.2 g
Ferric chloride (10% aqueous solution)
0.5 ml
Molybdenum trioxide (10% aqueous solution)
1 drop
Agar
15.0 g
Distilled water to
1000 ml
pH
7.2
0
Prepamtion: Steam sterilize at 121 C for 20 minutes.
Use: Used for the isolation of Azotobacter. Colonies appear flat, soft, milky and mucoid.
Azotobacter Medium
(Ashby's mannitol agar)
Composition:
Mannitol
20.0 g
K2HPO4
0.2 g
MgSO4.7H2O
0.2 g
NaCl
0.2 g
K2CO4
0.1 g
CaCO3
5.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Sterile CaCO3 should be added after steam sterilization of remainder
medium at 1210C for 20 minutes.
Use: Used for isolation of Azotobacter.
Azotobacter Medium
(Burk's nitrogen free medium)
Composition:
Burk's salt
Fe Mo mixture
Sucrose
Distilled water to
pH
Burk's salt
MgSO4
K2HPO4
1.3 g
1.0 ml
20.0 g
1000 ml
7.0
20.0 g
80.0 g
KH2PO4
20.0 g
CaSO4
13.0 g
Fe Mo mixture
FeCl3
1.45 g
Na2NoO4
0.253 g
Distilled water to
1000 ml
Prepnration: Steam sterilize the medium at 11 5°C for 20 minutes.
Use: Used for isolation of Azotobactor.
Azotobacter Medium
(Jensen's medium)
Composition:
Sucrose
20.0 g
K2HPO4
1.0 g
MgSO4.7H2O
0.5 g
NaCl
0.5 g
FeSO4
0.1 g
Na2MoO4
0.005 g
CaCO3
2.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.0
Preparation: Add all components except CaCO3 and agar. Adjust the pH. Add agar.
Sterilize at 115°C for 30 min. (not sterilize if freshly used). Add sterile CaCO3 in melted
sterile agar medium.
Use: Used for isolation of Azotobacter.
Azospirillum Semisolid Medium
Composition:
K2HPO4
KH2PO4
MgSO4
NaCl
CaCl2
FeCl2
Na2MoO4
Sodium malate
Bromothymol blue (0.05% Ehanol)
Agar
0.1 g
0.4 g
0.2 g
0.1 g
0.02 g
0.01 g
0.002 g
5.0 g
5.0 ml
2.25 g
Distilled water to
1000 ml
pH
6.8
Preparation: Steam sterilize the medium in autoclave at 121°C for 20 min.
Use: Used for cultivation of Azospirillum.
Azospirillum Medium
Composition:
K2HPO4
0.50 g
MgSO4.7H2O
0.10 g
NaCl
0.02 g
MnSO4.H2O
0.01 g
KOH
4.00 g
FeSO4.7H2O
0.05 g
Na2MoO4
0.002 g
CaCl2
0.01 g
Malic acid
5.0 g
Distilled water to
1000 ml
pH
6.0 : 7.0
Preparation: Steam sterilize the medium in autoclave at 121°C for 20 min.
Use: Used for cultivation of Azospirillum.
Bacteroides Medium
(VL medium)
Composition:
Sodium azide
Sterile beef bile
Tryptone
NaCl
Meat extract
Yeast extract
Cysteine hydrochloride
Glucose
Agar
Distilled water to
pH
Preparation: Adjust the pH. Sterilize by autoclaving at 1150C for 20 minutes. Use: Used
for cultivation of Bacterioides fragilis from fecal matter. After anaerobic incubation for
48 hrs. The colonies appear round (1mm in diameter) translucent and are found at the
highest dilutions. Colonies are surrounded by a whitish precipitate of bile salts.
Bdellovibrio Medium
(Cation - supplemented yeast extract, peptone, Na acetate and cysteine agar1YPSC agar)
Composition:
Bacto yeast extract
0.1 g
Bacto peptone
0.1 g
Sodium acetate trihydrate
0.5 g
L-Cysteine hydrochloride
5.0 mg
CaCl2
0.002 .M
MgSO4
0.003 M
Distilled water to
1000 ml
pH
7.6
0
Preparation: Autoclave at 121 C for 15 minutes.
Use: It is a medium of choice for plaque counts in most combinations of Bdellovibrio
strains and associate bacterial strains.
Beijerinckia Medium
(Becking's medium)
Composition:
Socrose
KH2PO4
K2HPO4
MgSO4.7H2O
FeCl3
Na2MoO4
Agar
Distilled water to
pH
Reparation: Steam sterilize at 1210C for 20 minutes.
Use: Used for isolation of Beijerinckia.
20.0 g
0.8 g
0.2 g
0.5 g
0.1 g
0.005 g
20.0 g
1000 ml
6.5
Bioluminescent Bacteria Medium
Composition:
Basal medium
(Tris) Hydrozyrnethyl amino methane
12.1 g
NH4Cl
1.0 g
K2HPO4
0.075 g
FeSO4.7H2O
0.028 g
Artificial sea water
1000 ml
(NaCl -23.4 g, KCl 1.5 g, MgSO4.7H2O -24.6 g, CaCl2 -2.9 g, D.W. -1 litre)
pH
7.5
Biochemicals should be prepared in the above basal medium as follows
Amino acids
0.5%
Sugars
1%
Preparation: Dissolve Tris. Adjust pH. Then dissolve remaining ingredients. Steam
sterilize at 12 1 "C for 15 minutes.
Use: Basal medium is used for cultivation of bioluminescent bacteria.
Note: By adding 2% agar, prepare a solid medium.
Bordetella Medium
(Bordet Gengou medium)
Composition:
Horse blood (defibrinated)
50.0 ml
Potato slices
125.0 g
Sodium chloride
4.5 g
Glycerol
10.0 ml
Proteose peptone
10.0 g
Agar
22.5 g
Distilled water to
1000 ml
pH
7.0
Preparation: Boil the slices in 250 ml water. Dissolve agar in 750 ml water and add to
potato decoction, glycerol and peptone. Adjust the pH. Sterilize in autoclave at 115°C for
10 minutes. Add sterile defibrinated horse blood to the cooled agar. Mix and pour into
sterile
Use: It is used for isolation of Bordetella.
Bordetella Medium
(Chacol blood agar)
Composition:
Charcol agar base
Beef extract
10.0 g
Strach
10.0 g
Peptone
10.0 g
Charcoal (Bacteriological grade)
4.0 g
Yeast extract
3.5 g
Agar
15.0 g
Distilled water to
1000 ml
pH
7.4
Adjust the pH before addition of charcoal. Steam sterilize the medium 121°C for 20 min.
Complete medium
Charcoal agar base
100.0 ml
Horse blood (sterile, defibrinated)
10.0 ml
Penicillin (100 i.u./ml)
0.3 ml
Cool the charcoal agar base at 50°C. Add other ingredients aseptically. Pour plates.
Use: It is a selective medium of Bordetella pertussis.
Note: Some peptone brands are inhibitory to Bordetella, hence may be omitted from the
medium.
Brucella Medium
(Jones and Morgan medium)
Composition:
Basal medium
Peptone
Agar
NaCl
10.0 g
20.0 g
5.0 g
Meat extract
5.0 g
Distilled water to
1000 ml
0
Steam sterilize the medium at 121 C for 20 min.
Complete medium
Basal medium
100.0 ml
Sterile inactivated
Horse serum (sterile, inactivated)
5.0 ml
Glucose solution 25% (sterile)
4.0 ml
Bacitracin solution (2000 units/ml in sterile water)
1.25 ml
Polyrnyxin B solution (5000 units/ml in sterile water)
0.12 ml
Cyclohexamides solution 10 mg/ml
1.0 ml
(Dissolve in acetone dilute with sterile water)
Preparation: Cool the sterilized basal medium at 550C. Add remaining solutions
aseptically. Pour plates.
Use: Used for cultivation of Brucella.
Brucella Medium
(Liver infusion broth-Huddleson)
Composition:
Minced beef liver
8 lbs
Distilled water to
400 ml
Steam for 2 hrs with frequent stirring. Strain through lint and autoclave for 30 min. Then
add
Peptone
Sodium chloride
Distilled water
Steam for 30 min, cool and adjust the pH to 7.0.
Use: Used for cultivation of Brucells abortus.
10 g
5g
500 ml
Brucella Medium
(Filde's eatract)
Composition:
Defibrinated sheep blood
50.0 ml
Hydrochloric acid
6.0 ml
Pepsin
8.0 g
Normal saline
100 ml
0
Preparation: Heat at 65 C for 2 hrs. Add 20% NaOH, until a violet red colour develops
with cresol red indicator. Then add pure HCL drop by until a definite red tint develops
with phenol red.
Complete medium
Fildes extract
5.0 ml
Melted nutrient agar (Sterile)
100 ml
Use: Used for cultivation of Brucella.
Brucella selected medium
Composition:
Infusion from beef heart
500.0 g
Tryptone
10.0 g
NaCl
5.0 g
Glucose
2.5 g
Gelatin
1.0 g
Sheep blood
100.0 ml
Agar
15.0 g
Antibiotic solution
10.0 ml
Distilled water to
1000 ml
pH
7.4
Antibiotic solution
cycloheximide
1.0 g
Bacitracin
250,000 u
Circulin
250,000 u
Polymyxin B
100,000 u
Distilled water
100 ml
Filter sterilize the antibiotic solution.
Preparation: Add all components except blood and antibiotic solution in distilled water.
Steam sterilizer at 1210C for 20 minutes.
Use: Used for selective isolation Brucella species.
Campylobacter Medium
Composition:
Beef extract
10.0 g
Peptone
10.0 g
Charcoal
4.0 g
Casein hydrolysale
3.0 g
Sodium deoxycholate
1.0 g
FeSO4.H2O
0.25 g
Sodium pyruvate
0.25 g
Cefoperazone solution (0.32% in water)
10.0 ml
Agar
20.0 g
Distilled water to
1000 ml
pH
7.4
Preparation: Add all components except cefoperazone solution in distilled water. Steam
sterilize at 1210C for 20 min. Sterilize the cefoperazone solution by filtration. Mix
aspectically.
Use: Used for the selective isolation of Compylobacter species.
Caulobacter Medium
Composition:
Peptone
0.5 g
Agar
20.0 g
Sea water (filered)
1000 ml
pH
7.0
0
Preparation: Steam sterilize the medium at 121 C for 20 min.
Use: Used for cultivation of Caulobacter.
Cellulolytic Bacteria Medium
(Omeliansky’s medium)
Composition:
(NH4)2SO4
K2HPO4
MgSO4.7H2O
CaCO3
Nacl
1.0 g
1.0 g
0.5 g
2.0 g
trace
Distilled water to
1000 ml
pH
7.0
Preparation: Add all components except CaCO3. Adjust the pH. Steam sterilize at
1210C for 20 min. (not necessary to sterilize if freshly used.) Add sterile CaCO 3 in both.
Add
2-3 strips of filter paper to each tube, part of the paper remaining above the surface of the
medium. Cellulose decomposing organisms within 15-20 days.
Cellulytic Microorganism Medium
(Mandel and Reese medium)
Composition:
Ammonium sulphate
Potassium dihyydrogen
Phosphate
Magnesuim sulphate
Calcium Chloride
Ferrous sulphate
Manganese sulphate
Proteose peptone
Urea
Zinc chloride
Calcium chloride
Distilled water to
pH
Preparation: Sterilize the medium at 1000C for 1 hrs.
Use: Used for assaying cellulase.
1.4 g
2.0 g
0.3 g
0.3 g
0.005 g
0.0016 g
1.0 g
0.3 g
0.0017 g
0.002 g
1000 ml
5.3
Clostridim Medium
(Lowbury and Lilly’s medium)
Composition:
(A) Agar base
Peptone water (see indole test)
1000 ml
Agar
50.0 g
0
Autoclave at 121 C for 30 min. This is used as is lower layer of the medium for the
upper layer.
(B) Human serum
Serum is prepared by treating plasma with 5% of sterile 10% calcium chloride at 370C.
Complete medium
Agar base
100 ml
Flide’s peptic
6.5 ml
Digest of sheep blood
Human serum (sterile)
40 ml
Neomycin sulphate
1.5 ml
(Sterile aqueos solution 10,000 µg/ml)
Heat fields digest at 550C for 30 min. Melt the agar base & cool it to 550C. Add the
remaining ingradients. Pour this as upper layer on the layer of agar base. Spread 150 I.U.
of CL. Perfringenes antitoxin over half of each surface.
Use: It’s both selective & differential medium.
1. Neomycin inhibits aerobic forming lecithinase producing organisms.
2. Swarming growth of Proteus is inhibited by excess agar.
3. Cl. Perfringenes produce lecithinase differentiating the colonies.
Clostridium Medium
(Sulphite polymyxin sulphadiazine agar)
Composition:
Basal Medium
Tryptone
15.0 g
Yeast extract
10.0 g
Ferric citrate
0.5 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.0
Preparation: Dissolve the ingredients by steaming and adjust the pH to 7.0. Sterilize by
autoclaving at 1210C for 15 min.
Complete Medium
Sodium sulphate (10% solution)
5.0 ml
Polymixin B (0.1% solution)
1.0 ml
Sodium sulphadiazine (1.2% solution)
1.0 ml
Basal medium
100 ml
Sterilize other solution by filtration separately and add to basal medium melted and
cooled to 450C. Mix well and pour plates.
Use: The medium is used for the enumeration of sulphite reducing clostridia, e.g.
Clostridium perfringens. Sulphate reducing bacteria produce black coloured colonies.
Clostridium Medium
(Sulfite agar)
Composition:
Tryptone
10.0 g
Sodium sulphite
1.0 g
Ferric citrate
0.5 g
Distilled water to
1000 ml
pH
7.0
use: Cl. nigrificans, causeing sultlde spollage ofthe food, produce black coloured
colonies on the medium. (H2S producer).
Coliform Medium
(Briliant green lactose bile broth)
Composition:
Peptone
10.0 g
Ox bile
200 ml
Lactose
10.0 g
Brilliant green (0.1% aq. solution)
13 ml
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve the peptone in approx. 500 ml water. Then ad the ox bile and
lactose, dissolve. Adjust the pH. Add the brilliant green solution. Make up the volume 1
litre by addition of water Distribute in tubes. Autoclave at 1150C for 15 min.
Use: This medium is used to determine coliforms in water. (in MPN test)
Significance: It eliminates false positive due to anaerobes.
Coliform Medium
(Glucose broth)
Composition:
Nutrient broth
100 ml
Glucose
1.0 g
0
Preparation: Sterilize at 109 C for 30 min.
Use: Glucose acts as reducing agent. The medium is useful for the growth of facultative
eanaerobes like coliforms.
Coliform Medium
(Endo's agar)
Composition:
Lactose
Basic fuchsin (10% solution in 95% alcohol)
Sodium sulphite
(2.5% solution)
Distilled water to
pH
8.0 g
0.3 ml
about
5 to 10 ml
1000 ml
7.2
Preparation: Sodiu, sulphite is added until the hot mixture turns pink in colour. Steam
sterilize the medium at 1000C for 1 hr.
Use: Selective medium for coliform. Coliform produce red coloured colonies on the
medium.
Coliform Medium
(Eosin methylene blue agar/EMB agar)
Composition:
Peptone
10.0 g
Lactose
5.0 g
Sucrose
5.0 g
Dipotasium hydrogen phosphate
12.0 g
Eosin Y
0.4 g
Methylene blue
0.065 g
Agar
15.0 g
Distilled water to
1000 ml
pH
7.2
0
Preparation: Steam sterilize at 121 C for 30 min.
Use: This medium is used for distinguishing lactose fermenting gramnegative bacteris
from non-lactose fermenting types by their appearance. Colonies of Escherichia coil have
a characteristic metallic sheen.
(Cystine tellurite blood agar)
Composition:
(A) Beef heart infusion agar
Beef heart infusion from
500.0 g
Tryptone
10.0 g
Sodium chloride
5.0 g
Distilled water to
1000 ml
pH
6.8-7.3
0
Preparation: Sterilize the medium at 121 C for 15 minutes.
(B) Cystine tellurite solution
Potassium tellurite(0.3%) (sterile)
15.0 ml
L-cystine
5.0 mg
Mix thoroughly
5.0 ml
C. Sheep blood (sterile)
Preparation: Melt 100 ml of 2% beef heart infusion agar and cool to 50°C. Add
aseptically cystine tellurite solution and sheep blood. Mix & pour into sterile petriplates.
Use: It is used for isolation Corynebacterium diphtheriae. Cystine allows detection of
H2S producing colonies of C. diptheriae from other Coiynebacterizrm species by their
characteristic brown halo around black colonies.
Corynebacterium Medium
(Hoyle's medium)
Composition:
Nutrient agar (sterile, pH 7.8)
200 ml
Horse blood
10.0 ml
Potassium tellurite solution (0.7 gm in 20 ml)
2.0 ml
0
Preparation: Cool nutrient agar at 55 C. Add sterile blood laked by alternate freezingand thawing for 5 times. Then add potassium tellurite solution. Mix gently without
forthing and pour in plates.
Use: For isolation of Corynebacterium diphtheriae.
Corynebacterium Medium
(Loeffler's medium)
Composition:
Ox, sheep or horse serum
30 ml
1 per cent glucose broth
80 ml
(see page 72)
Preparation: Mix and distribute. Sterilize by heating at 75°C for 20 min on 3 successive
days.
Use: This is used for the cultivation of Corynebacterium diphtheriae.
Corynebacterium Medium
(Mueller-Hinton tellurite agar)
Composition:
Casein
Casamino acids
L-Tryptophan
Megnesium sulphate
Potassium dihydrogen phosphate
Agar
Tellurite serum(0.4% Pot. Tellurite in serum).
5.0 g
20.0 g
0.05 g
0.1 g
0.3 g
20.0 g
12.5 ml
Distilled water to
1000 ml
pH
7.4
Preparation: Sterike the medium without tellurite serum in autoclave at 121°C for 20
min. Add tellurite serum to cooled sterile malted medium aseptically.
Use: Used for isolation Corynebacterium diphtheriae. C. diphtheria produce black
coloured colonies. Black colouration is due to diffusion of tellurite ions and later
reduction to tellurium metal that precipitates inside the bacterial cells.
Cytopltaga Medium
Composition:
Sodium acetate
Casein
Yeast extract
Beef extract
Agar
Distilled water to
pH
Preparation: Steam sterilize the medium at 121°C for 20 min.
Use: Used for the cultivation of Cytophaga species.
0.5 g
0.5 g
0.5 g
0.2 g
4.0 g
1000 ml
7.2
Cytophaga Medium
Composition:
Casein digest
Yeast extract
Distilled water to
pH
Preparation: Steam sterilize at I2 1 OC for 20 minutes.
Use: Used for the cultivation of Cytophaga species.
Derxia Medium
(Campelo and Dobereiner medium)
Composition:
Starch
K2HPO4
KH2PO4
MgSO4.7H2O
CaCl2.6H2O
FeCl3.6H2O
Na2MoO4.2H2O
Bromothyrnol blue (0.5% in absolute alcohol)
NaHCO3
Agar
Distilled water to
Preparation: Steam sterilize at 121°C for 20 minutes.
0.05 g
0.05 g
1000 ml
7.0
20.0 g
0.05 g
0.15 g
0.20 g
0.02 g
0.01 g
0.002 g
0.05 ml
1.0 g
20.0 g
1000 ml
Use: Use for isolation of Derxia.
Denitrifying Baceria Medium
(Asparagine nitrate citrate solution)
Composition:
Solution A
KNO3
1.0 g
Asparagine
1.0 g
Distilled water
250 ml
Solution B
Neutral sodium citrate
8.5 g
KH2PO4
1.0 g
MgSO4.7H2O
1.0 g
CaC12.6H2O
0.2 g
FeC13.6H2O
trace
Distilled water
250 ml
Solution C
Agar
20.0 g
Distilled water to
500 ml
Dissolve by heating.
Complete medium
Solution A
250 ml
Solution B
250 ml
Solution C
500 ml
Preparation: Steam sterilize solution A, B and C separately at 1150C for 20 minutes. Mix
aseptically.
Use: To test denitrifying activity of bacteria.
Enteric Bacteria Medium
(D.E.C. medium)
Composition:
Lab-Lemco
Peptone
Sodium taurocholate
Ferric chloride
Lactose
Agar
Neutral red (0.25 % aqueous solution)
Distilled water to
05.0 g
0.5 g
0.85 g
0.3 g
1.25 g
2.5 g
1.5 ml
100 ml
pH
7.4
Preparation: The medium is sterilized by boiling at 100°C for 1 hr.
Use: It is a selective medium for the isolation of intestinal bacteria.
Enteric Bacteria Medium
MacConkey's broth (Single strength)
Composition:
Peptone
20.0 g
Sodium taurocholate
5.0 g
Neutral red solution (2% in 50% ethanol)
3.5 ml
Lactose (10% aqu. solution)
100 ml
Distilled water to
1000 ml
pH
7.5
MacConkey's broth (Double strength)
Prepare as above but use twice the quantity of all ingredients except the distilled water. It
is required to accommodate 10 ml water during MPN determination.
Preparation: Add all components except indicator. Dissolve. Adjust the pH. Add
indicator. Autoclave at 1 15OC for 20 min.
MacConkey's agar
Add 2.0 per cent agar to single strength MacConkey's broth prior to sterilization.
Use: The medium is selective for isolation of enteric bacteria. The bile salt inhibits the
growth of non-intestinal organisms. It is also a differential medium. The medium
differentiate coliform from noncoliforms. Coliforms colonies appear pink in colour as
they ferment lactose, producing acid. The noncoliform's colonies are colourless.
Erwinia Selective Medium
Composition:
(NH4)2SO4
K2HPO4
Eosin Y
Methylene blue
Glycerol
Antibiotic solution
A gar
Distilled water to
Antibiotic solution
Cycloheximide
Novobiocin
Neomycin sulfate
Distilled water to
5.0 g
2.0 g
0.4 g
0.055 g
10.0 ml
10.0 ml
20.0 g
1000 ml
2.5 g
0.4 g
0.4 g
100 ml
Prepamtion: Add all components in distilled water except antibiotic solution. Steam
sterilize the medium at 121°C for 1.5 minutes. Sterilize antibiotic solution by filtration.
Add aseptically. Pour plates.
Use: Used for selective isolation of Erwinia.
Flat Sour Bacteria Medium
(Dextrose tryptone bromocresol purple agar)
Composition:
Tryptone
2.5 g
Glucose
5.0 g
Agar
20.0 g
Bromocresol purple
0.04 g
Distilled water to
1000 ml
pH
7.2
0
Preparation: Steam sterilize at 115 C for 20 minutes.
Use: Used for the detection of flat sour bacteria. Acid producing coloured colonies are
detected on the medium.
Frankia Medium
(BAP medium)
Composition:
K2HPO4
0.591 g
KH2PO4
0.952 g
NH4Cl
0.267 g
MgSO4.7H2O
0.095 g
CaCl2.2H2O
0.010 g
FeNa EDTA
0.010 g
Na propionate (filter sterilize)
0.480 g
Trace elements solution
1 ml
Vitamin solution
1 ml
Distilled water to
1000 ml
pH
7.0
Trace elements solution
H3BO4
2.86 g
MnCL2.4H2O
2.27 g
ZnSO4.7H2O
0.22 g
CuSO4.5H2O
0.08 g
NaMoO4.2H2O
0.02 g
CoSO4.7H2O
0.001 g
Distilled water to
1000 ml
Vitamin solution
Thiamine HCl
10 mg
Nicotinic acid
50 mg
Pyridoxine HCl
50 mg
Biotin
225 mg
Folic acid
10 mg
Ca Pantothenate
10 mg
Riboflavin
10 mg
Preparation: Steam sterilize at 121°C for 20 minutes. Sterilize the phosphate separately.
Mix.
Use: Used for isolation of Frankia.
Fusobacterium Medium
(Qmata and Disraely medium)
Composition:
Casitone
15.0 g
Yeast extract
5.0 g
Glucose
5.0 g
NaCl
5g
L-cysteine
0.75 g
Crystal violet
0.01 g
Streptomycin
0.01 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Sterilize the medium at 11 5°C in autoclave for 20 min.
Use: Used for cultivation of Fzlsobacteriunt.
General Medium
(Blood agar)
Composition:
Nutrient agar (PH 7.2)
500 ml
Defibrinated blood
25 ml
0
Preparation: Autoclave the nutrient agar at 12 1 C for 15 minutes. Cool to 45-50°C and
add 25 ml of sterile blood aseptically. Rotate to mix thoroughly avoiding accumulation of
air bubbles and pour immediately into sterile tubes or plates, i.e., before solidification.
Use: It is differential medium for haemolytic organisms. Three patterns of hemolysis can
occur on blood agar plate.
a) Beta hemolysis (Complete hemolysis), where formation of a clear zone with a
clear edge around the colony takes place.
b) Alpha hemolysis (Incomplete hemolysis), where production of methemoglobin and
a green cloudy zone around the colony is formed.
c) Gamma hemolysis (No hemolysis), where no change in colour surrounding the
colony on blood agar takes place.
General Medium
(Chocolate agaraysed blood agar/Heated blood agar)
Nutrient agar (pH 7.2)
100 ml
Defibrinated blood
5 ml
Preparation: Sterilize agar at 121°C for 15 minutes. Cool to 50°C and aseptically add the
sterile blood and mix thoroughly. Gradually heat to 75OC in water bath by mixing the
blood and agar by gently agitation until the blood begins to coagulate and become
chocolate brown in colour.
Use: (1) Used for identification of Neisseria. Pour tetramethly-p-Phenylene diamine
hydrochloride over inoculated chocolate agar plate and observe for the pink to purple
colour to the colonies. Neisseria develop above colour.
General medium (for pathogens)
(Hiss serum water)
Composition:
It consists of 1 part of sterile ox serum and 3 parts sterile of water.
Use: It is used in biochemical media of Neisseria, Corynebacterium etc. requirig serum
for growth.
General Medium
A. Nutrient agar
Composition:
Peptone
1.0 g
Sodium chloride
0.5 g
Beef extract
0.3 g
Agar
2.0 g
Distilled water to
100 ml
pH
6.8
Preparation: Dissolve all components in distilled water except agar. Adjust the pH. Add
the agar. Sterilize in autoclave at 121°C for 20 min.
B. Nutrient broth
Preparation: Same as nutrient agar except agar is omitted.
Use: Used for cultivation of heterotrophic microorganisms.
General Medium
(Plate count agar)
Composition:
Tryptone
5.0 g
Yeast extract
2.5 g
D-Glucose
1.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.0
Prepnration: Sterilize the medium in autoclave at 100°C for 1 hr.
Use: For counting the bacteria from soil samples.
General Medium
(Tryptone yeast glucose agar)
Composition:
Tryptone
5.0 g
Yeast extract
2.5 g
Glucose
1.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
0
Preparation: Sterilize the medium in autoclave at 115 C for 20 minutes.
Use: This is a general media used for cultivation of microorganisms.
General Medium
(Tryptone agar)
Composition:
Tryptone
10.0 g
Agar
20.0 g
Distilled water to
1000 ml
0
Preparation: Sterilize the medium at 121 C for 20 min.
Use: Used as a general media for isolation and cultivation of microorganisms.
General Medium
(Yeast extract agar)
Composition:
Yeast extract
Peptone
Agar
3.0 g
5.0 g
20.0 g
Distilled water to
1000 ml
pH
7.2
Prepamtion: Dissolve the ingredients by heating. Steam sterilize at 1310C for 20 minutes.
(Yeast extract milk agar)
The medium is prepared in the same way as yeast extract agar but 10 ml of skim or whole
milk is added per litre of broth.
Use: This medium is used for making plate counts of viable bacteria in milk.
Note: Plates should be used fresh because its pH tends to fall on keeping.
Gram-negative Bacteria Medium
(Penicillin agar)
Composition:
Plate count agar fortified with 10-35 units penicillin per ml of agar nay be used in place
of EMB agar to detect gram-negative bacteria n given sample.
Gram-negative Broth
Composition:
Peptone
Dextrose
D-mannitol
Sodium citrate
Sodium desoxycholate
Dipotassium hydrogen Phosphate
Sodium chloride
Potassium hydrogen phosphate
Distilled water to
pH
Preparation: Sterilize the medium at 121°C for 15 minutes.
Use: It is an enrichment medium for Salmonella and Shigella.
20.0 g
1.0 g
2.0 g
5.0 g
0.5 g
4.0 g
5.0 g
1.5 g
1000 ml
7.0
Haemophilus Medium
(Levinthal's medium)
Composition:
Sterile nutrient agar
100 ml
Sterile rabbit or human blood
5 ml
Preparation: Melt the agar, add the blood and heat mixture in boiling water. Allow tlie
deposit to settle and distribute the clear supernateants in petriplates.
Use: Used for cultivation of Haemophilus.
Haemophilus Medium
(Bacitracin heated blood agar)
Composition:
Chocolate blood agar (see page 82)
100 ml
Add bacitracin to get the concentration 10 I.u./ml in the medium.
Use: Used for isolation and cultivation of Haemophilus species.
Halophiles Medium
(Abram and Gibbon's medium)
Composition:
Casmino-acid
Yeast extract
Propeose-peptone
Na citrate
KC1
MgSO4.7H2O
FeSO4.7H2O
NaCl
Distilled water to
pH
Preparation: Steam sterilize at 1210C for 20 min.
Use: Used for cultivation of extreme halophiles.
0.5 g
1.0 g
0.5 g
0.3 g
0.2 g
2.0 g
0.005 g
20-30 g
100 ml
7.0
Halophiles Medium
(Eimhjellen medium)
Composition:
Yeast extract
MgSO4.7H2O
CaC13.2H2O
NaCl
Distilled water to
pH
Preparation: Steam and sterilize at 1210C for 20 minutes.
Halophiles Medium
(Milk medium of Dussault and Lachance)
Composition:
MgSO4.7H2O
Mg(NO3)2.6H2O
FeCl3.7H2O
0.5 g
2.0 g
0.5 g
25.0 g
100 ml
7.2
5.0 g
1.0 g
0.025 g
Peptone
5.0 g
Glycerol
10.0 g
NaCl
200 g
Distilled water to
1000 ml
Preparation: Dissolve the ingredients except salt in 500 ml of water. Add 30 g of agar.
Sterilize at 121°C for 20 min. Sterilize the slightly moist salt and 50 g of skimmed milk
powder dissolved in 500 ml water in separate vessels. Add the hot agar solution to the
salt and dissolve as much of the salt as possible. Add the milk. Mix to dissolve
the remaining salt.
Use: Used for isolation of halophiles.
Halophiles Medium
Composition:
MgSO4.7H2O
Casamino acids
2.5 g
1.0 g
Yeast extract
Peptone
Trisodium citrate
KCl
NaCl
Distilled water to
pH
Preparation: Steam sterilize at 1210C for 20 minutes.
Use: Used for isolation of halophilic microorganisms.
1.0 g
0.5 g
0.3 g
0.2 g
25.0 g
1000 ml
7.2
Lactic Acid Bacteria Medium
(APT agar)
Composition:
Yeast extract
Tryptone
Glucose
Na citrate
NaCl
K2HPO3
MnCl2.H2O
MgSO4.7H2O
FeSO4
Na2CO3
7.5 g
12.5 g
10.0 g
5.0 g
5.0 g
5.0 g
0.14 g
0.8 g
0.04 g
1.25 g
Tween-80
Distilled water to
pH
Preparation: Sterilize in autoclave at 1210C for 20 min.
Use: Used for cultivation of lactic acid bacteria.
Lactic Acid Bacteria Medium
(Neutral red chalk lactose agar/NRCLA)
Composition:
Peptone
Meat extract
Yeast extract
Lactose
CaCO3
Neutral red (1%)
Agar
Distilled water to
pH
Preparation: Steam sterilize at 109°C for 1 hr.
Use: Used for cultivation of lactic acid bacteria.
0.2 g
100 ml
6.1
3.0 g
3.0 g
3.0 g
10.0 g
15.0 g
5.0 ml
25.0 g
1000 ml
6.8
Lactobacilli Medium
(DeMan, Rogosa and Sharpe's medium)
Composition:
Peptane
10.0 g
Meat extract
10.0 g
Yeast extract
5.0 g
Glucose
20.0 g
Tween 80
1 ml
Dipotassium hydrogen Phosphate
2.0 g
Sodiuin acetate
5.0 g
Triammonium citrate
2.0 g
Magnesium sulphate
200 mg
Manganese sulphate
50 g
Agar
20.0 g
Distilled water to
1000 ml
pH
6.0
Preparation: Dissolve the ingredients. Adjust the pH and autoclave at 1210C for 20 min.
Use: This is a selective medium supports good growth or Lactobacillus.
Lactobacilli Medium
(Tomato juice agar)
Composition:
Peptone
1.0 g
Casein hydrolysate
1.0 g
Tomato juice
40 ml
Agar
2.0 g
Distilled water to
60 ml
Preparation: Dissolve all components except agar in tomato juice. heat gently. Adjust to
pH 5.0 wit11 lactic acid. Dissolve the agar in the 60 ml water and mix both solutions
while they are hot. Filter through thin layer of cotton and autoclave at 1150C for 10 min.
Use: It is selective medium for the growth of Lactobacili.
Significance: Tomato juice provides growth factor and pH 5.0, makes the medium
selective.
Precaution: At pH 5.0, agar tends to be hydrolysed on heating and repeated melting
should be avoided.
Leuconostoc Medium
(Sucrose gelatin agar)
Composition:
Tryptone
10.0 g
Yeast extract
5.0 g
NaCl
5.0 g
K2HPO4
5.0 g
Glucose
1.0 g
Sucrose
50.0 g
Gelatin
50.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Take 500 ml water. Dissolve all ingredients except geltain and agar. Adjust the pH. Add gelatin. Mix 4% agar solution (500 ml). Steam sterilize at 1210C for 20
min.
Note: Addition of 0.02% sodium azide makes the medium more selective.
Use: Sucrose inhances capsule or slime production. Used for cultivation of Leuconostoc
mesenteroides and better slime formation.
Leptospira Medium
(Dinger's modification of Noguchi's medium)
Composition:
(a) Semi solid medium
Nutrient agar, 3%
6 ml
Distilled water
100 ml
Sterile inactivated serum
10 ml
Preparation: Mix the agar and water and sterilize by autoclaving at 1210C for 20 min.
Cool and add the serum with sterile precautions, distribute in tubes.
(b) Solid medium
Tryptose-phosphate broth (Dehydrated)
0.2 g
Agar
1.0 g
Distilled water to
90 ml
pH
7.5
0
Preparation: Sterilize mixture by autoclaving at 121 C for 15 min. After cooling add 10
ml sterile rabbit serum and I mi hemoglobin solution prepared by lysing washed and
packed sheep erythrocytes in 20 vol cold distilled water. Sterilize by filtration. Heat
mixture at 560C for 30 min and pour plates.
Use: Used for isolation of Leptospira
Leptospira Medium
(Korthof's medium-modified)
Composition:
(i) Peptone salt solution
Peptone
0.8 g
NaCl
1.4 g
NaHCO3
0.02 g
KCl
0.04 g
CaCl2
0.04 g
KH2PO4
0.24 g
Na2HPO4.2H2O
0.88 g
Distilled water to
1000 ml
pH
7.2
Preparation: Steam the ingredients at 100°C for 20 min and filter through Whatman No. 1
filter paper. Adjust the pH. Bottle in 100 ml amounts and autoclave at 115OC for 15 min.
(ii) Blood serum
Rabbit's serum is inactivated by heating at 56°C for 30 min & sterilized by seitz
filtration.
(iii) Hemoglobin solution
Hemoglobin is prepared from blood clot after removal of serum & then adding equal
volumes of distilled water and then freezing & thawing repeatedly. It is sterilized by seitz
filtration.
Complete medium
Peptone salt solution
100 ml
Sterile blood serum
8ml
Sterile hemoglobin solution
0.8 ml
Use: Used for cultivation of Leptospira.
Leptospira Medium
(Modified Stuart's medium)
Composition:
(i) Stock solutions
(a) L-asparagine (dextro-rotatory)
1.3%
(b) NH4CI
0.54%
(c) MgC12.6H2O
2.03%
(d) Thiamine hydrochloride
0.1%
(e) Sodium chloride
0.58%
(f) Phenol red
0.02%
Preparation: Prepare the stock solutions with distilled water and sterilize by autoclaving
at 115°C for 15 min.
(ii) Phosphate buffer solutions of pH 7.6
Sterilize by autoclaving at 121°C for 30 min.
(iii) Blood serum
Serum is prepared as for Korthofs medium.
Complete medium
L-asparagine solution
Ammonium chloride solution
Magnesium chloride solution
Sodium chloride solution
Thiamine hydrochloride solution
Phenol red solution
Distilled water
Phosphate buffer, pH 7.6
Sterile inactivated rabbit s e m
Preparation: Mix all ingredients except the rabbit serum and autoclave at 11 5°C for 15
min. Add the serum with sterile precautions, distribute and use.
Use: Used for the cultivation of Leptospira.
Manganese bacteria medium
(Bromfield medium)
Composition:
KH2PO4
0.005 g
MgSO4 .7H2O
0.002 g
(NH4)2SO4
0.010 g
Ca3(PO4)2
0.010 g
Yeast extract
0.005 g
MnSO4.4H2O
0.005 g
Distilled water to
100 ml
pH
6.0
Preparation: Medium is autoclaved at 11 5°C for 15 min. To solidify the medium add
2% agar.
Use: Used for isolation of (Manganese bacteria) bacteria capable of
converting (oxidizing) mn++ (mangnus) to mn+++ (mangnic) ions.
Minimal Mediuru
Composition:
Potassium dihydrogen phosphate
3.0 g
Disodium hydrogen phosphate
6.0 g
Sodium chloride
5.0 g
Ammonium chloride
2.0 g
Magnesium sulphate
0.1 g
Glucose
8.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.0
Preparation: Magnesium sulphate should be separately autoclaved and added to the rest
autoclaved constituents at 1210C for 20 minutes.
Use: It is a synthetic medium allow the growth of only prototrophs.
Minimal Medium
Composition:
Glucose
(NH4)2SO4
K2HPO4
MgSO4
Agar
Distilled water to
pH
Preparation: Sterilize medium at 1210C for 20 minutes.
2.0 g
1.0 g
7.0 g
0.5 g
20.0 g
1000 ml
6.8
Use: It allows the growth of only prototroplls of Escherichia coli.
Minimal Medium
(Davis and Mingioli-synthetic medium)
Composition:
Glucose (sterile 10% soln)
20.0 ml
K2HPO4
7.0 g
KH2PO4
3.0 g
Sodium citrate
0.5 g
(Na3C6H2O7.2H2O) MgSO4.7H2O
0.1 g
(NH4)2SO4
1.0 g
Trace element solution
5.0 ml
Agar
20.0 g
Distilled water to
1000 ml
PH
7.1
Trace element solution
Composition:
FeSO4.7H2O
0.5 g
ZnSO4.7H2O
0.5 g
MnSO4.3H2O
0.5 g
H2SO4
0.1 ml
Distilled water to
1000 ml
Preparation: Glucose is added with sterile precaution, as a sterile solution after the
remainder ofthe medium has been autoclaved, other sugar may be substituted for the
glucose, particular amino acids or growth factors may be added. The citrate may be
omitted.
Use: Medium allows the growth of only protoroph.
Mutant Isolation Medium
(Streptomycin agar)
Composition:
To 1 litre of sterile liquidnutrient agar (50°C), aseptically add 100 mg of streptomycin
sulphate. Pour directly into sterile petri plates.
Use: To isolate streptomycin resistant mutants, only streptomycin resistant organism can
form colony on the medium.
Mycobactericrrn Medium
(Dorset's egg medium)
Composition:
Beaten egg (213 hen's eggs)
7.5 ml
Sterile nutrient broth
2.5 ml
Preparation: Prepare the beaten eggs. Mix and add in the nutrient broth with sterile
precaution. The medium is solidified in inspissator at 75°C.
Use: For the isolation of Mycobacteriuin tuberculosis.
Precaution: It is advised to add malachite green solution (2%), 1.25 ml per 100 rnl for
the isolation of Mvcobacterium tuberctrlosis.
Mycobacteriurn Medium
(Dubo's medium)
Composition:
Casein hydrolysate (20 % solution)
10 ml
NaH2PO4
6.25 g
M2PO4
1.00 g
Sodium citrate
1.5 g
Ferric citrate
0.1 g
Magnesium sulphate (7H2O)
0.6 g
Tween 80 (10% solution)
5.0 ml
Distilled water to
1000 ml
pH
7.2
Preparation: Add all the components in water. Autoclave at 1150C for 20 min. Before
use add 0.1 ml of 9% solution of bovine albumin. Albumin solution is sterilized by
filtration.
Use: Used for cultivation of Mycobacterium tuberculosis.
Mycobacteriurn Medium
(Loewenstein and Jensen medium)
Composition:
(a) Mineral salt solution
KH2PO4
MgSO4
Magnesium citrate (. 14 H2O)
Asparagine
Glycerol
Distilled water to
Boil the solution for 2 hrs and cool overnight.
(b) Salt-starch solution
Mineral salt solution
Starch
2.4 g
0.24 g
0.6 g
3.6 g
12.0 ml
600 ml
600 ml
30 g
Mix. Heat in a water bath at 56°C with constant stirring for 15-20 min. Keep in
water bath for I hr. 1000 ml.
(c) Egg fluid
Obtained by beating 20-13 eggs aseptically. (see page 13)
Complete medium
Salt-starch solution
600 ml
Egg fluid
1000 ml
Mix. Strain through sterile gauze.
Then add Malachite green (sterile) (2 % aqueous solution)
20 ml
Preparation: Distribute in tubes. Sterilize by keeping in inspissator at 85°C for 30 min.
on first day and at 75OC for 30 min. on second day, without disturbing the tubes placed
in slanting position. The tubes are stored in the refrigerator and used within a months.
Use: Used in cultivation of Mycobacterium tuberculosis.
Note: According to Jensen 1955, starch may be omitted because (1) It makes the medium
more difficult to prepare. (2) It is unnecessary for the growth of A4ycohacteriunt
tuberculosis.
Myxobacteria Medium
(CT Agar)
Composition:
Casein digest
20.0 g
MgSO4.7H2O
2.0 g
Potassium phosphate
500 ml
buffer (0.02 M, pH 7.6)
Agar
20.0 g
Distilled water to
1000 ml
pH
7.6
Preparation: Add casein digest and NgSO4.7H2O in distilled water. Adjust the pH.
Autoclave at 121°C for 20 minutes. Steam sterilize the phosphate broth at 121°C for 20
minutes, separately, mix both solutions aseptically. Pour plates.
Use: Used for cultivation of Myxobacteria.
Myxobacteria Medium
(Dowrkin's defined medium)
Composition:
Tryosine
0.6 g
Asparagine
0.5 g
Leucine
1.0 g
Isoleucine
0.5 g
Proline
0.5 g
Arginine
0.1 g
Histidine
0.05 g
GI ycine
0.05 g
Lysine
0.25 g
Methionine
0.05 g
Phenylalanine
0.15 g
Tryptophan
0.05 g
Serine
0.1 g
Threonine
0.1 g
Valine
0.1 g
Djenkolic acid
0.1 g
Alanine
0.05 g
Glycogen
3.0 g
MgSO4.7H2O
1.0 g
Distilled water to
1000 ml
Make the medium 0.01 M in K2HP0, -KH,P02 buffer (pH 7.6).
Use: Used for the cultivation of Myxobacteria. Omission of Phenylalanine and
tryptophan initiate the fruiting stage.
Myxobacteria Medium
(McCurdy's medium)
Composition:
Raffinose
Sucrose
Galactose
Soluble starch
Casitone
MgSO4.7H2O
K2HPO4
Agar
Distilled water to
pH
Use: Used for cultivation of Myxobacteria.
1.0 g
1.0 g
1.0 g
5.0 g
2.5 g
0.5 g
0.25 g
20.0 g
1000 ml
7.4
Myxobacteria Medium
(Noren's medium)
Compsotion:
Casein hydrolysate
Asparagines
2.5 g
2.5 g
K2HPO4
NaCl
MgSO4.7H2O
CaC12.2H2O
MnSO4.4H2O
Ferric citrate
Distilled water to
pH
Use: Used for cultivation of fruiting Myxobacteria.
2.0 g
1.2 g
0.01 g
0.01 g
0.001 g
0.003 g
1000 ml
7.5
Myxobacteria Medium
(Stanier medium)
Composition:
KNO3
1.0 g
K2HPO4
0.2 g
MgSO4.7H2O
0.1 g
CaCl2.2H2O
0.1 g
FeC12.6H2O
0.02 g
Agar
10.0 g
Distilled water to
1000 ml
0
Preparation: Sterilize at 121 C for 20 min. Pour plates. Place sterilize piece of filter
paper on the surface of agar.
Use: Used to isolate cellulolytic Myxobacteria.
Mycoplnsma Medium
(Specimen collection media/SCM)
Composition:
Trypticase soy broth
3.0 g
Bovine albumin powder
0.3 g
Phenol red 20%
0.5 ml
Distilled water to
1000 ml
Penicillin
2000 µ/ml
Thallium acetate
1:2000
Preparation: Suspend the trypticase soy broth in water. Mix thoroughly and warm gently
till solution is complete, add phenol red and autoclave at 100°C for I5 min. Add the test
at temperature of 50°C or below. Pass through seitz filter, distribute in 2 to 3 ml
quantities in sterile capped vial. Store in the refrigerator.
Use: SCM for mycoplasmas.
Mycoplasma Broth
Composition:
Mycoplaslrza broth base
100 ml
Horse serum
10 ml
Fresh yeast extract
5.0 g
Dexyribonucleic acid (Calf thymus DNA)
0.02 g
Phenol red
0.002 g
Glucose
0.1 g
Arginine
0.1 g
Preparation: Mix all the constituents in mycoplasma broth base except serum and
autoclave at 121°C for 15 minutes. Allow the medium to cool to 45-50°C and add horse
serum aseptically.
PPLO agar: Add 2% agar.
Use: Used for isolation of mycoplasmas. They appear as round colonies 250-750 pm in
diameter with dense centre and less dense periphery giving the fried egg appearance.
My coplastnu broth
(Liquid medium)
Composition:
PPLO broth without crystal violet pH 7.8
70.0 ml
Yeast extract pH 7.0
10.0 ml
Horse serum (unheated)
20.0 ml
Glucose solution, 10 % w/v Sodium deoxyribonucleate 10.0 ml
(calf thymus) solution in 80 w/v 0.2 %
1.0 ml
Thanllous acetate solution in 80 w/
1.0 ml
K2HP04. 1 M Pencillin solution
2.0 ml
50,000 units per ml
2.0 ml
Phenol red solution, 0.2 % w/v
1.0 ml
Yeast extract solution
Baker's yeast
1.0 kg
Dejonized water
1000 ml
HCL, Analar grade about
6.5 ml
Preparation: Add the yeast to 500ml water at 50°C. Mix well. Add the
remaining water. Warm to 80°C and add acid to adjust the pH to 4.5.
Mix well and heat at 80°C for 20 min. Allowthe cells to settle and filter
the supernatant through seitz filter. Adjust the pH 7.0 before use.
Sterilize the solutions by filtration and add them to the sterilize broth.
Use: This medium is used for primary isolation of Mycoplasmas.
Mycoplasma Medium
Composition:
PPLO broth or PPLO agar without violet pH 6.0 70.0 ml
Yeast extract pH 7.0 (see page 102)
10.0 ml
Horse serum (unheated)
20.0 ml
Urea solution, 20 % w/v
5.0 ml
Phenol red solution 0.2 % w/v
1.0 ml
Penicillin solution, 200,000 units per ml
0.25 ml
pH
6.0
Preparation: Sterilize the solutions by filtration and add to sterilize broth. Check the pH.
Use: Used for cultivation of T-strain mycoplasmas.
Mycoplasma Medium
(Sloppy agar medium)
Composition:
PPLO broth without crystal violet pH 7.8
70.0 ml
PPLO agar base without crystal violet pH 7.8
10.0 ml
Yeast extract pH 7.0 (see page 102)
10.0 ml
Horse serum (unheated)
20.0 ml
Sodium deoxyribonucleate (calf thymus) solution 0.2 % w/v
1.0 ml
Thallus acetate solution, 1 in 80 w/v
1.0 ml
Dipotassium hydrogen phosphate solution, 1M
2.0 ml
Penicillin solution, 50,000 unit per ml
0.2 ml
Preparation: Sterilize the solutions by filtration and add to the sterile broth. Add the 10
1n1 sterile molten PPLO agar base. Distribute in sterile tube. Prepare slopes.
Use: Used for cultivation of mycoplasmas.
Mycoplasmn Medium
(Transport medium - Staurt 1959)
Composition:
Anaerobic salt solution
Thioglycollic acid
Distilled water (pass through anion exchange
resin column to remove chlorine, if present)
IN NaOH
Sodium glycerophosphate (20% aqueous)
Calcium chloride (1 % aqueous)
Agar solution
Agar
2 ml
900 ml
12-15 ml
100 ml
20 ml
6.0 g
Distilled water to (Chlorine free) Dissolve by heating
1000 ml
Complete medium
Melt the agar solution and add the anaerobic salt solution. Adjust the pH to 7.2. Add 4 ml
methylene blue solution (0.1%). Mix. Distribute in tubes. Autoclave at 121°C for 15 min.
Use: Used as transport medium when delay for isolation of mycoplasmas/gonococci
from specimen is unavoidable.
Mycoplasma Medium
(Kelton medium)
Composition:
Heart infusion broth
1000 ml
Yeast extract
10.0 g
Peptone
10.0 g
pH
7.9
0
Preparation: Autoclave at 12 1C for 20 minutes and before use add 100 ml horse serum.
Use: For the storage of mycoplamas.
Mycoplasma Medium
[Chalquest and fabricant medium - modified)
Composition:
(1) Solid phase
PPLO agar, dehydrated
39.6 g
Soluble starch
5.0 g
Trypticase
5.0 g
Thallium acetate Nicotinamide adenine
0.25 g
dinucleotide WAD)
0.1 g
Swine serum (inactivated)
100 ml
Penicillin
1,000,000 units
Distilled water to
1000 ml
Preparation: Dissolve the agar by heating. Add trypticase, starch, thallium acetate and
phenol red. Autoclave at 121°C for 20 min. Cool to 50°C. Add swine serum, penicillin
and the NAD (prepare in 10 ml quantities). Sterilize by filtration.
( 2) Liquid phase
PPLO borth dehydrated, without crystal violet
18.9 g
Cysteine violet HCl
0.1 g
Phenol red
0.025 g
Thallium acetate
0.25 g
NAD
0.1 g
Swine serum (inactivated)
100 ml
Penicillin
1,000,000 units
Distilled water to
1000 ml
Use: This medium is also very suitable for the propagation of M. pneumnoniae. And M.
synoviae.
Mycoplasma Broth
Composition:
Bacto tryptose
20.0 g
Dextrose
5.0 g
NaCl
5.0 g
Na2HPO3
2.5 g
Glycerol
5.0 g
Yeast extract (see page 102)
1.0 g
Distilled water
1000 ml
0
Pig serum (inactivated at 56 C for 30 min)
100 ml
Penicillin
100 units/ml
Preparation: The complete medium is sterilized by seitz.
Use: This medium is used for all routine isolation, filtration and vaccine production of M.
mycoides var mycoides.
Mycoplasma Medium
(Hayflick medium)
Composition:
(a) Horse serum broth
Bacto PPLO broth (without crystal violet)
70 ml
Horse serum
20 ml
Yeast extract (see page 102)
10 ml
ThaIIous acetate (I % w/v)
2.5 ml
Penicillin 100,000 units per ml
0.2 ml
0
Preparation: Sterilize the PPLO broth at 121 C for 20 min Sterilize other solutions by
filtration. Add to broth aseptically. Distribute in sterile tubes.
(b) Horse serum agar
Use bacto PPLO agar instead of broth in above medium.
Use: Used for cultivation of niycoplasnias.
Mycoplasma Medium
(A2 medium)
Composition:
Trypticase soy broth powder
30.0 g
Distilled water to
pH
Bottle in 76 ml amounts
Autoclave at 1210C for 15 min. Before use add____
Unheated horse serum (Sterile, pH -6)
Yeast extract (see page 102)
Benzyl penicillin
Use: Used for cultivation of T-strain mycoplasmas.
1000 ml
7.0
20 ml
5 ml
1000 units/ml
Neisserin Transport Medium
(Amies transport medium)
Composition:
Potassium chloride
0.2 g
Calcium' chloride
0.1 g
Sodium chloride
0.3 g
Magnesium chloride
0.1 g
Disodium hydrogen phosphate
1.15 g
Potassium phosphate
0.2 g
Sodium thioglycollate
1.0 g
Charcoal
10.0 g
Agar
4.0 g
Distilled water to
1000 ml
pH
7.3
0
Preparation: Dissolve the ingredients in water. Sterilize at 121 C for 20 minutes.
Use: It is used for transporting swab specimens especially suspected for N. gonorrhoeae.
Nitrosomonas Medium
(Ammonium medium)
Composition:
(NH4)2SO4
2.0 g
MgSO4.7H2O
0.5 g
FeSO4.7H2O
0.03 g
NaCl
0.3 g
MgCO3
10.0 g
K2HP04
1.0 g
Distilled water to
1000 ml
pH
7.3
Preparation: Add all ingredients in water. Adjust the pH. Sterilization is not necessary if
the inoculations are made as soon as the medium is made LIP. Sterilization at 121°C for
30 min. is desirable for storage.
Use: It is used for the isolation of Nitrosomonas, i.e , bacteria causing conversion of
ammonia to nitrite.
Pathogen Preservation Medium
(meat extract broth)
Peptone
10.0 g
Meat extract
10.0 g
NaCl
5.0 g
Distilled water to
1000 ml
pH
7.4
Preparation: Dissolve the ingredients by heating. Broth may be filtered if precipitate
appears. Sterilize by autoclaving at 121°C for 20 min.
Use: This broth is good for preservation of stock culture of pathogens.
Composition:
(NH4)2SO4
1.0 g
K2HPO4
0.5 g
MgSO4.7H2O
0.2 g
NaCl
2.0 g
NaHCO3
5.0 g
Yeast extract
0.1 g
Organic substrate
1.5 g
Tap water to
1000 ml
pH
7.0
Organic substrate may be malate, succinate or fumarate.
Preparation: Steam sterilize the medium at 121°C for 30 minutes.
Use: Used for cultivation of photosynthetic bacteria from Athiorhodaceae family.
Photosynthetic Bacteria Medium
Composition:
(NH4)Cl
1.0 g
K2HPO4
1.0 g
MgCl2
1.0 g
NaHCO3
1.0 g
Na2S.9H2O
1.0 g
Tap water to
1000 ml
pH
8.0
0
Preparation: Steam sterilize the medium at 121 C for 30 minutes.
Use: Used for cultivation of photosynthetic bacteria from Thiorhodaceae family.
Photosynthetic Bacteria Medium
Composition:
(NH4)Cl
1.0 g
KH2PO4
1.0 g
MgCl2
0.5 g
NaCl
0.3 g
NaHCO3
2.0 g
Na2S.9H2O
1.0 g
Fe
500 µg
Tap water to
7.3
pH
7.3
Preparation: Steam sterilize the medium at 121°C for 30 minutes.
Use: Used for cultivation of photosynthic bacteria from Chlorobeacae family.
Pneumococcal Medium
(Meat infusion broth)
Composition:
Lean meat, ox heart or beef
500.0 g
water
1000 ml
Peptone
10 to 20g
Sodium chloride
5.0 g
pH
7.4
Preparation: Remove all fat from the fresh meat and mince it. Add the minced meat to
the water and extract by keeping it in the refrigerator for 24 hrs. Strain through muslin.
Pseudomonas Agar F
Composition:
Peptone
20.0 g
Maltose
10.0 g
K2HPO4
1.5 g
Magnesium sulphate
0.73 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
0
Preparation: Steam sterilize the medium at 121 C for 20 minutes.
Use: Used for cultivation of Pseudomonas.
Pseullomonas Medium
(Cetrimide agar)
Composition:
Peptone
20.0 g
Potassium sulphate
10.0 g
Magnesium chloride
1.4 g
Cetyl trimethy ammonium bromide
0.3 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2 - 7.4
Use: It is a selective medium used for isolation for Pseudomonas. It inhibits the growth
of coliforms and Staphylococcus. Colonies of Pseudomonas are green due to the water
soluble pigment that diffuses into the medium.
Pseudomonas Medium
(Cetrimide agar)
Composition:
Basal medium
Peptone
20.0 g
Glycerol
10.0 ml
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve the ingredients. Adjust pH. Sterilize by autoclaving at 1210C for
30 min.
Complete medium
Melted basal medium
1000 ml
K2HPO4 (15%)
10.0 ml
MgSO4.7H2O
10.0 ml
Cetrimide solution (2%)
15.0 ml
K2HPO4, MgSO4.7H2O, and cetrimide solution are sterilized by filtration.
Use: It is a selective medium for isolation of Pseudomonas aeruginosa. Cetrimide 0.03%
make the medium selective. The yellow fluorescence of the colonies of Pseudornonas
may be enhanced by examining the culture plates in a dark box fitted with a source of
UV
irradiation.
Psedomonas Medium
(King, Ward and Raney's medium)
Composition:
Medium A - Pyocayanin
Peptone
20.0 g
Glycergl
10.0 ml
MgC12 (anhydrous)
1.4 g
K2S04 (anhydrous)
10.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
Medium B - Fluorescein
Peptone
20.0 g
Glycerol
10.0 ml
K2HPO4 (anhydrous)
1.5 g
MgSO4.7H2O
1.5 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve the ingredients by heating. Adjust the pH. Sterilize in autoclave at
121°C for 15 min.
Use: For the cultivation of Psezidonionas aeruginosa. Plates of medium A are incubated
at 370C for 48 hrs or longer. Plates of medium B are incubated at 370C for 24 hrs and
then at room temperature for 2-3 days.
Note: Medium A enhances pyocyania (blue) pigment whereas medium B enhances
fluorescein (yellow) pigment.
Rhizobium Medium
(Bergersen's synthetic medium)
Composition:
Mannitol
10.00 g
Na2HPO4.12H2O
0.45 g
Sodium glutamate
1.10 g
MgSO4.7H2O
0.10 g
FeCl3.6H2O
0.02 g
CaC12.6H2O
0.04 g
Thiamine
100 µg
Biotin
200 µg
Distilled water to
1000 ml
pH
7.0
Preparation: Vitamin solutions are sterilized by filtration and added to the medium after
sterilization 115°C for 20 minutes.
Use: Used for isolation of Rhizobium.
Rhizobium Medium
(Congored yeast extract mannitol agar)
Composition:
Mannitol
10.0 g
K2HPO4
0.5 g
MgSO4.7H2O
0.2 g
NaCl
0.1 g
Yeast extract
1.0 g
Agar
20.0 g
Distilled water to
1000 ml
Congored 1% solution
2.5 ml
Preparation: Dissolve weighed amounts of all constituents in distilled water and
autoclave at 121°C for 20 minutes. Congored solution is to be sterilized separately and
added in the medium at the time of pouring in petriplates.
Use: Used for isolation of Rhizobium.
Rhizobium Medium
(Glucose peptone agar)
Composition:
Glucose
5.0 g
Peptone
10.0 g
Agar
20.0 g
Bromo cresol purple (1 % alcoholic solution)
10.0 ml
Distilled water to
1000 ml
pH
7.0
Preparation: Steam sterilize at 121°C for 20 minutes.
Use: Rhizobium rows poorly in this medium and causes little change of pH.
Rumen Bacterial Medium
(Bryant and Robinson medium)
Composition:
Glucose
Cellobiose
Soluble starch
Agar
Resazurin
Minerals 1
0.25 g
0.25 g
0.05 g
2.0 g
0.0001 g
3.75 ml
Minerals 2
3.75 ml
Na2CO3
0.2 g
Cysteine HCl –Na2S (of a solution containing 2.5% w/v of each)
Centrifuged rumen fluid
20 ml
Distilled water to
100 ml
1 ml
Mineral 1
K2HPO4
Distilled water to
0.6 g
100 ml
Mineral 2
KH2PO4
0.6 g
(NH4)2SO4
1.2 g
NaCl
1.2 g
MgSO4.7H2O
0.25 g
CaCl2
0.6 g
Distilled water to 100 ml
Preparation: The medium is sterilized in autoclave at 115°C for 20 min. The medium is
equilibrated with and incubated under a gas phase of 50% CO2 and 50% H2.
Use: Used for isolation of rumen bacteria.
Rumen Bacteria Medium
Composition:
Casitone
Yeast extract
Mineral I
Mineral I1
Centrifuged (clarified) rumen fluid
Agar
Resazurin
Sodium lactate (70% w/v)
Glucose
Maltose
Cellobiose
Crysteine HCl
1.0 g
0.25 g
15 ml
15 ml
20 ml
2.0 g
0.0001 g
1.0 g
0.2 g
0.2 g
0.2 g
0.05 g
Sodium bicarbonate
Distilled water to
0.4 g
100 ml
K2HPO4
Distilled water to
3.0 g
1000 ml
Mineral I
Mineral II
KH2PO4
3.0 g
(NH4)2SO4
6.0 g
NaCl
6.0 g
MgSO4.7H2O
0.6 g
CaCl2
0.6 g
Distilled water to
100 ml
Preparation: Sterilize the medium at 100°C for 1 hr in autoclave. The medium is
equilibrated with and incubated under 100% CO2.
Use: Used for isolation of rumen bacteria.
Salmonella - Shigella Agar
Composition:
Peptone
5.0 g
Beef extract
5.0 g
Lactose
10.0 g
Bile salt mixture
8.5 g
Sodium citrate
8.5 g
Ferric citrate
1.0 g
Brilliant green
0.033 g
Neutral red
0.025 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.0
Preparation: Add all components except agar and indicator. Adjust the pH. Add
indicators then agar. Sterilize at 115OC for 30 min.
Use: Used for selective isolation of Salnronella and Shigella.
Salmonella Medium
(Bismuth sulphite agar)
Composition:
Peptone
Beef extract
Disodium phosphate
Ferrous sulphate
Bismuth sulphite
Brilliant green
Agar
Distilled water to
10.0 g
5.0 g
4.0 g
0.3 g
8.0 g
0.025 g
20.0 g
1000 ml
pH
7.5
Preparation: Dissolve by boiling the ingredients in water. Do not 1 sterilize. Pour in
sterile petriplates.
Use: It is a selective medium used for cultivation of Sabnonella typhi. The colonies of
Saln1017ella appear black.
Salmonella Medium
(Brilliant green agar)
Peptone
10.0 g
Yeast extract
3.0 g
Lactose
10.0 g
Sucrose
10.0 g
NaCl
5.0 g
Agar
20.0 g
Brilliant green
0.0125 g
Phenol red
0.08 g
Distilled water to
1000 ml
pH
7.2
Preparation: Steam sterile at 115°C for 30 min.
Use: Used for isolation' of Salntonella, as Salmonella is non lactose fermenting, colonies
will be red.
Scrlmonella and Shigella Medium
(Dexycholate citrate agar - Hyne's modification of Leifson)
Composition:
Medium A
Meat extract
5.0 g
Peptone
5.0 g
Agar
22.0 g
Neutral red solution [2% in 50% ethenol]
1.25 ml
Lactose
10.0 g
Distilled water to
1000 ml
pH
7.4
Preparation: Dissolve the meat extract in 50 ml water. Make just alkaline to
phenolphthalein with 50% NaOH. Boil at 100°C and filter. Adjust the pH. Make the
volume 50 ml and add peptone. Dissolve the agar in 950 ml water by steaming at 100°C
for 1 hour. Mix two solutions. Add neutral red and lactose. Sterilize at 115°C for 15
minutes.
Solution B
Sodium citrate
Sodium thiosulphate
Ferric ammonium citrate
Sterile water
Dissolve by heating at
17.0 g
17.0 g
4.0 g
100 ml
60°C.
Solution C
Sodium deoxycholate
10.0 g
Sterile water
100 ml
Dissolve by heating at
60°C.
Complete medium
Medium A
100 ml
Solution B
5 ml
Solution C
5 ml
Melt the medium A and add solution B and C in this order under sterile condition.
Precaution: The medium should be poured immediately otherwise it
tends to become very soft in presence of deoxycholate.
Use: It is a selective medium for isolation of Salmonella and Shigello.
Salmonella Medium
(Glycerol saline transport medium)
Composition:
Glycerol
300 ml
NaCl
4.2 g
Na2HP04, anhydrous
10 g
Phenol red, (0.02% aqueous) About
15 ml
Distilled water to
700 ml
Preparation: Dissolve the sodium chloride in the water and add the glycerol. Add the
phosphate and heat to dissolve. Then add phenol red to give a purple-pink colour.
Distribute in tubes.
Use: It is used as transport medium for carrying specimens of faeces to laboratory for
isolation of enteric fever bacilli. The medium supresses the growth of other intestinal
bacteria.
Salmorzella Medium
(Kauffmann - Muller tetrathionate broth)
Composition:
(a) Thiosulphate solution
Sodium thiosulphate Na2S2O3.5H2O
50.0 g
Sterile water
Preparation: Same as in tetrathionate medium.
(b) Iodine solution
Potassium iodide
Iodine
Sterile water to
Preparation: Same as in tetrathionate medium.
(c) Ox bile solution
Desiccated ox bile
Sterile water
Dissolve with sterile precautions.
Complete medium
Nutrient broth (pH 7.4)
CaCO3
Brilliant green (1 in 1000 aq. solution)
Thiosulphate solution
Iodine solution
Ox bile solution
Preparation: Same as in tetrathionate medium.
Use: Used for selective isolation of Salmonella.
100 ml
25.0 g
20.0 g
100 ml
0.5 g
5 ml
90 ml
5.0 g
1 ml
10 ml
2 ml
5 ml
Salmonell Medium
(Selenite F broth)
Composition:
Sodium acid selenite [NaHseO3]
4.0 g
Peptone
5.0 g
Lactose
4.0 g
Disodium hydrogen phosphate
9.5 g
Sodium dihydrogen phosphate
0.5 g
Sterile distilled water to
1000 ml
pH
7.1
Preparation: Dissolve the ingredients with sterile precautions in water. Adjust the pH
and distribute in sterile tubes. Autoclave at 100°C for 30 min.
Precaution: Excessive heat is detrimental to the medium. Salts of selenium are very toxic
for animals and man.
Use: For the enrichment of typhoid and paratyphoid group from faeces.
Significance: This medium intibits coliform bacilli. But it may permits the growth of
Proteus.
Salmonella Medium
(Selenite cystine broth)
Composition:
Tryptone
5.0 g
Lactose
4.0 g
Na2HPO4
10.0 g
Sodium acid selenite
4.0 g
L-cystine
0.01 g
Distilled water to
1000 ml
pH
7.0
Preparation: Dissolve ingredients in sterile water aseptically. Steam at 100°C for 30 min.
Use: This is an enrichment medium for salmonellae, add 1 ml o sample to 9 ml of
selenite cystine broth.
Precaution: Salts of selenium are very toxic for man and animal.
Salmonella Medium
(Terathionate broth)
Composition:
(a) Thiosulphate solution
Sodium thiosulphate Na2S2O3.5H2O
24.8 g
Sterile water to
100 ml
Add the salt in water with sterile precautions. Steam at 100°C for 30 minutes.
(b) Iodine solution
Potassium iodide
20.0 g
Iodine
15.7 g
Sterile water to
100 ml
With sterile precautions dissolve the potassium iodide and iodine.
Complete medium
CACO3
2.5 g
Nutrient broth (pH 7.4)
78 ml
Thiosulphate solution
15 ml
Iodine solution
4 ml
Phenol red (0.02 % in 20% ethanol)
3 ml
Add the CaCO3 to the nutrient broth. Sterilize it by autoclaving at 121°C for 20 min.
Cool and add the thiosulphate, iodine and phenol red solutions with sterile precautions.
Distribute in 10 ml amounts in sterile tubes.
Use: The tetrathionate broth inhibits the coliform bacilli and allow the growth of the
typhoid - paratyphoid group. However, it can’t inhibit the growth of Proteus groups.
Salmonella Medium
(Wilson and Blair's medium)
Composition:
Solution A-Bismuth sulphite glucose phosphate mixture
Bismuth ammonio citrate
3.0 g
Sodium sulphite
10.0 g
Disodium hydrogen phosphate (12 H2O)
10.0 g
Glucose
5.0 g
Sterile water to
100 ml
With sterile precautions dissolve the bismuth ammonio-citrate in 25 ml boiling water and
sodium sulphite in 50 ml boiling water. Mix two solutions and boil. Add the sodium
phosphate crystals while boiling the mixture. Cool. Add the glucose dissolve in 25 ml
boiling water and cooled.
Solution B - Iron citrate brilliant green mixture
Ferric citrate solution (1% in sterile distilled water)
200 ml
Brilliant green solution (1% in sterile distilled water)
25 ml
Mix together
Complete medium
Sterile 3% nutrient agar
100 ml
Solution A
20 ml
Solution B
4.5 ml
Melt the agar and cool to 60°C. Add both solutions with sterile precautions and pour
plates.
Use: It is selective medium for the isolation Salmonella. Briliant green makes the
medium selective. It is also a differential as 5. paratyphi A produce green coloured
colonies while S. typhi and S paratyphi B produce black coloured colonies by reducing
sulphite to sulphate.
Salmonella Medium
(Wilson and Blair's medium-Reed's modification)
Composition:
Peptone (2% solution)
8.8 ml
Sea salt mixture
1.2 ml
Mannose (10%)
1.0 ml
Liquid bismuth
0.12 ml
Sodium sulphite (20%)
1.2 ml
Absolute alcohol
0.2 ml
Mercuric perchloride (1 : 10,000)
0.8 ml
Preparation: Sterilize the medium without alcohol at 121°C for 30 min. Add alcohol.
Sea salt water
CaC12
27.0 g
KC1
01.0 g
MgC12.6H2O
03.0 g
MgSO4.7H2O
1.75 g
Distilled water to
100 ml
Use: It is liquid enrichment medium for V. cholerae.
Note: Instead for seat salt mixture bazaar common salt may be used.
Soil Microorganisms Medium
(Asparagine mannitol agar)
Composition:
Disodium hydrogen phosphate
Potassium nitrate
MgSO4.7H2O
1.0 g
0.5 g
0.2 g
CaCl2.6H2O
0.1 g
Sodium chloride
0.1 g
FeCl2.6H2O
Trace
Asparagine
0.5 g
Agar
15.0 g
Distilled water to
1000 ml
Preparation: Add the agar and salt. Dissolve by heating. Add the mannitol. Adjust the
pH. Sterilize in autoclave at 121°C for 20 min.
Use: For the isolation of soil microorganisms.
Soil Microorganisms Medium
(Soil extract agar)
Composition:
Glucose
1.0 g
K2HPO4
0.5 g
Agar
20.0 g
Soil extract (stock)
100 ml
Distilled water to
900 ml
pH
7.2
Preparation: 1000 g of sieved garden soil is mixed thoroughly with 1000 ml of tap
water. A small amount of CaCO3 is added. Mixed throughly and filtered through paper.
Dissolve the agar in 900 ml by heating. Add other components. Adjust the pH and steam
sterilize at 1210C for 20 minutes.
Use: Used for isolation of microorganisms from soil.
Spirochete Medium
(Noguchi's medium)
Composition:
Sterile normal saline
80 ml
Fresh rabbits serum
10 ml
Nutrient agar (sterile melted 3%)
10 ml
Rabbits haemoglobin
12 ml
Procedure: Add rabbits serum to saline at 50°C. To the mixture add sterile nutrient agar
cooled to 50°C. Add rabbits haemoglobin. (3 ml of rabbits blood to 9 ml of sterile
distilled water.) Mix. Distribute in tubes and place at once in the refrigerator until the
agar gels. Incubate to test the sterility.
Use: Used for cultivation of spirochetes.
Staphylococcus Medium 110
Composition:
Protein digest
1.0 g
Yeast extract
0.25 g
Gelatin
3.0 g
D-mannitol
1.0 g
Lactose
0.2 g
NaCl
7.5 g
K2HPO4
0.5 g
Agar
2.5 g
Distilled water to
1000 ml
pH
7.0
Use: It is a selective medium for the isolation of Staphylococcus aureus. Phenol red may
be added as an indicator. Staphylococcus ferment mannitol and produce acid.
Staphylococcus Medium
(Baird Parker medium)
Composition:
Tryptone
Beef extract
Yeast extract
Sodium pyruvate
Glycine
Lithium chloride
Agar
10.0 g
5.0 g
1.0 g
10.0 g
12.0 g
5.0 g
20.0 g
Distilled water to
1000 ml
pH
7.0
Egg yolk emulsion Potassium tellurite
50 ml
solution (3.5%)
3.0 ml
Preparation: Dissolve all the ingredients in 950 ml distilled water. Srerilize by autoclave
at 121°C for 15 minutes. Cool to 50°C and add septically egg yolk emulsion and sterile
potassium tellurite solution. Mix and pour into plates.
Use: It is a selective medium for isolation of coagulase positive staphylococci from pus
and wou?d. The colonies of staphylococci appear black owing to reduction of potassium
tellurite to metallic telllurium (black).
Staphylococcus Medium
(Brain heart infusion broth)
Composition:
Infusion from calf brains
200.0 g
Infusion from beef heart
250.0 g
Peptone
10.0 g
Dextrose
2.0 g
NaCl
5.0 g
Na2HPO4
2.5 g
Distilled water to
1000 ml
pH
7.4
Preparation: Autoclave at 1 1 5OC for 30 minutes.
Use: Used for cultivation of staphylococci specifically when the culture is to be used for
performing coagulase test.
Staphylococcus Medium
(a) (Glycerol monoacetate agar)
Composition:
Heart infusion broth
100 ml
Glycerol monoacetate
1.0 g
Agar
2.0 g
Preparation: Dissolve the agar in broth by heating. Add the glycerol monoacetate and
autoclave at 1210C for 15 min.
Use: For the cultivation of Staphylococci.
Significance: Colonies of coagulase position staphylococci on this medium are orange,
yellow or buff whereas coagulase negative strains are porcelain white. There is good
differentiation after 48 hr incubation.
(b) (Salt glycerol monoacetate agar)
5% NaCl is .added hl Glycerol monoacelate agar.
Significance: Staphylococci grow in sodium chloride concentration that are high enough
to be inhibitory to many other bacteria. Thus it makes the medium selective.
Staphylococcus Meduim
(Mannitol salt agar)
Composition:
Mannitol
10.0 g
Peptone
10.0 g
Sodium chloride
75.0 g
Beef extract
1.0 g
Phenol red
0.025 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.4
Preparation: Autoclave at 115°C for 3 minutes.
Use: It is useful for the selective isolation of pathogenic staphylococci. Since most other
bacteria are inhibited by the high salt concentration. Colonies of pathogenic
staphylococci are surrounded by a yellou halo indicating mannitol fermentation.
Staphylococcus Medium
(Milk agar)
Composition;
Fresh milk (Sterile) Sterile nutrient agar
100 ml
(3.5% agar)
200 ml
pH
7.2
Preparation: Sterilize the milk by autoclaving at 121°C for 20 min. Melt the sterile agar.
Cool to 50°C. Mix with the milk and pour plates.
Use: For the isolation of Staphylococczrs.
Significance: Pigmentation of staphylococcal colonies is marked and easily recognised
against opaque white background.
Staphylococcus Medium
(Salt milk agar)
Milk agar (given above) with 7-10% sodium chloride.
Use: It is a selective medium for staphylococci. High salt inhibit the other organisms.
Staphylococcus Medium
(Sodium chloride selective medium)
Composition:
Peptone
1.0 g
Meat extract
0.3 g
NaCl
10.0 g
Distilled water to
1000 ml
pH
7.2
Preparation: Steam sterilize the medium at 121°C for 20 minutes.
Use: The medium is selective for the isolation of Staphylococcus aureus.
Staphylococcus Medium
(Vogel-Johnson agar)
Composition:
LiCl
Tryptone
Yeast extract
Mannitol
K2HPO4
Glycine
Agar
Phenol red
Distilled water to
Preparation: Steam sterilize at 121°C for 20 minutes.
Use: Used for isolation of Staphylococcus.
5.0 g
10.0 g
5.0 g
10.0 g
5.0 g
10.0 g
15.0 g
0.025 g
1000 ml
Streptococcus Medium
(Crystal violet blood agar)
Composition:
Sterile nutrient agar
90 ml
Sterile horse blood
10 ml
Crystal violet (1 in 1000 aq. solution)
0.2 ml
Preparation: Melt the agar, cool to approx. 50°C. Add the blood and crystal violet with
sterile precaution.
Use: For the cultivation of Streptococcus pyogenes.
Significance: Crystal violet inhibits the growth of some bacteria like Staphylococci.
Streptococcus Medium
(Glucose sodium azide glycerol agar)
Composition:
Tryptone
D-Glucose
20.0 g
5.0 g
Potassium dihydrogen phosphate
1.5 g
Dipotassium hydrogen phosphate
4.0 g
Sodium azide
0.5 g
Sodium chloride
5.0 g
Bromocresol purple
0.032 g
Distilled water to
1000 ml
pH
7.0
0
Preparation: Sterilize the medium at 121 C for 20 minutes.
Use: It is used for selective isolation for Streptococcus faecalis.
Streptococcus Medium
(Hartley's broth)
Composition:
Pancreatic extract
Fresh pig pancreas
50.0 g
Distilled water
150 ml
Absolut&lcoho
l50 ml
Concentrated hydrochloric acid about
2 ml
Preparation: Remove fat, mince the pancreas and mix it with the water and alcohol.
Shake the mixture thoroughly in a large stoppered bottle. Allow it to stand for 3 days at
room temperature, shaking occasionally. Strain through muslin and filter through paper.
Measure the volume ofthe filtrate and add 0.1% hydrochloric acid. This extract
can be stored for about two months in stopped bottles in the refrigerator.
Complete medium
Lean meat, ox heart or beef
150 g
Distilled water to
250 ml
Sodium carbonate 0.8% solution
250 ml
Pancreatic extract
5 ml
Chloroform
5 ml
Concentrated HCl
4 ml
Preparation: Mix the minced meat and water and heat them at a temperature of 80°C.
The add the sodium carbonate. Cool to 450C and add pancreatic extract and chloroform.
Incubate the mixture at 45°C for 3 hr. stirring frequently. Add acid, heat at 100°C for 30
min. Filter. Store the broth in an acid condition in bottle with 0.25% chloroform. Shake
vigorously and frequently in the next two or three days. Store in cool, dark place. Before
use, adjust the pH to 8.0 and heat at 100°C for 1 hr. to precipitate phosphate. Filter while
hot and allow to cool. Adjust the pH to 7.6, distri~utean d autoclave at 11 5OC
for 20 minutes.
Note: Used for obtaining luxuriant growth of exacting organisms. Streptococci grow well
in this medium. The medium is used specially for the production of diphtheria toxin.
Streptococcus Medium
Composition: (Horse flesh digest medium)
Horse flesh
90.0 g
Water
350 ml
Sodium carbonate
1.2 g
Pancreatin
1.75 g
Concentrated hydrochloric acid
2.0 ml
Peptone
3.5 g
Calcium chloride
0.44 g
Sodium biocarbonate
0.7 g
pH
8.0
Preparation: Mince the meat and mix it with 150 ml of cold water, raising the
temperature to 80°C. Add the remainder of the cold water and the sodium carbonate.
Adjust the pH. Add the pancreatic. Heat the mixture at 56°C for 6 hr. Then add the acid
and again boil at 100°C for 30 min. Filter, add the peptone and filter. Add the sodium
bicarbonate Sterilize by filtration.
Use: It is used for cultivation of haemolytic streptococci.
Streptococcus Medium
(Sodium azide medium)
Composition:
Peptone
10.0 g
Sodium chloride
5.0 g
Dipotassium hydrogen phosphate
5.0 g
Potassium dihydrogen phosphate
2.0 g
Glucose
5.0 g
Yeast extract
3.0 g
Sodium azide, (NaN3)
0.25 g
Bromocresol purple (1.6% solution in ethanol)
2.0 ml
Distilled water to
1000 ml
pH (self adjusted)
6.8
Preparation: Dissolve the ingredients in water. Sterilize in the autoclave at 121°C for 15
min.
Use: This medium is used for the isolation of Streptococcus fecalis.
Streptococcus Medium
(Todd-Hewitt broth)
Composition:
Fat free minced beef
450 g
Distilled water to
1000 ml
pH
7.6
Keep in the refrigerator overnight. Heat at 85°C. Filter. Then add 2% peptone. Adjust the
pH. Add
Glucose
0.2%
NaCl
0.2%
Preparation: Boil the mixture for 20 min. Filter. Autoclave at 115 0C for 20 min. The
final pH should be 7.8.
Use: This broth is used for grouping and typing Streptococcus pyogenes.
Sulphate Reducing Bacteria Medium
Composition:
KH2PO4
0.5 g
NH4Cl
1.0 g
Na2SO4
4.5 g
CaCl2.6H2O
0.06 g
MgSO4.7H2O
0.06 g
Sodium lactate
6.0 g
FeSO4.7H2O
0.004 g
Yeast extract
1.0 g
Sodium citrate 2H2O
5.0 g
Distilled water to
1000 ml
pH
7.5 ± 0.2
0
Preparation: Sterilize by autoclaving 20 minutes at 115 C..
Use: Particularly useful for growing cells in continuous culture, for biochemical work
and for manometric studies. It contains no sediment and ferrous sulphate is not
precipitated during growth. Normally used in liquid form.
Sulphate Reducing Bacteria Medium
(Medium E-Postage 1966)
Composition:
KH2PO4
0.5 g
NH4Cl
1.0 g
Na2SO4
1.0 g
CaC12.6H2O
1.0 g
MgSO4.7H2O
2.0 g
Sodium lactate
3.5 g
FeSO4.7H2O
0.5 g
Yeast extract
1.0 g
Thioglycollic acid
1.0 g
Ascorbic acid
1.0 g
Agar
20.0 g
Tap water to
1000 ml
pH
7.6
Preparation: Ingredient dissolve by boiling, adjust pH. Sterilize by autoclaving for 20
min. at 1150C. The thioglycollic acid should be sterilized by membrane filtration.
Use: Used for enrichment of.sulphate reducing bacteria. For enumeration, used in agar
form.
Sulphate Reducing Bacteria Medium
(A.P.I. medium-American Petroleum Institute, 1965)
Composition:
K2HPO4 (anhydrous)
0.01 g
NaCl
10.0 g
MgSO4.7H2O
0.2 g
Sodium lactate
4.0 ml
FeSO4(NH4)2SO4.6H2O
0.2 g
Ascorbic acid
0.1 g
Yeast agar
1.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.3
Use: Dissolve ingredients with heating. Adjust the pH. Sterilize by autoclaving at 121°C
for 10 min.
Sulphate Reducing Bacteria Medium
(Asparagine sodium lactate gelatine medium)
Composition:
Asparagine
1.0 g
Sodium lactate
5.0 g
K2HPO4
5.0 g
MgSO4.7H2O
1.0 g
FeSO4(NH4)2.6H2O
Trace
Gelatine
120 g
Distilled water to
1000 ml
Preparation: Sterilize in an autoclave at 1090C for 20 minutes.
Use: Used for isolation of sulphate reducing bacteria.
Sulphur Oxidizing Bacteria Medium
(Thiosulphate agar)
Composition:
Na2S2O3.SH2O
K2HPO4
NaHCO3
NH4Cl
Agar
Distilled water to
Preparation: Steam and sterilize at 121°C for 20 minutes.
sterilized separately.
Use: Used for isolation of sulphur oxidizing bacteria.
5.0 g
0.1 g
0.2 g
0.1 g
20.0 g
1000 ml
Add an excess of CaCO3
Sulphur Oxidizing Bacteria Medium
(Sulphur phosphate medium)
Composition:
(NH4)2SO4
0.20 g
KH2PO4
3.00 g
MgSO4 .7H2O
0.50 g
CaC12.6H2O
0.25 g
FeSO4.7H2O
trace
Sulphur powdered
10.00 g
Distilled water to
1000 ml
Preparation: The medium is sterilized in flowing steam for 3 consecutive days. The pH
of medium is about 4.0.
Use: Used for isolation of sulphur oxidizing bacteria.
Treponema Medium
(a) (Ascitic fluid agar)
Composition:
Glucose agar
100 ml
Ascitic fluid
20 ml
Preparation: Sterilize the glucose agar at 121°C for 20 min. Cool at 45°C. Add sterile
ascitic fluid.
(b) Hydrocele fluid agar
Composition and preparation: Same as above except use hydrocele fluid instead of
asciticfluid.
Use: Used for isolation of a virulent strain of Treponema pallidum.
Vibrio Medium
(Alkaline peptone water)
Composition:
Peptone
10.0 g
NaCl
5.0 g
Distilled water to
1000 ml
pH
9.0
Preparation: Add the components in distilled water. Steam sterilize the medium at 1210C
for 20 min.
Use: Used for cultivation of alkaliphilic bacteria (including Vibrio species.)
Vibrio Medium
(Aronson's medium)
Composition:
(a) Agar base
Peptone
Beef extract
Sodium chloride
Distilled water to
pH
Preparation: Sterilize the medium at 121°C for 20 minutes.
10.0 g
30.0 g
5.0 g
1000 ml
7.2
Complete Medium
Anhydrous sodium carbonate (10% solution)
6 ml
Sucrose (20% solution)
5 ml
Glucose (20% solution)
5 ml
Sodium sulphite (10% solution)
2 ml
Basic fuchsine
0.4 ml
(Saturated alcoholic solution) Melted sterile agar base
100 ml
Preparation: Except agar base all the solutions are sterilized by steaming for 30 min for
three successive days.
Add carbonate solution to melted agar base. Heat for 10 min. at 100°C. While hot
a add other solutions. Heat for 20 min. more. The mixture is allowed to stand to settle the
precipitate. Pour plates.
Use: V. cholerae grow with formation of colony, pouched egg in appearance with reddish
centre and paler periphery. The medium is alkaline, it is selective for the cultivation of V.
cholerae.
Vbrio Medium
(Bile peptone transport medium)
Composition:
Peptone
1.0 g
Sodium chloride
1.0 g
Sodium taurocholate
0.5 g
Distilled water to
1000 ml
pH
8.5
Preparation: Dissolve the ingredients, adjust the pH. Autoclave at 121°C for 15 minutes.
Precaution:
1) Usually subcultures should be made within 6 hours.
2) In order to make medium more selective for the vibriob, sterile potassium tellurite
solution may be added after autoclaving to give a final concentration of 1 in
2,00,000 as for Monsur's medium.
Use: Useful for maintaining the viability of Vibrio cholerae and preventing overgrowth
by other organisms during transportation of clinical sample.
Vibrio Transport Medium
(Carry-Blair medium)
Composition:
Sodium thoglycollate
0.75 g
Disodium hydrogen phosphate
0.55 g
Sodium chloride
2.5 g
Calcium chloride (1%)
4.5 ml
Distilled water to
1000 ml
pH
8.4
Preparation: Heat the medium in boiling water bath. Cool to 50°C and add 4.5 ml of
freshly prepared calcium chloride solution. Adjust the pH. Sterilize by autoclaving at
1210C for 15 minutes.
Use: It is used as transport medium for Vibrio, Salnronella and Shigella.
Vibrio Medium
(Gelatin agar)
Composition:
Trypticase
Sodium chloride
Gelatin
Agar
1.0 g
1.0 g
3.0 g
1.5 g
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve the ingredients by heating. Adjust the pH. Autoclave at 121°C for
20 minutes.
Use: This medium is complementary to Monsur's medium. But it is not inhibitory to
coliform bacilli. Used for primary isolation of Vibrio cholerae.
Vibrio Medium
(Monsur’s medium)
Composition:
(a) Bile Salt gelatin agar
Tryptose
1.0 g
Sodium chloride
1.0 g
Sodium taurocholate
0.5 g
Sodium carbonate
0.1 g
Gelatin
3.0 g
Agar
2.0 g
Distilled water to
100 ml
pH
8.5
Preparation: Dissolve the ingredient by heating. Adjust the pH. Sterilize by autoclaving
at 121°C for 20 minutes.
(b) Stock potassium tellurite solution
Potassium tellurite
0.5 g
Distilled water
100 ml
0
Autoclave at 115 C for 20 min.
Complete medium
Bile salt gelatin agar
100 ml
Potassium tellurite solution
1 ml
Preparation: Make 1 in 10 dilution of the stock potassium tellurite solution by using
sterile distilled water. Add it to the melted and cooled agar medium. The final pH of the
medium must be 8.5 to 9.3.
Precaution: Use the medium when fresh because its pH drops after storage.
Use: This medium is used for the selective isolation of cholera and other vibrios. A
medium without gelatin and agar is used for enrichment. 'The potassiun~te llurite inhibits
most coliforrn bacilli.
Vibrio Medium
Composition:
Boric acid, H3BO3
3.101 g
KCl
3.728 g
NaOH (0.2 M solution)
133.5 ml
Dried sea salt
200 g
Distilled water to
1000 ml
pH
8.5
Preparation: Dissolve the boric acid and potassium chloride in 20 ml hot water. Cool
and make up the volunie to 250 ml. Add the sodium hydroxide, make up the volume to 1
litre and add the salt. Filter the solution through paper and autoclave at 121°C for 20 min.
Use: Used for maintaining viability of V. cholerae.
Vibrio Medium
(Thiosulphate citrate bile sucrose agar/TCBS medium)
(1) Basal agar medium
Composition:
Yeast extract
5.0 g
Peptone
10.0 g
Sodium chloride
10.0 g
Agar
20.0 g
Thymol blue solution (2% in 50% ethnol)
2.0 ml
Sucrose
10.0 g
Water
800 ml
pH
8.5
Preparation: Dissolve the yeast extract, peptone and salt in 200 ml water and adjust the
pH to 8.5. Ilissolve the agar in 600 ml water by heating. Add the indicators and sucrose
mixing again and steam sterilize at 1150C for 15 minutes.
(2) Solution A
Sodium citrate Na3C6H5O7.H2O
10.0 g
Sodium thousulphate Na2S2O3.5H2O
10.0 g
Ferric citrate
1.0 g
Distilled water (sterile)
100 ml
(3) Solution B
Ox bile, desiccated
8.0 g
Distilled water (sterile)
100 ml
Prepare these solutions with sterile precautions, heating if required.
Preparation of complete medium
Basal agar medium
80 ml
Solution A
10 ml
Solution B
10 ml
Melt the agar and add solution A and B in this order, with sterile precaution. Four plates
immediately.
Use: It is highly selective for V. parahaemolyticus and may be used for the isolation of
V. chloerae.
Significance: It is inhibitory to gram-positive organisms, most coliforllls and many
strains of Proteus. V. paraheamolyticus from colonies with green or blue centre.
Xanthomonas Medium
(Beef peptone agar)
Composition:
Beef extract
Peptone
Dextrose
Yeast extract
Agar
Distilled water to
pH
Use: Used for isolated of Xanthomonas.
3.0 g
5.0 g
10.0 g
5.0 g
20.0 g
1000 ml
7.2
Xanthomonas Medium
Composition:
Chalk
4.0 g
Glucose
0.5 g
Yeast extract
0.5 g
Agar
2.0 g
Distilled water to
100 ml
0
Preparation: Steam sterilize the medium at 121 C for 20 minutes.
Use: For cultivation of Xanthomonas.
Plant Tissue Culture Medium
[Murashige Skoog (MS) tissue culture medium]
Composition:
Macronutrients:
NH4NO3
KNO3
CaCl2.2H2O
MgSO4.7H2O
KH2PO3
Micronutrients:
1650.0 mg
1900.0 mg
440.0 mg
370.0 mg
170.0 mg
KI
0.83 mg
H3BO3
6.2 mg
MnSO4.4H2O
22.3 mg
ZnSO4.7H2O
8.6 mg
Na2MoO4.2H2O
0.025 mg
CuSO4.5H2O
0.025 mg
CoCl2.6H2O
2.025 mg
Fe-Versenate (EDTA)
43.0 mg
Vitamins and hormones
Inositol
100.0 mg
Nicotinic acid (Vitamin 9,)
0.5 mg
Pyridoxine-HC1
0.5 mg
Thiamine-KC1
0.1 mg
Indole acetic acid (IAA)
1.30 mg
Kinetin
0.01-10.0 mg
Carbon source
Sucrose
30.0 g
Distilled water to
0.2 g
pH
0.2 g
Use: Commonly used medium for plant tissue cultures. For solidifying, agar is added at
the rate of 0.6%.
Seedlings Medium
(Jensen's medium-modified)
Composition:
CaHPO4
K2HPO4
MgSO4.7H2O
NaCl
FeCl3
Trace elements solution
Distilled water to
pH
A stock solution of trace elements
Bo
Mn
Zn
Mo
Cu
1.0 g
0.2 g
0.2 g
0.2 g
0.1 g
1.0 ml
1000 ml
7.0
500 mg
500 mg
50 mg
50 mg
20 mg
Distilled water to
1000 ml
Add agar at the rate of 20 g/litre when solid medium is needed.
Use: Used to growing seedlings to test root nodulation in legumes.
2. Fungi
Alternaria Medium
(Lukens and Sisler synthetic medium)
Composition:
Glucose
20.0 g
Ammonium sulphate
3.0 g
Magnesium sulphate (MgSO4.7H2O)
0.25 g
Monobasic potassium phosphate
3.0 g
Glycine
1.0 g
Thiacinc HCI
2.0 µg
Niacin
30 µg
Biotin
1 µg
I-inositol
200 µg
Pyridoxine
10 µg
Folic acid
10 µg
pH
6.0
Preparation: Add Bo, Mn, Zn, Cu, Mo and Fe in trace. Steam sterilize at 100°C for 1 hr.
Use: Used for cultivation of Allernaria.
Ascomycetes Medium
(Claussen's medium)
Composition:
Potassium dihydrogen phosphate
Ammonium nitrate
0.05 g
0.05 g
Magnesium sulphate (MgSO4.7H2O)
Ferrous phosphate
Inulin
Agar
Distilled water to
Preparation: Steam sterilize at 121°C for 20 min.
Use: Used for cultivation of Asconrycetes.
0.02 g
0.001 g
2.0 g
3.0 g
1000 ml
Ascomycetes Medium
(Yeast extract agar)
Composition:
Yeast extract
Malt extract
Dextrose
Agar
Distilled water to
Use: Used for cultivation of Acomycete.
4.0 g
10.0 g
4.0 g
15.0 g
1000 ml
Aspergillus Medium
(Pailey, Stafamak, Olson and Johnson's medium)
Composition:
Glycerol
7.5 g
Cane sugar
7.5 g
Peptone
5.0 g
Magnesium sulphate
0.05 g
Potassium dihydrogen phosphate
0.06 g
Sodium chloride
4.0 g
Agar
20.0 g
Distilled water to
1000 ml
Preparation: Add glycerol, cane sugar, magnesium sulphate, potassium dihydrogen
phosphate to 200 ml water. Mix peptone and salt to a paste with 200 ml water, at 60°C
and add to the first mixtde. Dissolve agar in 600 ml, then mix all together, pH does not
need adjustment. Autoclave 121°C at for 20 min.
Use: Used for cultivation of AspergilIus auvezrs.
Aspergillzis Medium
Composition:
Glucose
(NH4)2NO3
20.0 g
1.0 g
KH2PO4
MgSO4
FeCl3
ZnSO4
Distilled water to
pH
Add CaCO3 toll effervescence stops.
Preparation: Steam sterilize at 1210C for 20 min.
Use: Used for cultivation of Aspergillus.
0.68 g
0.5 g
0.016 g
0.005 g
100 ml
5.4
Basdiomycetes Medium
Composition :
Basal medium
Potassium dihydrogen phosphate
Magnisum sulphate (MgSO4.7H2O)
Dextrose
Casein sulphate
Adenine sulphate
Cytosine
Guanine HCl
Hypoxanthine
Thymine
Uracil
Xanthine
Choline Cl
Orotic acid
Thiamine HCl
Riboflavin
Pyridoxine
Nicotinic acid
Calcium pantothenate
Para-aminobenzoic acid
m-inisitol
Folic acid
Biotin
Vitamin B12
B
Cu
Fe
1.5 g
0.5 g
20.0 g
2.0 g
9.09 mg
2.22 mg
4.11 mg
2.72 mg
0.25 mg
2.24 mg
0.06 mg
5.58 mg
0.62 mg
0.337 mg
0.376 mg
0.205 mg
0.123 mg
0.476 mg
0.137 mg
216.19 mg
1 mg
0.01 mg
0.01 mg
0.005 mg
0.02 mg
0.1 mg
Ga
0.01 mg
Mn
0.01 mg
Mo
0.01 mg
Zn
0.09 mg
Distilled water to
1000 ml
Neutralize with calcium carbonate (CaCO3)
Steam sterilize at 100°C for 1 hr.
Wood extract
Autoclave 500 g dry beech wood (ground in a mill) in 5 litre of distilled water. Reduce
the filtrate on a hot plate to 1 litre.
Tomato extract
Filter canned tomato juice through muslin.
Complete medium
Basal medium
100 ml
Wood/Tomato extract
8 ml
Use: Used for cultivation of basidiomycetes.
Botrytis Separation Agar
Composition
Potassium chloride
1.0 g
Potassium diflydrogen phosphate
1.5 g
Magnesium sulphate
0.5 g
Sodium nitrate
3.0 g
Casein hydrolysate
5.0 g
Yeast extract
3.0 g
Glycerol
5.0 g
L-Sorbose
2.5 g
Agar
20.0 g
Tap water to
1000 ml.
Preparation: Autoclave at 121°C for 20 minutes.
Use: A selective medium to distinguish between Botrytis allii and B. cinera on onion.
The former species is severely restricted by sorbose.
Candida Medium
(Corn meal agar)
Composition:
Corn meal
Agar
Water to
40.0 g
20.0 g
1000 ml
pH
6.8
Preparation: Heat the corn meal in the water at about 60°C for 1000 ml. Filter through
filter paper. Make the volume again 1000 ml by adding water. Add the agar. Autoclave at
121°C for 30 minutes. The pH is self-adjusted.
Use: This medium is used for the production of chlamydospores. In the case of Candida
albicans the appearance of the chlamydospores is diagnostic.
Candida Medium
(Molybdenum medium)
Composition:
Peptone
10.0 g
Sucrose
40.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
7.6
0
Preparation: Autoclave at 109°C for 20 minutes. Cool to 50 -55°C and 15 ml of a 12.5%
aclueous soln. of Merck phosphomolybdic acid. (Final concentration 1.9 mglml)
Use: Used for cultivation of Candida albican.
Candida Medium
(Rice starch agar)
Composition:
Rice flour
10.0 g
Agar
20.0 g
Tween 80
10 ml
Distilled water to
1000 ml
Preparation: Boil the water and mix with the rice flour. Boil for more 30 sec. Stand for a
few seconds and filter through cotton guaze. Add water to re-adjust the volume. Add the
agar and tween 80 and autoclave at 121°C for 30-minutes.
Use: This medium is used to stimulate chlamydospore formation by Candida albicans.
Celluloytic Fungal Medium
(Cellulose yeast extract agar)
Composition:
Filter paper
12.0 g
Yeast extract
4.0 g
Agar
10.0 g
Distilled water to
1000 ml
Preparation: Tear paper into small pieces and macerate in some of the water until the
fibers are separated. Add this to the remainder of the water and dissolve the yeast extract
and agar. Autoclave at 1210C for 20 minutes.
Use: Used for cultivation of cellulolytic fungi.
Cellulolytic Fungal Medium
(Czapek medium-modified)
Composition:
Sodium nitrate
2.0 g
Potassium chloride
0.5 g
Magnesium sulphate
1.5 g
Dipotassiuln hydrogen phosphate
1.0 g
Ferrous sulphate
0.01 g
Cellulose
10.0 g
Distilled water to
1000 ml
pH
6.4 to 7.0
Preparation: Autoclave at 115°C for 15 minutes to prevent break down of cellulose.
Use: For assaying fungal cellulase.
Note: Substitute cellulose with pectin for assaying pectinase.
Chaetomium Medium
(Knop's Solution)
Composition:
Calcium nitrate
0.5 g
Potassium nitrate
0.125 g
Magnesium sulphate (7H2O)
0.125 g
Potassium phosphate
0.125 g
Ferrous chloride
0.005 g
Distilled water to
1000 ml
0
Preparation: Steam sterilize at 121 C for 20 minutes.
Use: Filter paper saturated with Knop's solution (with agar) is used for maintaining
chaeromium spp.
Chaetomium Medium
(Potato malt agar with cellulose)
Composition:
Potatoes
30.0 g
Malt syrup
5.0 g
Agar
20.0 g
Distilled water to
1000 ml
Preparation: Prepare potatoes extract by boiling sliced in water. Autoclave at 121°C for
20 minutes. Strips of sterilized paper are placed on the surface of the medium in petri
dishes or tubes.
Use: Used for cultivation of cellulolytic fungi (Chaetomium)
Dermatophyte Test Medium
Composition:
Phenol red solution
Phenol red
0.5 g
Sodium hydroxide (0.1 N)
15 ml
Distilled water to
85 ml
Complete medium
Peptone
10.0 g
Glucose
10.0 g
Agar
20.0 g
Phenol red solution
40 ml
Hydrochloric acid (0.8 N)
6 ml
Cycloheximide (25% w/v) in acetone
2 ml
Gentamycin sulphate (5% W/V aqu.)
2 ml
Chlorttracycline (0.4% wlv in sterile water)
25 ml
Distilled water to
1000 ml
Preparation: Dissolve the peptone glucose and agar in the water by heating. Add the
phenol red solution, acid, cyclohexmide and gentamycin to the hot medium. Steam
sterilize at 1150C for 15 min and cool. Add the chlortetracycline.
Use: The medium is highly selective for dematophytes. Significance: Antibiotics inhibit
most bacteria and cycloheximide restrains bacteria, other molds and yeasts.
Dermatophytes produce enough alkali to tun1 the medium red within 2 weeks at 280C.
Fungus Preservation Medium
Composition:
Peptone
Agar
Water to
pH
Preparation: Steam sterilize at 1210C for 20 minutes.
Use: Used as preservation medium for fungi.
Fungal Medium
(Bread crumb agar)
Composition:
30.0 g
20.0 g
1000 ml
5.4
Commercial bread crumbs (without preservations) 100.0 g
Agar
20.0 g
Distilled water to
1000 ml
Use: Used to permit the luminescence of various fleshy fungi.
Fungal Medium
(Carrot agar)
Composition:
Whole carrot
300.0 g
Agar
30.0 g
Distilled water to
1500 ml
Preparation: Cook carrot in 500 ml distilled water, macerate. Add 1000 ml distilled
water and 30 g. agar an4 dissolve. Autoclave at 121°C for 15 minutes.
Use: Used for cultivation of fungi.
Fungal Medium
(Cherry agar)
Composition:
Cherry extract
300 ml
Agar
20.0 g
Distilled water to
700 ml
pH
3.8 – 4.6
Preparation: Dissolve agar in water, add cherry extract. Distribute into pre-sterilized
bottles or tubes and sterilize at 102OC for 5 minutes. Overheating should be avoided, as
the acid reaction will prevent the setting of the medium.
Cherry extract
Stone chemes (red variety). To each 200 g pulp, add 1 litre water for 2 hr. Strain through
cloth and sterilize at 110°C for 1 hr.
Use: Used for cultivation of fungi.
Fungal Medium
[Cornmeal (maize) peptone yeast-extract agar]
Composition:
Cornmeal
Dextrose
Peptone
Yeast extract
Agar
Distilled water to
20.0 g
10.0 g
10.0 g
4.0 g
20.0 g
1000 ml
Preparation: Boil cornmeal in 700 ml of water for 10 minutes. Strain through cloth.
Dissolve peptone, dextrose, yeast extract and agar to filtrate and make the volume to 1 lit.
Autoclave at 121°C for 20 minutes. .
Use: Used for cultivation of b g i .
Fungal Medium
(Martin rose Bengal agar)
Composition:
Dextrose
10.0 g
peptone
5.0 g
Potassium dihydrogen phosphate
1.0 g
Magnesium sulphate (MgSO4.H2O)
0.5 g
Rose Bengal
0.035 g
Agar
20.0 g
Distilled water to
1000 ml
Aueomycin (chlortetracycline)
35 µg/ml
0
Preparation: Autoclave at 121 C for 20 minutes. Add antibiotic after sterilization of the
medium.
Use: Used for selective isolation of fungi.
Note: Auremycin may be replaced by streptomycin (30.0 mg).
Fungal Medium
(Elliott's agar)
Composition:
Potassium dihydrogen phosphate
Sodium carbonate
Magaesium sulphate
Dextrose
Asparagine
Agar
Distilled water to
Use: Used for cultivation of fungi.
Fungal Medium
(Emmon's culture medium for fungi)
Composition:
Glucose
Peptone
Agar
1.36 g
1.06 g
0.50-g
5.0 g
1.0 g
20.0 g
1000 ml
20.0 g
10.0 g
20.0 g
Chloramphenicol
40mg
Distilled water to
1000 ml
pH
7.2
Preparation: Dissolve the glucose, peptone, and agar by heating. Add the
chloramphenicol which has been suspended in 10 ml of 95% alcohol. Remove alcohol by
heating medium. Autoclave at 121°C for only 10 minutes.
Use: Selective medium for isolation of fungi, I
Significance: It’s principal advantage is that a neutral pH does not inhibit certain molds
that have difficulty in growing at pH 5.6.
Fungal Medium
(Fish broth medium)
Composition:
Fresh cod fish
1.0 kg
Peptone
5.0 g
Sodium chloride
200 g
Wheat flour
400-500 g
Distilled water to
1000 ml
Preparation: Macerate the fish in the water and allow to stand for 4 hr. Strain through
muslin. Add peptone and sodium chloride and autoclave at 10g°C for 20 minutes. Add
the flour and strain through muslin. Autoclave at 1210C for 20 minutes.
Use: Used for cultivation of fungi.
Fungal Medium
(Hay extract agar)
Composition:
Hay
200.0 g
Glucose
5.0 g
Agar
20.0 g
Distilled water to
l000 ml
Preparation: Boil the hay for 30 minutes in 500 ml of the water. Filter and add the
glucose. Make up to 1 litre, then add the agar and heat until dissolved. Sterilize at' 1210C
for 20 minutes.
Use: Used for cultivation of fungi.
Fungal Medium
(Honey peptone medium)
Composition:
Honey
60.0 g
Peptone
10.0 g
Agar
20.0 g
Water
1000 ml
Preparation: Dissolve the agar before addition of honey. Steam sterilize at 100°C for 1
hr.
Use: The pH is favorable to fungal growth and inhibitory to most bacteria.
Fungal Medium
(Kauffman's agar)
Composition:
Maltose
5.0 g
Magnesium sulphate
0.10 g
Calcium nitrate (4H2O)
0.50 g
Potassium dihydrogen phosphate
0.25 g
Agar
20.0 g
Distilled water to
1000 ml
Preparation: Dissolve the ingredients in water. Sterilize at 109°C for 20 minutes.
Use: Used for cultivation of fungi.
Fungal Medium
(Malt salt agar)
Composition:
Malt extract
100.0 g
Sodium chloride
100.0 g
Agar
20.0 g
Distilled water to
1000 ml
Preparation: Dissolve be ingredients in the water and autoclave at 1210C for 20 minutes:
Use: For halophilic yeast and molds.
Fungal Medium
(Malt agar)
Composition:
Malt extract powder
30.0 g
Glucose
16.0 g
Agar
15.0 g
Distilled water to
1000 ml
Melt and acidify to pH 3.5 with sterile lactic acid solution prior to use.
Use: Medium is selective for mold growth.
Fungal Medium
(Oatmeal agar)
Composition:
Oatmeat
20.0 g
Agar
18.0 g
Trace salt solution
1.0 g
Distilled water to
1000.0 ml
Trace salt solution
FeSO4.7H2O
0.1 g
MnCl2.4H2O
0.1 g
ZnSO4.7H2O
0.l.g
Distilled water
1000.0 ml
0
Preparation: Steam sterilize at 121 C for 20 minutes.
Use: Used for cultivation of fungi.
Fungal Medium
(Onion-asparagine agar)
Composition:
Onion
100.0 g
Asparagines
0.25 g
Peptone
0.5 g
Agar
20.0 g
Distilled water to
1000 ml
Preparation: Peel and cut up onions and boil in water bath in 500 ml of the water. Strain
and add agar and remainder of ingredients dissolved in the other 500 ml water. Autoclave
at 115°C for 15 min. Use: Used for cultivation of fungi.
Fungal Medium
(Potato dextrose agar)
Composition:
Potato (peeled)
200.0 g
Dextrose
20.0 g
Agar
15.0 g
Distilled water to
1000 ml
Preparation: Peel off the skin of potatoes, cut into small pieces and boil in 500 ml. of
water till they are easily penetrated by a glass rod. Filter through cheesecloth. Add
dextrose to the filtrate. Dissolve agar in water and bring upto the required volume by the
addition of water. pH is self adjusted. Sterilize at 121°C for 20 minutes.
Use: Medium is selective for isolation of fungi.
Fungal Medium
(Prune agar)
Composition:
Prunes
30.0 g
Sucrose
40.0 g
Agar
30.0 g
Distilled water to
1000 ml
Preparation: Boil prunes in water, then pass through sieve to get extract. Add agar, boil
to dissolve, add sucrose and stir until dissolved. Autoclave at 121eC for 20 minutes.
Use: Used for cultivation of fungi.
Fungal Medium
(Sabouraud's glucose agar)
Composition:
Glucose
4.0 g
Peptone
1.0 g
Agar
20.0 g
Water to
100 ml
pH
5.4
Preparation: Dissolve the glucose and peptone in water. Adjust the pH. Add agar.
Autoclave at 115°C for 15 minutes.
Use: For the isolation of fungi.
Significance: The low pH arid high sugar content prevent the bacterial growth. It is
useful to add cyclohexiplide for the isolation of dermatophytes.
Fungal Medium
(Tellurite malt agar)
Composition:
(a) Basal medium
Malt extract
4.0 g
Agar
2.0. g
Water to
100 ml
pH
5.4
Preparation: Dissolve the malt extract and agar by heating. Adjust the pH. autoclave at
115°C for 20 minutes.
(b) Potassium tellurite solution
Potassium tellurite
0.5 g
Distilled water to
25 ml
Preparation: Dissolve the potassium tellurite. Autoclave at 115°C for 20 minutes. Store
in the dark.
Preparation of complete medium
Basal medium
100 ml
Potassium tellurite solution
1-3 ml
Cool the melted basal medium to 5S°C. Mix the tellurite solution and distribute.
Use: It is a selective medium for the isolation of fungi.
Fungal Medium
(Waksman's medinm)
Composition:
Glucose
10.0 g
Peptone
5.0 g
Potassium dihydrogen phosphate
1.0 g
Magnisum sulphate (MgSO4.7H2O)
0.5 g
Agar
25.0 g
Distilled water to
1000 ml
pH
4.0
Adjust pH 4.0 by addition of 1 N H2SO4
Use: Used for isolation of soil fungi. (plate count agar for fungi.)
FusmonumM edium
(Bihi medium, modification by Joffe)
Composition:
Potassium dihydrogen phosphate
1.0 g
Potassium nitrate
1.0 g
Magnisum sulphate
0.5 g
Potassium chloride
0.5 g
Starch powder
0.2 g
Glucose
0.2 g
Sucrose
0.2 g
Distilled water to
1000 ml
Use: Strips of cellulose lens paper are added. This medium always induces conidial
formation in Fusarium.
Fusarium Medium
(Coon's medium)
Composition:
Saccharose
Dextrose
7.2 g
3.6 g
Magnesium sulphate
1.23 g
Potassium dihydrogen sulphate
2.72 g
Potassium nitrate
2.02 g
Agar
20.0 g
Distilled water to
1000 ml
Preparation: Add to this malachite green to make 1: 40,000 solution or gentian violet to
make 1 :26,000 solution.
Use: Used for cultivation of Fusarium.
Fusmiwn Medium
(Malehite greencaptan medium, a modification of Czapek-Dox)
Composition:
Sodium nitrate
2.0 g
Dipotassium hydrogen phosphate
1.0 g
Magnesium sulphate (7H2O)
0.5 g
Potassium chloride
0.5 g
Ferrous sulphate
0.01 g
Sucrose
30 g
Distilled water to
1000 ml
Malachite green
50 mg
Captain
100 mg
Dicrysticin (mixture of Streptomycin sulphate
Procain pencillin G and Sodium penicillin G)
0.75 mg
Preparation: Add fiesh solutions of malachite green, captan and dicrysticin after
autoclaving and before pouring the plates.
Use: Used for selective isolation of Fusarium fiom soil.
Fusarium Medium
(Potato sucrose agar)
Composition:
Potato extract (see page 162)
Sucrose
Agar
Distilled water to
pH
Preparation: Autoclave at 121°C for 20 minutes.
Use: Used far cultivation of fungi. (Fusarium)
Fusarium Medium
500 ml
20.0 g
20.0 g
500 ml
6.5
(Park's medium/Liquid)
Composition:
Glucose
Magnesium sulphate
Potassium dihydrogen phosphate
Ammonium nitrate
Distilled water to
Preparation: Steam sterilize at 1 l5°C for 30 ininutes.
Use: Used for cultivation of Fusarium.
0.7.g
0.5 g
0.2 5
0.1 g
1000 ml
Fusarium Medium
(PCNB medium-modified)
Composition:
Peptone
15.0 g
PCNB (Terraclor, a commercial product is 75% active) 0:5 g
Oxgall
0.5 g
Agar
20.0 g
Chlorotetracycline HCl
50.0 mg
Streptomycin sulphate
100.0 mg
Distilled water to
1000 ml .
Preparation: Steam, sterilizer at 121°C for 20 minutes.
Note: Chlorotetracyline and streptomycin sulphates are heat labile. Sterilize separately.
PCNB: Penta chloronitrobenzene.
Use: To isolate fusaria from soil. It need not to be sterilized if made up fiesh.
Lignolytic Fungi Medium
Composition:
K2HPO4
1.0 g
KCl
0.5 g
MgSO4.7H2O
0.5 g
FeSO4
0.1 g
Lignin
25.0 g
Agar
20.0 g
Distilled water to
1000 ml
pH
5.4
Preparation: Steam sterilize at 121°C for 20 minutes.
Note: By substituting cellulose 'for lignin, the same medium can be used for cellulolytic
fungi.
Use: Used for isolation of lignolytic fungi.
Mucor-synthetic Medium
(Hesseltine, 1954)
Composition:
Dextrose
Asparaghe
Potassium dihydrogen phosphate
Magnesium sulphate
Thiamine chloride
Agar
Distilled water to
Use: Used for cultivation of Mucor.
40.0 g
2.0 g
0.5 g
0.25 g
0.005 g
20.0 g
1000 ml
Neurospora Minimal Medium
(Beadle and Tatum, 1945)
Composition:
Ammonium tartrate
Ammonium nitrate
Potassium dihydrogen phosphate
Magnesium sulphate
Sodium chloride
Calcium chloride
Sucrose
Biotin
Bo
Cu
Fe
Mn
Mo
Zn
Distilled water to
Use: Used for cultivation of Neutospora.
5.0 g
1.0 g
1.0 g
0.5 g
0.1 g
0.1 g
15.0 g
5 x 10-6g
0.01 mg
0.1 mg
0.2 mg
0.02 mg
0.02 mg
2.0 mg
1000 ml
Neurospora 'complete' Medium
(Beadle and Tatum, 1945)
Composition:
Glucose
Sucrose
Hydrolized casein
5.0 g
5.0 g
5.0 ml
Yeast extract
2.5 g
Spray-dried malt syrup
5.0 g
Vitamin solution
10 ml
Agar
20 g
Distilled water to
1000 ml
Vitamin solution
Thiamin
100 mg
Riboflavin
50 mg
Pyridoxin
50 mg
Pantothenic acid
200 mg
p-Aminobenzoic acid
50 mg
Nicotinarnide
200 mg
Choline
200 mg
Inositol
400 mg
Alkali hydrolysed yeast nucleic acid
500 mg
Folic acid
4 µg
Distilled water to
1000 ml
The casein hydrolysate in prepared by HC1 hydrolysis and made up to the
equivalent of 50 mg casein per litre.
Use: Used for cultivation of Neutospora.
Penkillium Medium
(Glucose asparagine agar-Krainsky's medium)
Composition:
Glucose
10.0 g
Asparagine
0.5 g
Potassium hydrogen phosphate
0.5 g
Agar
15.0 g
Distilled water to
1000 ml
Preparation: Dissolve glucose, asparagines and potassium phosphate in the water and
add agar. Boil until dissolve. Autoclave at 1150C for 30 minutes.
Use: Used for stimulating production of perithecia in Penicilfum. Also used for
cultivation of actinomycetes.
Penicillium Medium
(Fermentation medium)
Composition:
Lactose
Glucose
30.0 g
10.0 g
Starch
15.0 g
Ammonium sulphate
5.0 g
Ethyl mine
3.0 g
Citric acid
10.0 g
Acetic acid
2.5 g
Phenyl acetate
0.5 g
Distilled water to
1000 ml
pH
6.8
Preparation: Steam sterilize at 121°C for minutes.
Use: Used for penicillin production by Penicillium chrysogenum.
Penicillum Medium
(Orange fluid medium)
Composition:
Glucose
50.0 g
Ammonium citrate
1.9 g
Potasium dihydrogen phosphate
1.0 g
Potassium chloride
0.5 g
Magnesium sulphate
0.5 g
Ferrous sulphate
0.01 g
Extract of 1 orange to
1000 ml
pH
4.5
Orange extract: For 1 litre medium, macerate one good sized orange in 250 ml water.
Make up to 500 ml and boil for ten minutes with constant stirring. Strain through muslin.
Stir up pulp with another 250 mi water and strain again. Make the volume 1 litre.
Use: Used for cultivation of fungi.
Pythium Medium
(Glucose nitrate medium)
Composition:
Glucose
Sodium nitrate
Potassium dihydrogen phosphate
Magnesium sulphate (MgS0,.7H,O)
Agar
Thiamine hydrochloride
Distilled water to
pH
Preparation: Adjust pH. Autoclave at 1210C for 10 minutes.
5.4 g
1.5 g
1.0 g
0.5 g
20.0 g
(2ml of 1000 ppm stock sol)
1000 ml
6.0
Use: Used for reproduction of Pythium aad Phytophthora species.
Phyfophora Medium
(Petri solution)
Composition:
Calcium nitraSe
0.4 g
Magnesium sulphate
0.15 g
Potassium dihydrogen phosphate
0.15 g
Potassium chloride
0.06 g
Distilled water to
1000 ml
Preparation: The solution be kept in a refrigerator and used unsterile.
Use: Used for,the induction of sporangia of phytophthore.
Rhizoctonia Agar
Composition:
Saccharose
Potassium hydrogen phosphate
Agar
Distilledwaterto
Preparation: Steam sterilize at 115°C for 30 minutes.
Use: Used for isolation of Rhizoctonia.
10.0 g
1.0 g
20 g
1000 ml
Sclerotium Medium
(Garlic agar)
Composition:
Garlic
300.0 g
Agar
20.0 g
Distilled water to
1000 ml
Preparation: Peel and cut up garlic and boil in the water for 1 hr. Filter and restore to
original volume with more distilled water. Add agar and sterilize at 121°C for 20
minutes.
Use: Used for cultivation of Sclerotium.
Slime Mold Medium
(Peptone-yeast-extract dextrose agar)
Composition:
Peptone
Yeast extract
Dextrose
2.0 g
2.0 g
5.0 g
Agar
15.0 g
Distilled water to 1000 ml
1000 ml
pH
6.4 – 7.0
Preparation: Steam sterilize at 121°C for 20 minutes.
Use: Slime mold grow forming translucent patches and producing wheel like
psuedoplasmodia. Slime molds are phagotrophic. Bacterial colonies grow in the
petriplates and slime molds grow on this.
Semisynthetic Fungal Medium
(Czapek-Dox agar)
Composition:
Sodium nitrate
2.0 g
Dipotassium hydrogen phosphate
1.0 g
Magnesium sulphate
0.5 g
Potassium chloride
0.5 g
Ferrous sulphatb.
0.01 g
Sucrose
30.0 g
Zinc sulphate
Trace
Copper sulphate
Trace
Agar
20.0 g
Distilled water to
1000 ml
pH
6.4 – 7.0
Preparation: Dissolve all the ingredients except phosphate in half of the water and the
sucrose added. Dissolve the phosphate separately and add to the rest. Make volume to
1000 ml. Sterilize by autoclaving at 121°C for 15 minutes.
Use: Used for isolation of fungi. Czapek's broth is a synthetic medium.
Yeast Medium
(Penicillin streptomycin blood agar)
Composition:
Nutrient agar
90.0 ml
Blood
10.0 ml
Pencillin
300 units
Streptomycin
300 µg
Preparation: Prepare sterile solutions of penicillin and of streptomycin. Melt the sterile
nutrient agar, cool it to 550C. Add the blood collected aseptically and the antibiotic
solutions. Pour plates.
Use: The medium is used for selective cultivation of pathogenic yeast and fungi.
Yeast Medium
(Malt extract agar)
Composition:
Malt
Dipotassium hydrogen phosphate
Ammonium chloride
Citric acid (0.1 N)
Agar
Distilled water to
Preparation: Steam sterilize at 1210C for 20 minutes.
Use: Used for cultivation of yeast and Acetobacter.
15.0 g
1.0 g
1.0 g
15.0 ml
20.0 g
1000.0 ml
Yeast Medium
(Cysteine glucose blood agar)
Composition:
Nutrient agar (2.5% agar sterile)
83 ml
Glucose (20% sterile)
5 ml
Cystine solution (5% sterile)
2 ml
Blood (sterile)
10 ml
Preparation: Melt the agar, cool to 50°C add the remaining ingredients and pour plates.
Cystine solution is sterilized by filtration.
Use: This medium is useful for cultivating the yeast forms of dimorphic fungi.
Yeast Medium
(Hansen's medium)
Composition:
Peptone
Maltose
Potassium dihydrogen phosphate
Magnesium sulphate (MgSO4.7H2O)
Distilled water to
Preparation: Steam sterilize at 109°C for 20 minutes.
Use: Used for cultivation of yeasts.
1.0 g
5.9 g
0.3 g
0.2 g
1000 ml
Yeart Medium
(Malt carrot agar)
Composition:
Carrot
Malt extract
250.0 g
5.0 g
Agar
25.0 g
Distilled water to
1000 ml
Preparation: Boil carrot until soft. Pass through a fine sieve,
squeezing through as much pulp as possible. Dissolve agar by heating.
Add malt extract. Autoclave at 121°C for 20 minutes.
Use: Used for cultivation of yeast.
3. Algae
Algal Medium
Composition:
Sodium citrate
Dipotassium hydrogen phosphate
Potassium dihydrogen phosphate
Ammonium nitrate
Calcium chloride
Ferric chloride 6H2O
Magnesium sulphate
Trace metal solution
Distilled water to
pH
Trace metal solution
Ethylenediamintetra acetic acid
Managanese sulphate
Zinc sulphate
Ferrous ammonium sulphate
Cobalt chloride
Calcium sulphate
Potassium iodide
Distilled water to
pH
Preparation: Autoclave at 1210C for 15 minutes.
0.5 g
0.1 g
0.1 g
0.3 g
0.04 g
0.01 g
0.3 g
10 ml
1000 ml
7.0
0.5 g
6.0 g
11.0 g
2.0 g
0.5 g
0.05 g
0.02 g
1000 ml
7.0
Use: For isolation of algae fiom soil and water.
Note: Add agar 20gAitre to produce solid medium.
Algal Medium
(ASM-I medium)
Composition:
Sodium nitrate Dipotassium hydrogen
Phosphate
Disodium hydrogen phosphate
Magnesium chloride
Magnesium sulphate
Calcium chloride
Femc chloride
Sodium ethylene diarnine tetracetate (N% EDTA)
Boric acid
Manganese chloride
Zinc chloride
Cobalt chloride
Cupric chloride
Distilled water
pH
Preparation: Sterilize at 121°C in autoclave for 15 minutes.
Use: For cultivation of freshwater planktonic algae.
170.0 mg
17.4 mg
14.2 mg
40.7 mg
49.3 mg
22.2 mg
1.1 mg
6.7 mg
2.5 mg
1.4 mg
0.4 mg
0.02 mg
0.00014 mg
1000 ml
7.0 - 7.5
Algal Medium
(Beneck's broth)
Composition:
KNO3
MgSO4.7H2O
K2HPO4 or KH2PO4
CaCO3
FeCl3 (1%)
Distilled water to
Agar
pH
Preparation: Steam sterilize at 121°C for 15 minutes.
Precaution: Phosphate should be autoclaved separately from
mixed aseptically upon cooling.
Use: Used for isolation of algae from soil.
0.2 g
0.2 g
0.2 g
0.1 g
2.0 drops
1000 ml
25 g
7.0 – 7.5
the other components and
Algal Medium
(Chu's medium No. 10 modified)
Composition:
Calcium nitrate
Dipotassium hydrogen phosphate
Sodium carbonate
Magnesium sulphate 7H20
Sodium silicate
Ferric citrate
Citric acid
A5 trace elements stock solution (Optional)
Distilled water to
A5 Trace elements stock solution
Boric acid
Manganese chloride
Zinc sulphate
Molybdenum trioxide (85%)
Cupric sulphate
Distilled water to
pH
Preparation: Autoclave at 1210C for 15 minutes.
Use: For cultivation of common freshwater and soil algae.
Precaution: Sterilize phosphate separately.
0.04 g
0.01 g
0.02 g
0.025 g
0.25 g
0.003 g
0.003 g
1.0 ml
1000.0 ml
2.86 g
1.81 g
0.222 g
0.0177 g
0.079 g
1000.0 ml
8.5 - 9.0
Algal Medium
(Modified Bristol's medium)
KH2PO4
NaNO3
MgSO4.7H2O
CaCl2.6H2O
NaCl
FeCl3.6H2O
Distilled water to
Preparation: Steam sterilize at 12 1°C for 15 minutes.
Use: Fro isolation and cultivation of soil algae.
Blue Green Algal Medium
Composition:
0.50 g
0.50 g
0.15 g
0.05 g
0.05 g
0 0.01 g
1000 ml
K2HPO4
KNO3
Ca(NO3)2
MgSO4.7H2O
FeCl3
Tap water to
0.2 g
1.0 g
0.1 g
0.1 g
0.001 g
1000 ml
Blue Green Algal Medium
Composition:
K2HPO4
(NH4)2HPO4
MgSO4.7H2O
CaSO4
FeSO4.7H2O
Tap water to
0.4 g
0.8 g
0.4 g
0.4 g
0.001 g
1000 ml
Blue Green Algal Medium
Composition:
K2HPO4
MgSO4.7H2O
CaC12.6H2O
NaHCO3
Distilled water to
pH
0.2 g
0.2 g
0.116 g
0.2 g
300 ml
7.8
Blue Green Algal Medium
Composition:
KH2PO4
MgSO4.7H2O
NaCl
CaCO3
FeSO4.7H2O
Microelements
Distilled water to
pH
Microelements:
MnSO4.4H2O
Na2MoO4.2H2O
Distilled water to
Blue Green Algal Medium
(Allen and arnon's medium-modified)
Composition:
0.25 g
0.125 g
0.125 g
5.5 g
0.0025 g
1 ml
1000 ml
7.3
2.5 g
0.25 g
1000 ml
Magnesium sulphate
Calcium chloride
Sodium chloride
Dipotassium hydrogen phosphate
A5 trace elements stock solution (see page 178)
Distilled water to
PH
Use: For isolation of nitrogen fixing blue algae.
0.025 g
0.05 g
0.20 g
0.35 g
1.0 ml
1000 ml
8.5 - 9.0
Blue Green Algal Medium
(Gerloff medium)
Composition:
Ca(NO3)2
0.04 g
K2HPO4
0.01 g
MgSO4.7H2O
0.025 g
Na2CO3
0.02 g
Na2SiO3 .9H2O
0.025 g
Ferric citrate
0.003 g
Citric acid
0.003 g
Distilled water to
1000 ml
Chromatium Agar Shake Deeps
Composition:
Solution A
NH4Cl
0.1 g
KH2PO4
0.1 g
MgCl2
0.05 g
Agar
20.0 g
Distilled water to
780 ml
Solution B
NaHCO3
2.0 g
Distilled water to
100.0 ml
Solution C
Na2S.9H2O
1.0 g
Distilled water to
100.0 ml
Preparation: After steam sterilizing each of the three solutions at 121°C for 15 minutes,
cool them to 50°C and pour solutions B and C in to flask A. Dipense aseptically to sterile
tubes.
Use: For purification of Chrimatium.
The use of enrichment media never produces a pure culture. To get pure isolates it
is necessary to serial dilute the mixed cultures 1 with sterile agar shake tubes.
Chlorobium Enrichment Media
Composition:
Solution A
NH4C1
1.0 g
KH2PO4
1.0 g
MgC12
0.5 g
Distilled water to
700 ml
Solution B
NaHCO3
2.0 g
Distilled water to
100 ml
Solution C
Na2S.9H2O
1.0 g
Distilled water to
100 ml
Solution D
FeCl3.6H2O
5.0 g
Distilled water to
100 ml
Solution E
1 N H3PO4
Reparation: Sterilize all five solution at 1210C for 15 minutes. Cool them to 50°C. Add
solution B, C and D into solution A. Adjust the pH at 7.3 by using solution E. Dispense
aseptically in to tubes.
Use: For enrichment of Chlorobiurn.
Mastigocladus Agar
Composition:
MgSO4.7H2O
K2HPO4
NaHCO3
CaC13
Soil extract
Ferrous ammonium citrate (0.1 %)
Glass distilled water to
0.02 g
0.01 g
10 g
1.0 ml
100 ml
Phormidium Agar
Compositwn:
Distilled water
Soil extract
100 ml
10 ml
KNO3
K2HPO4
Sodium glutamate
Glycine
Liver infusion (Dehydrated)
Trace elemet stock solution
Artificial sea water (double strength)
Trace element stock solutions
Na2B2O7.10H2O
CaSO4.5H2O
Fe3PO4
MnSO4.4H2O
Na2MoO4.2H2O
ZnSO4.7H2O
Distilled water to
Preparation: Steam sterilize at 1210C for 3 minutes.
Use: Used for cultivation of Phormidium.
0.02 g
0.002 g
0.05 g
0.05 g
0.002
0.1 ml
36 ml
20 mg
100 mg
150 mg
20 mg
10 mg
100 mg
1000 ml
Porphyridium Agar
Composition:
Yeast extract
0.1 g
Tryptone
0.1 g
K2HPO4
0.002 g
MgSO4.7H2O
0.002 g
KNO3
0.02 g
Soil extract
10 ml
Artificial sea water (double strength)
36 ml
Distilled water to
100 ml
Preparation: Autoclaved at 121°C for 3 minutes.
Use: Used for cultivation of Porphyridium.
Note: Longer autoclaving has been found to result in detractive changes in the media and
resulting solutions do not permit the optimal growth.
4. Protozoa
Entamoeba medium
(Balamuth's medium)
Composition:
Liver, infusion from
27.2 g
Rice powder
1.42 g
Agar
1.1 g
Peptone
0.55 g
Sodium glycerol phosphate
0.3 g
NaCl
0.27 g
Horse serum (sterile)
5.0 ml
Distilled water to
100 ml
Preparation: Add all components, except horse serum and rice powder in water.
Dissolve by heating. Steam sterilize at 121°C for 20 minutes. Sterilize the rice powder at
160°C for 60 minutes. Add (0-lg) rice powder and (0.35ml) horse serum in tubes
containing
medium (7.0ml).
Use: Used for cultivation of Entamoeba species.
Trichomonas Medium
(Diamond's medium - modified)
Composition:
Casein digest
Yeast extract
20.0 g
1.0 g
L-Cysteine HCI.H20
0.5 g
Maltose
0.5 g
L-Ascorbic acid
0.02 g
Horse serum, inactivated
100 ml
Antibiotic solution
10 ml
Distilled water to
1000 ml
pH
6.5
Antibiotic solution
Streptomycin sulphate
1.5 g
Amphotericin B
2.0 mg
Penicillin G
10,00,000 units
Distilled water to Sterilize by filtration.
100 ml
Preparation: Add all components except horse serum and antibiotic solution in distilled
water. Steam sterilize at 121°C for 20 minutes. Mix all solutions aseptically. Distribute in
sterile tubes.
Use: Used for the selective cultivation of Trichmonas.
Trichimonas Medium
(Modified CPLM medium - Cysteinej Peptone, Liver Infusion, Maltose medium)
Composition:
(a) Basal medium
Peptone
32.0 g
Maltose
1.6 g
Liver digest
20.0 g
Cysteine hydrochloride
2.4 g
Ringer’s solution to (1/4 strength)
1000 ml
NaOH
about 9 ml to adjust pH 6.0
Dissolve the ingredients. Adjust the pH. Steam sterilize at 100°C for 30 minutes
and filter with Whatman no. 1 paper. Autoclave at 115°C for 15 minutes.
(b) Pencillin streptomycin solution
Pencillin
1,00,000 units
Streptomycin
0.1 g
Sterile water
10 ml
(c) Nystatin solution
Nystatin
50,000 units
Sterile water
10 ml
Preparation of complete medium
Basal medium
90 ml
Sterile inactivated
10 ml
Serum (Horse/human/rabbit) Penicillin
Streptomycin solution
1 ml
Nystation solution
1 ml
Before use add the serum and antibiotics.
Use: Used for cultivation of Trichmonas vaginalis.
This medium support the growth of I: vaginalis under strictly anaerobic condition.
Leishmania Medium
(Tobie, Von Brand and Mehlman's medium)
Composition:
Solid phase
(a) Basal medium
Meat extract
Peptone
NaCl
Agar
Distilled water to
pH
Autoclave at 121°C for 25 minutes.
1.5 g
2.5 g
4.0 g
7.5 g
500 ml
7.2 – 7.4
(b) Citrated blood
Rabbits blood collected in sterile sodium citrate and inactivated at 56°C for 30 minutes.
Preparation of complete medium
Basal medium
75 ml
Blood
25 ml
0
Melt the basal medium cool to 45 C. Add the blood and distribute approx. 5 ml in
sterile tubes. Prepare slants.
Liquid phase
NaCl
KCl
CaCl2
KH2PO4
Glucose
Distilled water to
8.0 g
0.2 g
0.2 g
0.3 g
2.5 g
1000 ml
Dissolve the ingredients and autoclave at 121°C for 15 min. with sterile
precautions add 2 ml to test tubes containing the solid medium.
Use: Used for cultivation of Lekhmania and Trypanosomas.
5. Baceriophage
A great many culture media have been devised for the growth of bacteriophage. These
media have to satisfy two main criteria.
1. They must be adequate for the growth of the host bacteria.
2. They must provide suitable conditions for the attachment, penetration,
multiplication and release of the phase.
Soft Agar
Composition:
Agar
Distilled water to
Preparation: Sterilize at 121°C for 15 minutes.
Use: Used as a superimposed layer over firm agar.
0.7 g
100 ml
Phage Broth
Bactotryptone
0.5 g
Bactopepone
0.5 g
Glucose
0.1 g
Distilled water to
100 ml
pH
7.2
0
Preparation: Autoclave at 115 C for 30 minutes.
Use: Used for dilution of sample for isolation of bacteriophages.
Phage Agar
Composition:
Peptone
0.5 g
Beef extract
0.3 g
Agar
1.5 g
CaCl2
0.002 M
Note: NaCl is committed to prevent spreading of Proteus. Preparation: Previously
sterilized CaCl2 is to be added after autoclaving the medium 121°C for 20 minutes.
Use: For preparation of phage typing plates for Proteus.
Tryptone Broth
Composition:
Tryptone
10.0 g
Sodium chloride
5.0 g
Calcium chloride (1M) solution
1.0 ml
Distilled water to
1000 ml
pH
7.2
Preparation: Previously sterilized CaC12 is too added after autoclaving the medium
121°C for 20 minutes.
Use: Tryptone broth is used for dilution of samples for isolation of bacteriophages.
Tryptone Soft Agar
Composition:
Tryptone broth 1000 ml
Agar 11.0 g
Preparation: As above.
Use: Tryptone soft agar is used as a superimposed layer over firm agar for isolation of
bacteriophages.
Tryptone Yeast Extract Agar
Composition:
Tryptone
Yeast extract
Dipotassium hydrogen phosphate
Sucrose
Agar
Distilled water to
pH
Preparation: Steam sterilize the medium at 100°C for 1 hr.
10.0 g
5.0 g
3.0 g
50 g
15 g
1000 ml
7.4
Use: In phase typing, bacterial suspension to be tested is swabbed over an agar surface.
The bottom of the plate is marked off in squares and labelled to indicate which phase
types are going to be used. A small drop of each phase type is added to their respective
squares.
Deca Strength Phase Broth (DSPB)
Composition:
Peptone
100 g
Yeast extract
50.0 g
Sodium chloride
25.0 g
K2HP04
80.0 g
Distilled water to
1000 ml
pH
7.6
Preparation: It is sterilized in autoclave at 121°C for 20 minutes.
Use: 5 ml broth + 5ml Escherichia coli is added to 45 ml of sewage hr the enrichment of
bacteriophase during its isolation.
Significance: DSPB medium is ten times as strong as ordinary broth to accommodate
dilution with 45 ml of sewage.
"F" Medium (Anderson's)
(Defined lactate ammonium medium)
Composition:
NH4Cl
1.0 g
MgSO4.7H2O
0.1 g
KH2PO4
1.5 g
Na2HPO4
3.5 g
Lactic acid
9.0 g
Distilled water to
1000 ml
pH
6.5
Preparation: Autoclave at 121°C for 20 minutes. Magnesium sulphate and lactic acid
solution is sterilized separately and then added aseptically.
Use: Used for growing E. coli and the T-even phages.
"N" Medium (Anderson's)
Composition:
Difco nutrient broth
NaCl
Distilled water to
Preparation: Autoclave at 121°C for 20 minutes.
8.0 g
5.0 g
1000 ml
Use: Used for growing E. coli and T-even phages.
"M9" Medium (Adam's)
Composition:
NH4Cl
1.0 g
MgSO4.7H2O
0.13 g
KH2PO4
3.0 g
Na2HPO4
6.0 g
Glucose
4.0 g
Distilled water to
1000 ml
0
Preparation: Autoclave at 121 C for 20 minutes. Magnesium sulphate solution is
sterilized separately and then added aseptically.
Use: Used for growing E. coli and the T-even phases.
Peptone Broth (Hershey, Dixon and Chase)
Composition:
Peptone
10.0 g
Nacl
3.0
Glucose
1.0 g
MgSO4
1 mm
CaC12
0.1 mm
P (as phosphate buffer, pH 7.0)
5 mg
Distilled water to
4000 ml
pH
6.8
Preparation: Autoclave at 121°C for 20 minutes. Sterilize MgSO4
separately.
Use: Used for growing E. coli and the T-even phages.
MS Broth (Davis and Sinsheimer)
Composition:
Tryptone
Nacl
Yeast extract
Distilled water to
Glucose solution, (1 0%) sterile
CaCl2 (M) solution, sterile
Thiamine hydrochloride 10 mg/ml, sterile
Preparation: Autoclave at 1210C for 15 minutes.
Use: Used for preparing coliphases MS2.
10.0 g
8.0 g
1.0 g
1000 ml
10 ml
2 ml
1 ml
and glucose
H Medium (Maaloe and Hannawalt)
Composition:
Tris
KCl
MgCl2
MgCl2.6H2O
Na2HPO4
Na2SO4
HC1 (conc.)
Glucose
Distilled water to
Preparation: Autoclave at 121°C for 15 minutes.
Use: Used for growing filamentous coliphages M13.
12.0 g
2.0 g
2.0 g
0.5 g
0.05 g
0.02 g
7.5 ml
0.5 g
1000 ml
KC Broth (Sinsheimer et al)
Composition:
KCl
Tryptone
Cacl2 (0.01 M)
Distilled water to
Preparation: Autoclave at 121°C for 15 minutes.
Use: Used for growing coliphage X174.
MGM Medium (Lannni)
Composition:
Maleic acid
Glucose
NH4Cl
P (as orthophsphate)
S (as Na2SO4)
KCl
Gelatin
FeSO4.7H2O
MgSO4
Distilled water to
pH
Use: Used for growing phages T5.
L Broth (Davision and Freifelder)
100 mg
200 mg
1 ml
20 ml
5 x 10-2M
5.0 g
1.0 g
40 mg
27 mg
2.0 g
0.1 g
0.5 mg
10-4M
1000 ml
7.15
Composition:
Tryptone
1.0 g
Nacl
0.5 g
MgCl2
0.01 M
Distilled water to
100 ml
Use: Used for preparation of h phase.
"L" Broth (Levine)
Composition:
Trypone
1.0 g
Yeast extract
0.5 g
NaCl
0.05 g
Glucose
0.01 g
Distilled water to
100 ml
pH
7.0
Use: Used for growing Escherichia coli and for isolation of bacteriophage.
3
Staining Methods
Bacterial Staining
Simple Staining (Monochrome staining)
Solution required
(a) Loeffler' methylene blue
Methylene blue chloride
0.3 g
95% Ethanol
30 ml
0.1 % KOH
100 ml
Dissolve methlylene blue chloride in ethanol. Add 0.1% KOH. Filter the solution before
use. Store at room temperature.
OR
a. 1% Crystal violet
OR
a. Carbol fuchsin (see acid fast staining). Dilute 10 times before use.
Procedure
1. Prepare the smear and heat fix
2. Treat the smear with 5-7 drops of staining solution.
3. Allow the smear to react as follows
Loeffler's methylene blue for 120 to 150 seconds.
OR
per cent crystal violet for 60 to 120 seconds.
OR
Diluted carbol fuchsin for 15 to 30 seconds.
4. Pour off the staining solution and wash the slide by gentle
5. flow of tap water.
6. Dry the slide in air.
7. Examine under oil immersion lens.
Result: Bacteria appear blue or violet or red by respective stains.
Simple Staining Methods for Bacteria in Milk
(Breed's smear count method)
Solution Required
a. Xylene or chloroform
b. 95% ethyl alcohol
c. Breed's methylene blue.
Methylene blue chloride
0.3 g
95% Ethyl alcohol
30.0 ml
2% phenol in water
100.0 ml
Dissolve methylene blue chloride in ethanol. Then add above solution to phenol in
water. Mix
d. 90% alcohol.
Procedure
1. Mark a clean slide with glass marking pencil to make one centimetre square.
2. Place 0.01 ml of milk sample in the centre of the square.
3. Spread the sampfe with inoculating needle to form uniform smear covering the
square.
4. Heat fix the smear and treat the slide with solution a for about 1 minute.
5. Treat the smear with solution b for 3 minutes.
6. Treat the smear with solution c for 2 minutes.
7. Wash the smear with solution d till smear appears faintly blue. (approx 30
seconds)
8. Dry in air and examine under oil immersion lens.
Result: Bacteria appear blue in colour.
Staining of Azotobacter Cysts
Solution required
a. Staining solution
Glacial acetic acid
8.5 ml
Sodium sulphate (anhydrous)
3.25 g
Neutral red
200.0 g
Light green S.F. yellowish
200.0 mg
Ethanol
50.0 ml
After 15 minutes of incubation remove amorphous precipitate by filtering through
0.5 pm membrane filter.
Procedure
1. Suspend the growth of the bacteria in the solution a for wet mount preparation.
2. Observe under oil immersion lens.
Result
1. Vegetative cens appear light yellowish green.
2. The early stage of encystment shows dark green cytoplasm.
3. Cyst: Intine appears colourless
Exine appears brownish red.
Cytoplasm appears green.
Staining of Actinomycetes
Solutions required
a. Absolute methanol
b. Hucker's crystal violet (see grams staining)
Procedure
1. Treat the growth on the coverslip with few drops of solution a for 15 minutes.
2. Wash with tap water and blot dry.
3. Stain with solution b for 1 minute.
4. Wash with tap water, dry in air.
5. Observe under oil immersion lens.
Results: Mycelium and spores appears violet in colour.
Note: Slide cultures must first be dried by placing them over boiling water for about 5
minutes until agar has dried.
Staining of Actinomycetes
Solutions required
a. Staining solution
Bismark brown stain (0.1% w/v)
Toluidine blue stain (0.1% w/v)
Saturated ammonium sulphate solution Mix together
40 ml
40 ml
20 ml
Procedure
1. Grow the culture of actinomycetes on the surface of sterile cellophane placed on
placed on solidified nutrient agar medium.
2. Remove the cellophane bearing growth fiom the agar surface.
3. Treat the growth for 2 minutes with the solutions a.
4. Wash the slide with tap water.
5. Air dry and observe under oil immersion lens.
Result: Vegetative mycelium appears light yellow and the spores blue.
Staining of Mycoplasmas Colony
I. Dine's method
Solution required
a. Diene's stain
Methylene blue
2.5 g
Azure II
1.25 g
Sodium carbonate
0.25 g
Benzoic acid
0.20 g
Maltose
10.0 g
Distilled water to
100 ml
Procedure
1. Flood the plate containing suspected colonies of mycoplasmas with 1:9 dilued
solution a in distilled water.
3. Remove the stain.
4. Examine the plate under low power microscope.
Result: The colony appears granular, royal blue to greenish blue in colour.
II Staining with cresyl-fast violet
Solution required
a. Cresyl-fast violet solution
Stock solation
Cresyl - fast violet
1.0 g
Distilled water to (pH-3.7, adjusted with Glacial
acetic acid 1-5 drops/100 ml)
100 ml
Allow the solution to ripen for 48 hours.
Working solution
Stock solution
20.0 ml
Sodium chloride
0.05 g
Maltose
7.0 g
Mix sodium chloride in stock solution. Filter. Add maltose.
Procedure
Similar to that of Dienes method.
Result: Colonies of mycoplasmas appear red to purple.
Staining method for Bruce11 Abortus
Solutions required
a. Carbol fuchsin 10 times diluted (see acid fast staining ZNCF method.)
b. 0.5 acetic acide.
c. Loeffler's methylene blue (see monochrome staining).
Procedure
1. Prepare the smear do not heat fix.
2. Treat the smear for 15 min with solution a.
3. Drain the solution a treat with solution b for 1'2-20 seconds.
4. Wash with water and treat with solution c for 1-2 min.
5. Wash with water, dry and observe under oil immersion lens. .
Results: Brucella abortus appears red in colour. Other organisms appear blue in colour.
Acid Fast Staining
1. Ziehl-Neelsen method
Solutions required
a. Carbol fuchsin (Ziehl-Neelsen)
Basic fuchsin
1.0 g
95% ethanol
10 ml
5% phenol
100 ml
Dissolve basic fuchsin in ethanol then mix with phenol. Allow this solution to
ripen for 1-2 weeks.
b. Sulphuric acid (20% solution)
c. Leffler's methylene blue (see monochrome staining)
OR
1% Malachite green in water.
Procedure
1. Prepare a smear and heat fix.
2. Treat the smear with solution a and heat the slide by gentle flame for five minutes.
(The stain must not be allowed to evaporate and dry on the slide.)
3. Allow the slide to cool.
4. Wash with water.
5. Treat with solution b till red cnlnl~rn o longer comes out (usually for 90 secs.)
6. Wash with water.
7. Treat the smear with solution c for 20 to 30 seconds.
8. Wash, air dry and examine under oil immersion lens.
Results: Acid fast cells stain bright red, while non-acid fast are stained green or blue
colour according to solution c used.
Note:
a. Following decolourisers are usually used for different organisms.
1. 5 per cent sulphuric acid for M. leprae.
2. 3 per cent HCl in 95% ethanol for M. tuberculosis.
3. 1 per cent sulphuric acid to demonstrate acid fast clubs of Actinomyces and
Nocardia.
b. When malachite green is used as a counterstain (solution c), use deep green filter
in the light source for microscopic observasion.
2. Method of Gross
Solutions required
a. Basic fuchsin with Tween 80
Basic fuchsin chloride
2.0 g
Phenol
6.0 ml
95% ethanol
12.5 ml
distilled water to
150.0 ml
Tween 80
15 drops
Dissolve basic fuchsin chloride in phenol at 80°C. Add 95% ethyl alcohol by
stirring. Make the final volume to 150 ml with distilled water. Allow it to ripen for
1-2 weeks. Filter before use and add Tween 80.
b. 3% HCl in ethanol.
c. Loeffler's methylene blue (see monochrome staining).
Procedure
1. Prepare the smear and heat fix.
2. Treat the smear with solution a for 5-10 min.
3. Wash the smear with water.
4. Wash the smear with solution b till red colour not longer comes out (usually 120
seconds).
5. Wash the slide with water.
6. Treat the slide with solution c for 3 min.
7. Wash, air dry and examine under oil immersion lens.
Results: Acid fast organisms appear red. Non acid fast organisms and background appear
blue in colour.
3. Method of Trauant et al.
Solutions required
a. Staining solutions
Auramine "0"
0.3 g
Phenol
3.0 g
Distilled water
97.0 ml
Dissolve the phenol in water with gentle heat. Add the auramine slowly. Shake
vigorously until dissolved, filter and store in dark stopped bottle.
b. Traunt's decolourizer.
NaCl
0.5 g
HCl
0.5 ml
75% Ethyl alcohol to
100 ml
c. Potassium permanganate solution (1 to 1000) aqueous. Procedure
Procedure
1. Prepare the smear and heat fix.
2. Treat it with solution a for 15 minutes.
3. Wash the smear with water and treat the smear with solution b for 5 minutes.
4. Wash with water then treat the smear with solution c for 30 seconds.
5. Wash air dry & examine under 8 mm dry objective &a high power eye piece (20x).
Result: Tubercle bacilli appear luminescent yellow coloured. Background appears dark.
Negative Staining
Solution required
a. Nigrosin solution
Nigrison (G.T Gurr)
10 g
Distilled water to
1000 ml
Dissolve nigrosin in warm distilled water (require an hour) & filter. Add formalin
0.5 % (i.e., formaldehyde 0.19 %) as a preservative.
OR
2 % congored solution.
OR
India ink.
Procedure
1. Take a loopful bacterial suspension and a drop of solution a at one end of clean
glass slide. Mix.
2. Spread this mixture as a film using the another slide.
3. Allow it to air dry and examine under high power and oil immersion lens.
Result: Bacteria appear colourless with dark background, blue black with nigrosin, red
with congored and blue with India ink.
Note: Film should not be too thick or too thin.
Capsule staining
A. Positive staining methods
1. Method of Hiss (modification of Anthony)
Solution required
a. 1% Crystal violet
b. 20% CuSO4.5H2O (aqueous solution)
Procedure:
1. Prepare a smear. Do not heat fm.
2. Treat the slide with solution a for 2 min.
3. Remove the solution a by washing with solution b.
4. Dry and examine under oil immersion lens.
Result: cells appear dark purple, capsules appear pale blue.
2. Method of Moller
Solutions required
a. Moller's fixations (see fixatives)
b. Moller's crystal violet solution
Crystal violet chloride
0.5 g
95% ethyl alcohol
10 ml
Distilled water to
100 ml
Dissolve crystal violet chloride in 95% ethyl alcohol. Then add distilled water.
c. Saturated aqueous CuS04.5H20 solution
Procedure
1. Prepare the smear and treat the smear with solution a for 15 seconds. Pour off the
solution a and dry the smear.
2. Treat the smear with solution b for 2 minutes. Pour off the solution b.
3. Treat the smear with solution c for 10 seconds.
4. Dry the smear and examine under oil immersion lens.
Result: Capsules appear light purpule violet. Bacterial cells appear dark violet.
B. Negative Stain methods
1. Method of Howie an Kirkpatrick (Releif staining)
Solution required
a. Staning solution
10 per cent water soluble eosin, 'Yellowish' or
'bluish' or erythrosine in distilled water
40 ml
Serum (human, rabbit, sheep or ox heated at
56OC for thirty minutes)
100 ml
Crystal of thymol
Mix well. Allow the mixture to stand at room temperature for several days.
Centrifuge and store the supernatant fluid at room temperature.
Procedure
1. Take one drop of suspension and one drop of solution a at Y one end of slide. Mix
well. Wait for 1 minute.
2. Spread this mixture with the help of another slide as a thin
3. Dry without heating and examine under oil immersion lens.
Result: Background and bacteria appear red while capsule appear stained or lightly
stained.
2. Method of Maneval's
Solution required
a 1% congored
b. Maneval's stain I
5% aqueous solution of phenol
30.0 ml
20% acetic acid
10.0 ml
30% FeC12.6H2O
4.0 ml
1% acid fuchsin
2.0 ml
Mix thoroughly.
Procedure
1. Take one drop of bacterial suspension and one drop of solution a at one end of
slide. Mix. Spread over a slide as a thin film with the help of another slide.
2. Air dry the smear.
3. Treat the smear with solution b for 1 minutes.
4. Wash with distilled water, air dry and examine under oil immersion lens.
Dissolve the nigrosin in warm distilled water, add the formalin and filter.
b. Loeffler's alkaline methylene blue. (see monochrome staining).
Procedure
1. Take one looful of culture on a clean slide.
2. Add one looful of freshly filtered solution a.
3. Mix, allow to dry in air and fix with gentle heat.
4. Treat the smear with solution b for 30s.
5. Rinse rapidly in water, air dry and observe under oil immersion lens.
Result: Bacterial cell appear blue, capsule appear colourless unstained against a dark
grey background of nigrosin.
Note: Safranin may be used in place of the methylene blue. (solution b)
5. Dry India ink film method
Solutions required:
a. 6% glucose solution in water.
b. 8% aqueous solution of nigrosin
OR
Indian ink.
a. Methanol or Leishman stain
b. Methyl violet stain (1% aqueous solution).
Procedure:
1. Take one drop of bacterial suspension and one drop of solution a at one end of
slide.
2. Add one drop of solution b to it.
3. Mix and spread the mixture over a slide as a thin film with the help of another
slide.
4. Dry the smear in air.
5. Treat the smear by solution c. Pour off immediately, dry in air.
6. Treat the smear with solution d for 1-2 minutes.
7. Wash with water, dry and examine under oil immersion lens.
Result: Capsules appear colourless, cells appear purpule. Background appear grey6.India ink preparation (Wet film)
Solution required:
a. Indian ink
Procedure:
1. Place one loopful of solution a on a perfectly clean glass slide.
2. Emulsify a small portion of solid bacterial culture in the drop of solution a or mix
in a loopful of liquid culture.
3. Cover the mixture with a clean cover glass and press the latter down firmly, to
form a very thin film.
4. Seal the edges of the cover glass with paraffin wax or other suitable medium.
5. Examine the smear under oil-immersion objective.
Result: Bacteria highly refractile, surrounded by a clear zone against a dark grey
background of ink particles. Noncapsulated bacteria do not show this clear zone.
Sprirochete Staining
1. Fontana's method
Solutions required:
a. Fontana's fixative (see fixatives)
b. Absolute ethyl alcohol
c. Fontana's mordant
Phenol (melted)
Tannic acid
Distilled water to
Dissolve tannic acid in water then add melted phenol.
d. Fontana's stain.
Add 10 per cent ammonia solution to 0.5 per cent aqueous silver nitrate solution
dropswise, until the precipitate formed just redissolve. Slowly add more silver nitrate
solution until the light precipitate reformed and remain stable.
Procedure:
1. Prepare the smear and air dry.
2. Treat the smear with solution a for three times, each time for 30 seconds.
3. Wash the smear with solution b and treat with the same solution for 3 min.
4. Remove solution b and dry the slide in air.
5. Treat the smear with solution c and heat the slide from below i till steam rises for
30 seconds. Cool.
6. Wash the slide with distilled water and air dry.
7. Treat the smear with solution d and heat the slide until steam rises and smear
becomes brown in colour.
8. Wash, dry and examine under oil immersion lens.
Result: Spirochetes appear brownish black against yellow background.
Note: Some immersion oils, cause the film to fade at once. Use Canada balsam only.
2 Becker's method (modified)
Solution required:
a. Fontana's fixative (see fontana's method)
b. Mordant (as in fontana's method)
c. staining solution.
Basic fuchsin (Saturated alcoholic solution)
45.0 ml
Shunk's mordant B (95 or 100% ethanol and
aniline oil 4 ml)
18.0 ml
Distilled water to
100.0 ml
Mix the Shunk's mordant and the alcoholic fuchsin and then add the distilled
water. (the glassware should be dry). Filter before use.
Procedure:
1. Prepare the smear and dry in air.
2. Treat the smear with solution a for 1 to 3. minutes.
3. Wash in water.
4. Treat the smear with solution b for 3-5 minutes, wash in water.
5. Treat the smear with solution c for 3 to 5 minutes.
6. Wash in water and dry. Observe under oil immersion lens.
Result: Spirochetes appear red in colour.
3. Rue's method
Solutions required:
a. l%Formalin
b. 5% NaHCO,
c. Basic fuchsin
Basic fuchsin
95% ethyl alcohol 25.0 ml
Dilute the stain 1:9 before use.
Procedure:
1. Prepare a smear and air dry.
2. Treat the smear with solution a for 1 minute and dry.
3. Place a drop of solution b on the smear and then add 10 drops of solution c. Mix
well and treat the smear with this mixture for 3-5 minutes.
4. Wash with water, dry and examine under oil immersion lens.
Result: Spirochetes appear red in colour.
4. Levaditi's method (Staining of spirochetes in tissues) pyridine modification
Solutions required:
a. a.10% formalin
b. %-98% alcohol
c. Silver nitrate pyridine solution
i. Pure pyridine 10.0 ml
ii. Silver nitrate (1 % solution) 90.0 ml
d. d.10% pyriding solution
e. Reducing fluid
Acetone (pure)
10.0 ml
Pyridine (pure)
15.0 ml
Formalin (4%)
100.0 ml
Use immediately.
Procedure:
1. Keep the small piece of tissue (1 mm thick) in solution a for 24 hrs.
2. Wash the tissue for 1 hr in water and then place it in solution b for 24 hrs.
3. Treat the tissue by solution c at room temperature for 2 hrs and then at about 50°C
for 4 to 6 hrs.
4. Wash the tissue section with solution d.
5. Treat the tissue immediately by solution e for 48 hrs in the dark.
6. 'Wash with distilled water, dehydrate with increasing strength of alcohol.
7. Proceed for section cutting according to standard procedure.
Flagella Staining
Special care should be taken in cleaning slide and preparing bacterial smear in flagella
staining.
a. Cleaning the slide: Wash the slide with dichromic acid cleaning solution. Wash
with distilled water. Pass the slide through Bunsen flame until flame shows yellow
colour. I
b. Preparation 01 smear: Take agar slant having actively growing young culture. (1820 hrs old). Add slowly about 2-4 ml of sterile saline through the side of tube
keeping slant in upward position. Rotate the tube slowly between palms or 5
minutes. Incubate the tube at optimum temperature for 40 minutes. Take a small
drop of saline suspension from the tube by capillary pipette and put at the one end
of slide. Tilt the slide so that the drop will slowly run over the slide and form a
smear. Allow the slide to air dry. Do not heat fix the smear. Stain the slide the
suitable staining method.
1. Method of Bailey (modified)
Solutions required:
a. Bailey's staining solution a.
6% aqueous FeC13.6H2O
6.0 ml
10% solution of tannic acid Mix. Filter before use 18.0 ml
b. Bailey's staining solution b
Bailey's solution a 0.5% basic fuchsin
3.5 ml
in ethyl alcohol
0.5 ml
Concentrated HCl
0.5 ml
Formalin
2.0 ml
Mix. Filter before use. Use freshly.
c. Carbol fuchsin (see acid fast staining)
Procedure:
1. Prepare the smear as given above.
2. Treat the smear by solution a for about 3-5 minutes.
3. Remove the solution a and treat the smear by solution b for 7 minutes.
4. Wash with distilled water. Do not dry.
5. Treat the smear with solution c immediately and heat it from below till steam rises
for 1 min.
6. Wash the slide with water, air dry ad examine.
Result: Flagella and cell wall appear red in colour.
2. Method of Patel, Kulkarni and Gaikwad
Solutions required:
a. Tannic acid solution
Tannic acid
0.2 gm
Distilled water to
100 ml
b. Iodine solution
Iodine crystal
2.0 g
1 N NaOH
10.0 ml
c. Basic fuchsin solution
Basic fuchsin
Absolute alcohol
Distilled water
Procedure
1. Prepare a smear as mentioned earlier.
2. Treat the smear with 4-6 drops of solution a immediately followed by equal drops
of solution b.
3. Heat the smear from below gently for 1-2 minutes.
4. Add 2-3 drops of solution c to the smear, and again heat for
2. Minutes. (do not allow to dry the steam).
5. Wash the slide with water, air dry and examine tinder oil immersion lens.
Result: Bacteria appek red, flagella appear pink in colour.
3. Method of Gray
Solutions required:
a. Gray's staining solution
Solution I
Saturated solution of aluminium potassium sulphate
5.0 ml
20 per cent tannic acid saturated solution of
2.0 ml
Mercuric chloride
2.0 ml
Solution II
Basic fuchsin
6.0 g
Ethanol
100.0 ml
Complete Solution
Solution I
Solution II
9.0 ml
Prepare fresh
0.4 ml
Procedure:
1. Prepare smear as given earlier.
2. Treat the smear by solution a for 10 minutes.
3. Wash with water air dry and examine under oil immersion lens.
Result: Flagella appear light red and cells appear dark red in colour.
4. Method of Gray-modified
Solutions required:
a. Similar as Gray's staining solution except the concentration of basic fuchsin is doubled.
Procedure:
1. Prepare a bacterial suspension as given earlier.
2. Place a loopful of suspension on a clean slide and place a cover slip over it.
3. After ten minutes place two drops of solution a at one edge of coverslip. Capillary
action will such the solution below the coverslip.
4. Observe the slide after a minute.
Result: Similar as Gray's method.
Note: In this method it is possible to observe the m o w and presence of flagella.
5. Method of Leifson
Solutions required:
a. Staining solution
Solution I
Tannic acid
3.0 g
Phenol
0.2 ml
Distilled water to
100.0 ml
Solution II
Sodium chloride
1.5 g
Distilled water to
100.0 ml
Solution III
Basic fuchsin
1.2 g
Ethanol
100.0 ml
Adjust the pH of distilled water to 5 before use. Mix I, 11 & III solutions in equal
volumes in the given order. Store at 3-50C.
b. Loeffler's methylene blue. (see monochrome staining).
Procedure:
c. 1: Prepare the smear as given earlier.
3. Treat the smear with solution a for 5-10 minutes. (Allow to react till precipitates
are observed.)
4. Wash with water. Treat the smear with 1:10 diluted solution b and allow to react
for 1 minute.
5. Wash with water, air dry and examine under oil immersion lens.
Result: Flagella appear red while cells appear blue.
Metachromatic Granules (volutin) Staining
1. Method of Gohar
Solution required:
a. Loeffler's methylene blue (see monochrome staiing)
b. 0.1 % H2SO4
c. Gram's iodine (see gram staining)
d. 1% eosin Y.
Procedure:
1. Prepare the smear and heat fm.
2. Treat the smear with solution a for 5 minutes.
3. Wash with water. Treat the smear with solution b for 15-30 seconds
4. Wash with water. Treat smear with solution c for 1 minute.
5. Wash with water. Treat the smear with solution d for 1 min.
6. Wash with water, dry and examine under oil immersion lens.
Result: Metachromatic granules appear black. Cytoplasm appears pink.
2. Method of Pugh I
Solutions required:
a. Staining solution
Toluidine blue (C1 No. 52040)
1.0 g
Ethyl alcohol, absolute
20.0 ml
Acetic acid, glacial
50.0 ml
Distilled water
950.0 ml
Combine the acetic acid with the distilled water and dissolve the dye in the
alcohol. Mix. Filter.
Procedure:
1. Prepare a smear, air dry and heat fix.
2. Treat the smear with solution a for 2-3 minutes.
3. Wash with water, air dry and observe under immersion lens.
Result: Metachromatic granules appear reddish purple in colour. Remaining organism
appears light blue.
3. Method with methylne blue
Solution required:
a. Loffler's methylene blue (see monochrome staining)
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution a for 5 min.
3. Wash with water. Air dry and examine under oil immersion lens.
Result: Metachromatic granules appear red against pale blue cytoplasm.
4. Method of Albert (Laybourn modification)
Solutions required:
a. Albert's staining solution
Toiuidine blue-0
0.15 g
Methyl green or Malachite green
0.2 g
95% ethyl alcohol
2.0 g
Glacial acetic acid
1.0 ml
Distilled water
100.0 ml
Dissolve toluidine blue and methyl green in ethyl alcohol. Add this to distilled
water containing glacial acetic acid. Store the reagent for one day and then filter.
b. Lugol's iodine (Grams iodine) see gram staining method.
Procedure:
1. Prepare the smear and heat fix.
2. Treat the smear with solution a for 5 minutes.
3. Remove the solution a, do not wash-with water.
4. Treat the wet smear with solution b for 1 minute.
5. wash with water, air dry and examine under oil immersion lens.
Result: Metachromatic granules appear black. Cytoplasm appears light green.
Lipid Staining (Budon's method)
Solutions required:
a. Sudan black B staining solution
Sudan black B
70% ethyl alcohol
Shake before use.
b. Xylene
c. 0.5% safranin.
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution anfor about 10.15 minutes.
3. Remove solution a and dry it. Do not was with water.
4. Wash the smear with solution b.
5. Treat the smear with solution c for 5-10 seconds.
6. Wash yith water, air dry and examine under oil immersion lens.
Result: Lipid granules appear deep blue against light pink cytoplasm.
Cytoplasmic Membrane Staining
Solutions required:
a. Potassium nitrate
Distilled water to
b. Bouin's fixative (see fixatives)
c. Victoria blue solution
Victoria blue
0.4 g
Distilled water to
100.0 ml
Procedure:
1. Prepare a smear and heat fix..
2. Immerse the slide in solution a for 15 minutes.
3. Treat the slide with solution b for 15 minutes.
4. Wash the slide with water. Treat the slide with solution c for 30 seconds.
5. Wash the slide with water. Dry and examine under oil immersion lens.
Result: The cytoplasmic membrane appears as a deep blue outer layer covering a
contracted, irregular shaped body, the cytoplasm.
Cell Wall Staining
(1) Method of Webb
Solutions required:
a. Tannic acid solution
Tannic acid
5.0 g
Distilled water to
100.0 ml
b. Crystal violet solution
Crystal violet chloride
0.5 g
Distilled water to
100.0 ml
c. Congored solution
Congored
0.5 g
Dsitilled water to
100.0 ml
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution a for 30 minutes.
3. Wash the slide with water. Do not dry.
4. Treat the smear with solution b for 3 minutes.
5. Wash with water and treat the smear with solution c for 3 minutes.
6. Wash, dry and examine under oil immersion lens.
Result: Cell wall appear blue and cytoplasm appears colourless.
2. Method of Bourin
Solutions required:
a. Bouins fixative (see fixative)
b. Tannic acid solution
Tannic acid
7.0 g
Distilled water to
100 ml
c. Crystal violet solution
Crystal violet chloride
0.2 g
Distilled water to
1000 ml
Procedire:
1. Cut down agar slab (2mm thick) with growth of organisms.
2. Keep the covership in the centre of petriplate and place the agar slab on to it facing
growth side down.
3. Add solution a in to petri plate till the slab growth immerses in it. Treat it for 3 hrs.
4. Remove the solution a and then agar slab.
5. Wash the covership by allowing water flow in to petridish and overflow it for 2
hrs.
6. Treat the smear with solution b for 20-30 minutes.
7. Wash the covership and flood with solution c for 1 minute.
8. Mount in water and seal with nail varnish and examine under oil immersion lens.
Result: Bacterial cytoplasm appear pale purple while cell wall appear dark brownish
black.
3. Method of Chance
Solutions required:
a. 0.5% Basic fuchsin
b. 0.5% Congo red
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution a for 1 minute.
3. Wash with water.
4. Treat the smear with solution b for 5 minutes.
5. Wash with water.
6. Air dry and observe under oil immersion lens.
Result: Cell wall appears pink, cytoplasm appears colourless.
Spore Staining
1. Fuchsin - methylene blue spore staining
Solutions required:
a. Ziehl-Neelsen carbol fuchsin. (see acid fast staining method)
b. Ferric chloride, 30 per cent aqueous solution.
c. Sodium sulphite, 5 per cent aqueous solution.
d. Methylene blue 1 per cent aqueous solution.
Procedure:
a. Prepare a smear, air dry and heat fix.
b. Treat the smear with solution a for 3-5 min. heating the preparation until steam
water.
c. Wash the smear with water.
d. Treat the smear with solution b for 1-2 min.
5. Replace solution b with solution c and allow to act for 30s.
6. Wash with water, Treat the smear with solution d for 1 min.
7. Wash with water, air dry and observe under oil immersion lens.
Result: Spores appear bright red in colour. Vegetative cells appear blue.
2. Fuchsin-nigrosin spore staining (Fleming)
Solutions required:
a. Ziehl-Neelsen carbol -fuchsin. (see acid fast staining method)
b. (i) Nigrosin 1 per cent solution.
(ii) Sodium sulphik, 5 per cent solution.
c. Nigrosin 10 per cent solution.
Procedure:
1. Prepare a smear, air dry and heat fix.
2. Treat the smear with solution a for 5 min heating the preparation d l steam rises.
3. Wash with water, apply solution b (i) for 5-10 min or b (ii) for 5-30s.
4. Wash with water, air dry.
5. Place a small drop of solution c at one end of the slide and spread in an even layer
over the stained preparation with the edge of another slide.
6. Allow to dry and examine.
Result: Spore appears bright red in colour. Remainder of organism colourless against a
dark grey background of nigrosin.
3. Schaeffer and Fulton's method (modified by Ashby)
Solution required:
a. 5% aqueous solution of malachite green.
b. 0.5% S a b i n or 0.05% basic fuchsin.
Procedure:
1. Prepare the smear and heat fix.
2. Treat the smear with solution a steam heat the smear for 1 minute. (Do not allow
the smear to dry)
3. Wash the slide with water.
4. Treat the smear with solution b for 30 seconds.
5. Wash, dry and examine under oil immersion lens.
Result: Spores appear green, vegetative bacilli appear red.
4. 'Dorner's method (Synder's modification)
Solutions required:
a. Ziehl-Neelsen carbol fuchsion (see acid fast staining)
b. 95% ethanol.
c. Saturated aqueous solution of nigrosin.
Procedure:
1. Prepare a smear and dry it in air.
2. Treat the smear with solution a. Steam heat the smear for
a. 5-10 minutes. (Do not allow the smear to dry).
3. re at the smear by solution b for approx. 30 second.
4. Wash the slide.
5. Place a drop of solution c at one end of slide and spread it over the smear with the
help of another slide.
6. Dry the slide quickly using gentle heat and examine under oil immersion lens.
Result: Spores appear red, background appears black. Cells appear colourless.
5. Bartholomew and Mittwer's method. (Cold method)
Solutions required:
a. Saturated aqueous malachite green (approx. 7.6%)
b. 0.25% aqueous safranin.
Procedure:
1. Prepare the smear and heat fix the smear by passing through the flame 20 times.
2. Cool the slide and treat the smear with solution a for 10 minutes.
3. Wash the slide with water.
4. Treat the smear with solution b for 2 minutes.
5. Wash, dry and examine under oil immersion lens.
Result: Spores appear green. Cells appears red.
6. Acid fast staining for spores
Solution required:
a. Ziehl-Neelsen carbol fuchsin.(as in acid fast staining)
b. 2% solution of nitric acid in absolute ethyl alcohol.
c. 1% aqueous methylene blue.
Produce:
1. Prepare the smear and heat fix.
2. Treat the smear with solution a. Heat the smear for 3-5 min.
3. Wash with water.
4. Dip the slide rapidly in solution b and immediately wash with water.
5. Treat the smear with solution e for 3 minutes.
6. Wash, dry and examine under oil immersion lens.
Result: The spores appear bright red, cytoplasm appears blue.
Polysaccharide Staining
(1) Hotchkiss's method 1
Solution required:
a. Periodite solution
4% aqueous periodic acid solution
0.2 M aqueous sodium acetate
Ethyl alcohol
Mix well, store in brown bottle. Stable for several days.
b. 70% ethyl alcohol.
c. Reducing rinse solution
Potassium iodide
Sodium Thiosulphate pentahydrate
20.0 ml
10.0 ml
70.0 ml
10.0 g
10.0 g
Distilled water
200.0 ml
Ethyl alcohol
300.0 ml
2N HCI
5.0 ml
Dissolve potassium iodide and sodium thiosulphate pentahydrate in distilled water.
Add ethyl alcohol and HCl. Sulphur precipitates formed allowed to settle down.
d. Fuchsin sulphite solution
Basic fuchsin 2.0 g
Distilled water 400.0 ml
2N HCI 10.0 ml
Potassium metabisulphite 4.0 g
Activated charcoal 1.0 g
Dissolve basic fuchsin in distilled water. Dissolve by heating. Cool to 50°C and
filter. Add HCI and potassium metabisulphite to filterate. Incubate overnight in
dark and cool place. Add activated charcoal, mix and filter. Add more HCI
(approximately 10 ml) to this, till mixture when dried on glass does not turn pink.
Store in dark.
e. Sulphite wash solution
Potassium metabisulphite 4.0 g
Concentrated HCl 1.0 ml
Distilled water 100 ml
Prepare fresh.
f. 00.002% malachite green (aqueous)
Procedure:
1. Prepare a smear and heat fix.
2. Treat the slide with solution a for 5 minutes.
3. Wash the smear with solution b.
4. Treat the smear with solution c for 5 minutes.
5. Wash the smear with solution b.
6. Treat the smear with solution d for 15-45 minutes.
7. Wash the smear three times with freshly prepared solution e and then with water.
8. Treat the slide with solution f for few seconds.
9. Wash, dry and examine under oil immersion lens.
Result: Polysaccharide appears red. Cell appears green in colour.
2. Alcian blue staining method
Solution required:
a. Alcian blue stain
Alcian blue
1.0 g
95% ethanol
100.0 ml
Mix. Dilute ten times distilled water.
b. Carbol fuchsin (see acid fast staining). Dilute ten times with distilled water.
Procedure:
1. Prepare the smear and heat fix.
2. Treat the smear with solution a for 1 minute wash with water and aid dry the
smear.
3. Treat the smear with solution b and remove immediately with water.
4. Air dry and examine under oil immersion lens.
Result: Capsules and other polysaccharides appear blue, while cellular material appears
red.
3. Poly-P-Hydroxybutyrate Granules Staining
1. Prepare a smear of a 48-hour culture on a microscope slide.
2. Maintain a positive control by making smear of a known
3. Air dry the smear and then heat fix it.
a. Nile blue staining method.
1. Flood the smear with 1 % aqueous solution of Nile blue
2. Incubate for 10 min at 550C.
3. Drain off the staining solution, wash gently in running tap water and blot dry.
4. Cover the smear with 8% aqueous acetic acid for one minute at room
temperature.
5. Wash gently in running tap water and blot dry.
6. Examine the stained smear with an epifluorescence microscope at 450 nm
under oil immersion lens.
Result: PHB granules appear bright orange fluorescent.
b. Sudan black staining method:
1. Flood the fixed smear with 0.3% Sudan black B solution in 70% ethanol.
2. Incubate for 10 min at room temperature.
3. Wash with tap water and blot dry.
4. Dip the smear in xylol for half a minute and blot dry.
5. Flood the smear with 0.5% (w/v) aqueous safranin far 10 seconds at room
temperature.
6. Wash with tap water and blot dry. Apply a cover slip.
7. Examine the stained smear with a light microscope using transmitted light
under oil immersion lens
Result: PHB granules stain blue-black. The cell wall stains pink.
Nuclear Staining
1. Robinow's method
Solutions required:
a. Schaudinn's fixative (see fixatives)
b. 1 NHCI
c. Giemsa's stain.
(Stock Giemsa's stain-see protozoa stainirig)
Diluted 10 times with phosphate buffer. (pH 7.0)
Procedure:
1. Prepare a smead and air dry.
2. Treat the smear with warm solution a for 5 minutes.
3. Wash with water.
4. Place the slide in solution b at 60°C for 5-10 minutes.
5. Wash the slide with tap water three times and rinse with distilled water.
6. Treat the smear for 30 minutes at 37OC with solution c.
7. Wash, mount in water. Examine under oil immersion lens.
Result: DNA rich area appear red. Cytoplasm appear colourless.
2. Rapid method of Robinow
Solution required:
a. 0.2N HCl
b. 0.1 % Crystal violet
Procedure:
1. Treat unfixed and air dried smear with solution a at boiling temperature for 5
seconds.
2. Wash with water several times.
3. Teat with solution b for 1 minute.
4. Air dry and examine under oil immersion lens.
Result: Nuclear material appear purple in colour.
3. Feulgen method
Solution required:
a. Schaudinn's fixative (see fixatives)
b. lN HCl
c. Schiffs Reagent
Basic fuchsin
1.0 g
Thionyl chloride
1.0 ml
Distilled water
400 ml
Dissolve basic fuchsin in boiling distilled water. Cool and filter. Add 1 ml thionyl
chloride and allow to stand overnight. Clear the solution by shaking with activated
charcool. Filter and store in refrigerator.
Procedure:
1. This method is similar to Robinow method described above except only 6" step as
follows. Treat the smear for 60 minutes at 15 to 20°C. with solution c. (Schiffs
reagent).
Result: Nuclear material appears red in colour.
Viability Staining for Bacteria
Solutions required
a. Loeffler's methylene blue (see monochrome staining)
b. Dilute 1 : 10 carbol fuchsin (see acid fast staining)
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution a for 10 minutes. Wash the smear with distilled water
until the smear appears pale blue. (about 30 sec.)
3. Run solution b down the slide for a very short time.
4. Wash immediately with water. Air dry and examine under oil immersion lens.
Result:
1. Living bacteria appear purple and dead bacteria appear pink.
2. Living spores appear faintly pink and dead spores appear ' blue in colour.
Gram's Staining
1. Method of Hucker and Conn
Solutions required:
a. Hucker's crystal violet
Solution X
Crystal violet (90% dye content) chloride
2.0 g
Ethyl alcohol (95%)
20.0 ml
Solution Y
Ammonium oxalate
0.8 g
Distilled water
80.0 ml
Dissolve crystal violet in ethyl alcohol and the ammonium oxylate in
distilled water. Mix solutions X and Y.
b. Gram's iodine (Lugol's iodine)
Iodine
1.0 g
Potassium iodine
2.0 g
Distilled water
300.0 ml
Dissolve potassium iodide in distilled water then add iodine crystals. Dissolve.
c. Ethyl alcohol (95%)
Ethyl alcohol (1 00%)
95.0 ml
Distilled water
5.0 ml
d. Safranin
Safranin (2.5% solution in 95% ethyl alcohol
10.0 ml
Distilled water
100.0 ml
Filter the solution before use.
e. Basic fuchsin stain
Basic fuchsin
0.5 g
Distilled water
1000.0 ml
Note: Never use dilute carbol fuchsin because it tends to stain gram negative bacteria so
intensely that they may appear gram positive.
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution a for 1 minute.
3. Remove solution a by solution b and allow the smear to react with solution b for 1
minute.
4. Wash the smear with water.
5. Treat the smear with solution c for 10-1 5 seconds.
6. Wash the smear with water.
7. Treat the smear with solution d for 30 seconds. Wash air dry and examine.
Result: Gram positive bacteria appear violet, gram, negative appear pink in colour.
2. Method of Kopeloff and Beerman
Solutions required:
a. Methyl violet stain
Solution X1
Methyl violet 6B
10.0 g
Distilled water
1000.0 ml
Solution X2
Sodium bicarbonate
50.0 g
Distilled water
100.0 ml
Mix 600 ml of solution XI and 160 ml of solution X, Prepare freshly.
b. Iodine solution
Iodine
20.0 g
4% aqueous solution of NaOH
100.0 ml
Distilled water
900.0 ml
Dissolve the iodine in the NaOH solution and when it is dissolved add the distilled water.
b. Acetone (1 00%)
c. Basic fuchsin stain
Basic fuchsin
0.5 g
Distilled water
1000ml
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution a for 5 min.
3. Add solution b to remove solution a and allow the smear to react with solution b
for 2 min.
4. Wash the slide with water.
5. Wash the smear with solution c for only 2-3 seconds.
6. Wash the smear with water immediately.
7. Treat the smear with solution d for 30 seconds.
8. Wash with water, dry, examine under oil immersion lens.
Result: Same as Hucker's method.
3. Method of Jensen
Solutions required:
a. Methyl violet stain
Methyl violet 6B (or crystal violet)
5.0 g
Distilled water to
1000.0 ml
Filter before use.
b. Iodine solution (see Hucker's modification)
c. Absolute ethyl alcohol
d. Neutral red solution
Neutral red
1.0 g
1 % acetic acid
2.0 ml
Distilled water to
1000 ml
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution a for 30 seconds.
3. Add solution b to remove solution a and allow the smear to react with solution b
for 30 seconds.
4. Wash the slide with water.
5. Wash the smear by solution e for only 10-15 seconds.
6. Wash the smear with water.
7. Treat the slide with solution d for 1 minute.
8. Wash with water, dry and examine under oil immersion lens.
Result: Same as in Hucker's method.
Note: This method is used especially in examination of exudates for gonococci arid
meningococci. When gonococci and meningococci are scantly, Sandiford's solution
instead of neutral red solution is useful.
Sandifords stain
Malachite green
0.05 g
Pyronine
0.15 g
Distilled water to
100 ml
cells and nuclei stain bluish green. Gram positive organisms are purple black and
gonococci red.
4. Method of Preston and Morrell
Solutions required:
a. Hucker's crystal violet (see Hucker and Conn method)
b. Gram's iodine (see Hucker and Conn method)
c. Iodine - acetone mixture.
Liquor - iodine fortis
7.0 ml
Acetone to make the volume
200.0 ml
Liquor iodine fortis (BP) Iodine crystals
10.0 g
Potassium iodine
6.0 g
Methanol
90.0 ml
Distilled water
10.0 ml
Dissolve iodine crystals and potassium iodine in methanol. Add 10 ml distilled
water. Mix.
d. Basic fuchsin (see Hucker and Conn method)
Procedure:
1. Prepare a smear and heat fix.
2. Treat the smear with solution a for 30 seconds.
3. Add solution b to remove solution a and allow the smear to react with solution b
for 30 seconds.
4. Wash the smear with solution c.
5. Treat the smear the same solution c for 30 seconds.
6. Wash with water and treat with solution d for 30 seconds.
7. Wash dry and examine under oil immersion lens.
Result: Same as in Hucker's method.
5. Rapid method for Gram stain
Solutions required:
a. Crystal violet solution (see Hucker and Conn method)
b. Iodine solution (see Hucker. and Conn method)
c. Alcohol-acetone mixture (1 : 1)
d. Safranin (see Hucker and Conn method)
Procedure:
1. Prepare the smear and heat fix.
2. Wash the smear with solution a.
3. Then wash the smear with solution b.
4. Wash the slide rapidly with solution c.
5. Wash with water.
6. Wash the smear with solution d for 5 seconds.
7. Wash, dry and examine under oil immersion lens.
Result: Same as in Hucker's method.
Staining of Virus Elementary Bodies and Rickettsiae
1. Method of Gutstein
Solutions required
a. Methyl alcohol
b. Gutsteins stain
Solution (a)
Methyl violet
1.0 g
Distilled water
100.0 ml
Solutions (b)
Sodium carbonate
2.0 g
Distilled water to
100.0 ml
Mix (1) and (2). Prepare freshly.
Procedure:
1. Prepare thin film of smear by using scrapping of skin lesions.
2. Wash with saline and then with distilled water.
3. Treat the smear in solution a for 30 minutes.
4. Treat the smear by solution b for 20-30 minutes at 370C.
5. Wash the slide with distilled water, air dry and examine under oil immersion lens.
Result: Virus elementary bodies appear violet in colour.
Note: This method is useful for staining the elementary bodies of variola - vaccinia group
of viruses.
2. Method of Castaneda (Bedson's modification)
Solutions required:
a. 1.0 N HC1
b. Azure II Solution
Stock Azure II:
Azure II (Gurr)
1.0 g
Distilled water to
100.0 ml
Filter. Dilute 1:10 with formaldehyde buffer before use.
Formaldehyde buffer:
Commercial formalin
40.0 ml
Sorensen's M/1 5 phosphate
960.0 ml
buffer pH
7.2
Neturalize the formalin with 1.0 N NaOH in the presense of phenol red. Then mix.
c. 0.25% Aqueous solution of safranin.
Procedure:
1. Treat the smear in solution a for 2 min wash with distilled water.
2. Treat the smear with solution b for 20 min.
3. Wash with distilled water.
4. Treat with solution c for 6-8 seconds.
5. Wash with water, air dry and observe under oil immersion lens.
Result: Rickettsiae are stained blue, while cell nuclei and cytoplasm appears red.
Note: This procedure is also useful for elementary bodies of psittacosis.
3. Method of Nicholau
Solutions required:
a. Nicholau's staining solution
Solution 1
Isamine blue
1.0 g
95% ethanol
10.0 ml
Solution 2
Phenol
3.0 g
Distilled water to Mix solution 1 and 2 Store in
brown bottle.
100.0 ml
Procedure:
1. Prepare the smear and heat fix.
2. Treat the smear with solution a and heat the slide from below for 5 minutes.
Solution should not be boiled.
3. Wash with water, dry water, examine under oil immersion lens.
Result: Rickettsiae appear blue.
4. Method of Macchiavello (for rickettsia)
Solutions required:
a. Staining solution
Basic fuchsin
0.25 g
Distilled water to
100 ml
Adjust the pH 7.3 with alkali. Filter the stain before use.
b. 0.5% citric acid.
c. ' 1% aqueous methylene blue.
Procedure:
1. Prepare the film from tissue and air dry.
2. Heat the slide gently and treat for 4 minutes with solution a.
3. Wash with solution b then wash with water.
4. Treat the smear with solution c for few seconds, Blot dry and examine under oil
immersion lens.
Result: The rickettsiae appears red, tissue cells blue.
5. Method of Giemsa
Solutions required:
(see Giemsa stain for protozoa)
Procedure: Similar like protozoal staining by Giemsa's method.
Result: Virus elementary bodies appear purple in colour.
Note: This method is satisfactory for the elementary bodies of vaccinia and psittacosis.
6. Brucella differential staining method
Solutions required:
(see Brucella monochrome staining)
Procedure: Similar like Brucella staining and useful for staining the elementary bodies of
chlamydia.
7. Seller's stain for Negri bodies
Solutions required:
a. Seller's stain
Saturated solution of basic fuchsin in absolute
methyl alcohol
24.0 ml
Saturated solution of methylene blue in methyl
alcohol
15.0 ml
Absolute methyl alcohol (acetone free)
25.0 ml
Mix these solutions.
b. W/50 Phosphate buffer solution with pH 7.0
Procedure:
1. The smear while still moist, is immersed in the solution a in a coplin jar for 5
seconds.
2. Wash with solution b or with distilled water (pH 7)
3. Dry and examine under oil immersion lens.
Result: Negri bodies are stained cherry red and the inner granules deep blue. Cytoplasm
of the nerve cells appear blue to purplish blue whereas nerve fibres are coloured deep
pink but neural sheaths are not stained.
Note: A properly stained smear in transmitted. light should appear reddish violet in thin
portions whereas purpulish blue in thick portions.
8. Paschen's Staining method
Solutions required:
a. Absolute alcohol
b. Loeffler's. mordant
20% aqueous solution of tannic acid
100.0 ml
Saturated aqueous solution of ferrous sulphate
50.0 ml
Saturated aqueous solution of basic fuchsin
10.0 ml
c. Ziehl's fuchsin (see acid fast staining)
Procedure:
1. Prepare films from the infected material or tissue and allow it to dry.
2. Keep in distilled water for minutes and air.
3. Treat the film with solution a for 5 minutes and air dry.
4. Treat with solution b. Gently heat for one minute and allow the solution b to act on
the slide for 10 min.
5. Wash off the solution b with distilled water and stain with solution c, heating
gently for one minute.
6. Rapidly wash the film with water, dry and examine under oil emmersion lens.
Staining of Virus Inclusion Bodies
1. Giemsa staining method
The procedure and staining reagents are the same as described for protozoa. This method
is useful for demonstration of basophilic inclusion bodies.
2. Staining with methylene blue - Eosin
Solutions required:
a. Bouin's fixative (see fixatives)
OR
Zenker's fixative (see fixatives)
b. Mam's methylene blue - Eosin stain
1 per cent aqueous solution of methylene blue
35.0 ml
1 per cent aqueous solution of eosin
45.0 ml
distilled water
100.0 m ml
c. Solution C
Orange - G
0.5 g
70% ethanol
100.0 ml
Procedure:
1. Fix tissues in solution a and cut paraffin sections in routine way.
2. Wash with water.
3. Treat with soiution b for 12 hrs in the incubator at 370C.
4. Wash the section in water, differentiate under the micro scope in solution c.
Dehydrate the sections in alcohol clear in xylene. Mount in oil and observe.
Result: Negri bodies appear red. Nuclei and central granules of Negri bodies appear blue.
Note: This method is specifically useful for staining the specimen for diagnosis of rabies.
3. Lendrum's method
Solutions required:
a. Formal - saline (see saline)
OR
Fromal - corrosive (see fixative)
b. Mayer's haemalum
Haematoxylin
0.1 g
Sodium iodide
0.02 g
Aluminium potassium sulphate
5.0 g
Distilled water to
100.0 ml
Keep the solution overnight then add
Chloral hydrate
5.0 g
Citric acid
0.1 g
Mix well. Boil for few minutes.
c. Staining solution
Phloxine B or Rose Bengal
0.5 g
CaCl2
0.5 g
Distilled water to
100.0 ml
Dissolve CaC12 in water then add phloxine B or rose Bengal.
d. Saturated solution of tetrazine in 2-ethoxy ethanol.
e. 95% ethanol.
Procedure:
1. Treat tissues by solution a for 30 minutes and take paraffin sections in routine way.
Wash with water.
2. Treat the sections with solution b for five minutes and then differentiate in water
till they appear blue.
3. Treat the section with solution c for 30 minutes.
4. Wash with water and differentiate by adding dropwise n solution d.
5. Wash with solution e, dehydrate, clear, mount in oil and observe.
Result: Inclusion bodies appear red against yellow background.
Note: This method is specifically applied for staining acidophilic inclusion bodies.
Staining of Fungi
1. Periodic acid - Schiff's stain method (modified) for fungi in tissue sections
Solutions required:
a. Periodic acid solution (see polysaccharide staining)
b. Schiff's reagent (see nuclear staining)
c. Sulphite wash solution (see polysaccharide staining). Freshly prepare.
d. 0.002 per cent malachite green or light green.
Procedure:
1. Wash the section with distilled water and immerse in the solution a for 5 minutes.
2. Wash the sections in tap water for 15 minutes and then wash with distilled water.
3. Immerse the sections in solution b for 15 minutes.
4. Wash three times with solution c for 5 minutes each.
5. Wash with water for 10 minutes.
6. Wash with distilled water.
7. Treat the solution with solution d for 30-60 seconds.
8. Wash with water, dehydrate in absolute alcohol, clear in xylene and mount in oil.
9. Observe under high power microscope.
Result: Fungi appear red in colour. Cytoplasm appears green in colour.
2. Lactophenol cotton blue staining
Solutions required:
a. Lactophenol cotton blue solution
Lactic acid
20.0 ml
Phenol crystals
20.0 g
Glycerol
40.0 ml
Distilled water
20.0 ml
Cotton blue (or methyl blue)
0.075 g
Dissolve the phenol crystal in the liquids by gentle warming. Add the dye.
Procedure:
1. Take a drop of solution a in the centre of clean slide.
2. Transfer a small growth of the fungus with spore in to the drop, using a flamed but
cooled needle.
3. Tease slowly the fungal growth using two needles.
4. Put a coverslip over the preparation without entering air bubbles in the stain.
5. Observe under microscope. (high power)
Result: The fungal cytoplasm appear light blue in colour inside the unstained cell wall of
hyphae. Spores and spore bearing structure is surrounded by a light blue background.
Note: If the mycelia are dark coloured, the use of cotton blue in mounting medium may
be omitted.
Staining of Vesicular Arbuscular Mycorrhizae
(VAM fungi)
Solutions required:
a. Formlin acetic acid - alcohol mixture (see fixatives)
b. b.10% KOH
c. Staining solution
80% lactic acid
10.0 ml
Glycerine
10.0 ml
Chlorazol black E
0.01 g
Distilled water
10.0 ml
Dissolve chlorazol black E in distilled water. Then add lactic acid and Glycerine to it.
d. Mounting fluid
Chloral hydrate
20.0 g
Gum Arabic
20.0 g
Glycerine
20.0 g
Glucose syrup
Basic fuchsin (3% in 95% ethyl alcohol)
Distilled water
3.0 ml
10 drops
35.0 ml
Procedure:
1. Wash VAM infected material with water and treat over night with solution a.
2. Wash with water for several times.
3. Place the sample in to solution-b.
4. Steam sterilise the sample at 121°C for 15 minutes.
3. Wash several times with tap water, wash with distilled water.
5. Treat sample by solution c for 1 hr at 90°C.
6. Keep sample in to glycerine overnight. Mount sample on slide using solution d.
Result: VAM fungi appears brown black.
Note:
1. Prepare reagents several hours before use and keep undistributed to settle
undissolved particles.
2. Do not breath dust from stain.
Nuclear Staining of Filamentous Fungi
Solutions required:
a. Fixative
Ethyl alcohol
30.0 ml
Acetic acid
10.0 ml
OR
Ethyl alcohol
30.0 ml
Acetic acid
10.0 ml
Lactic acid
10.0 ml
b. 95% ethyl alcohol
c. 70% ethyl alcohol
d. 1M HCl
e. Phosphate buffer (pH 6.9) - see buffers
f. Dilute Giemsa stain
Stock Giema stain (see protozoa staining) Giemsa stain 1.0 ml
Phosphate buffer (pH 6.9)
15.0 ml
Procedure:
1. Treat spores and mycelia present on thin agar film on glass slide for 10-12 minutes
with solution a.
2. Wash the preparation with solution b, transfer the preparation in solution c (can be
stored for several days in solution c).
3. Wash with distilled water.
4. Treat the preparation for 5 minutes with solution d at room temperature. Transfer it
to solution d at 60°C and keep for 7 minutes.
5. Wash first with water and then with solution e for five times in each.
6. Treat the preparation with solution f for 2 hrs. (Do not allow to dry the smear).
7. Again wash with solution e.
8. Allow the preparation to air dry.
9. Mount in euparal. Cover the preparation with a coverslip.
10.Observer under high power lens.
Result: The nuclei are seen as rose to pink surrounded by colourless to light blue
cytoplasm.
Staining of Protozoa
A. For Protozoa in faeces
1. Iron haematomylin staining method
Solutions required:
a. Schaudinn's fixative (see fixatives)
b. 50% ethyl alcohol.
b. Grams iodine (see Gram staining by Hucker's method)
c. Weigert's iron haematomylin
Solution 1
Haematoxylin
1.0 g
Absolute alcohol
100.0 ml
Solution 2
Liquor - fem perchlorate (30%)
4.0 ml
Concentrated HCl
1.0 ml
Distilled water
100 ml
Complete solution
Mix equal parts of solutions 1 and 2. Prepare freshly.
Procedure:
1. Prepare wet smear and treat with solution a for 5 minutes.
2. Wash the smear with solution b and apply solution c for 2 minutes.
3. Wash with solution b for 30 seconds.
4. Wash with distilled water and treat the smear with solution d for 12 minutes.
5. Wash with water, dry and observe under oil immersion lens.
Result: Protozoa are stained blue-black in colour.
2. Method of Dobell
Solutions required:
a. Schaudinn's fixative (see fixative)
b. 2% Aqueous ammonium molybdate
c. 0.2% Iron haematoxylin solution (aqueous)
Procedure:
1. Treat at the smear with solution a for 5 minutes.
2. Wash with distilled water.
3. Treat the smear with solution b for 10 minutes.
4. Wash with distilled water.
5. Treat the smear for 10 minutes with freshly prepared solution C.
6. Wash with distilled water and transfer the smear into tap water for about 30
minutes, until the smear appear blue in colour.
7. Mount in water.
Result: Protozoa appear blue in colour.
B. For Protozoa in blood sample
Preparation of blood film
Gently touch a fresh drop of blood on to one end of clean grease free slide. Allow the
drop to spread along it. Holding the slide at 45°C push the spreader along the slide,
drawing the blood behind it, until the whole of the drop has been smeared. The thickness
and even distribution on the cells plays an important part in getting correct result.
1. Leishman's method
Solutions required:
a. Leishaman’s stain
Leishaman's power
0.15 g
Methanol pure (acetone free)
100.0 ml
Grind the crystals to in a clean dry mortar with little amount of methanol and add
the alcohol a little at a time until all stain is dissolved. The final volume is then
made up with methanol. (Prepared stain is easily available in market.)
b. Buffer solution (pH 7.0)
Na2HPO4 (anhydrous)
5.447 g
KH2PO4 (anhydrous)
4.752 g
Dissolve the phosphate in 2 litre distilled water. This gives a pH of 7.0.
Procedure:
1. 1 . Prepare the blood film. Do not heat fix and treat with solution a for 1-2 minutes.
2. Then it is diluted with an equal volume of solution b (or distilled water of 7.0).
Mix by gentle rocking the slide.
3. Allow diluted solution to react for 10 minutes. Wash the slide with distilled water
until the smear appears pink.
4. Air dry and examine under oil immersion lens.
Result: Cytoplasm appears blue. Nuclear material appears red.
2. Giemsa's method
Solutions required:
a. Methyl alcohol, absolute
b. Giemsa stain
Giemsa powder
Methanol (acetone free)
Glycerol 25.0 in1 I
Dissolve Giemsa stain in methanol and constant stirring for several hours. Dilute 1 : 10 in
buffer solution before use. (Ready Giemsa stain in available in the market.)
c. Buffer solution (pH 7) (see Leishman staining)
Procedure:
1. Prepare a blood film, air dry.
2. Treat with solution a for 3 min.
3. Treat the smear for 10 minutes with solution b.
4. Wash and differentiate with buffer solution (pH 7).
5. Air dry and observe under oil immersion lens.
Result: Chromatin of malarial parasite and trophonuclei of trypanosomes seen red purple.
Cytoplasm of protozoa appears pale blue.
Note: This method gives excellent results with thick blood films.
3. Field's method
Solutions required:
a. Field's solution a
Methylene blue
0.8 g
Azure I
0.5 g
Na2HPO4 (anhydrous)
5.0 g
KH2PO4
6.25 g
Distilled water to
500 ml
b. Field's solution b
Eosin (yellow)
1.0 g
Na2HPO4 (anhydrous)
5.0 g
KH2PO4
6.25 g
Distilled water to
500 ml
The phosphate salts are first dissolved in water and then stains are added. Use the
stain after 24 hrs. Filter before use.
Procedure:
1. Prepare a thick blood film and dry in air. Do not heat fix.
2. Dip the film for one second in solution a.
3. Wash the slide gently with distilled water until stain ceases. to flow from the film.
(for few seconds)
4. Dip the slide for one second in solution b.
5. Rinse by washing gently for 2-3 sec in clean water.
6. Place vertically against a rack to drain and dry.
7. Examine under oil immersion lens.
Result: Same as in Leishman staining method.
4. Simeon's modification of Boye's and Stevenel's method
Solutions required:
a. Methyl alcohol absolute
b. Stevenel's solution
(I) Medicinal ethylene blue
1.0 g
Distilled water to
75.0 ml
(II) Potassium permangnate
1.5 g
Distilled water to
75.0 ml
Mix I and II in a flask. A heavy precipitate forms at once. Heat for 30 minutes to
dissolve the precipitate. Filter and use.
c. Eosin solution
Eosin pure
1.0 g
Distilled water to
1000.0 ml
Procedure:
1. Prepare thick blood film. Do not heat fix.
2. Treat with solution a for 1 min. Wash with water for 4 seconds.
3. Treat the film with solution c for 10 seconds wash with water for 4 seconds.
4. Treat the film with solution b for 15 seconds. Wash with water for 4 seconds.
5. Treat the film with solution c for 5 seconds.
6. Wash with water for 4 seconds. Air dry and examine under oil immersion lens.
Result: Same as in Leishman staining method.
5. Wright's staining method
Solutions required:
a. Methyl alcohol absolute
b. Wright's stain
Wright's powder
0.2 g
Methanol (pure)
100.0 ml
Prepare the solution few before use.
c. Buffer solution (pH 7.0) (see Leishman staining method)
Procedure:
1. Prepare a blood film air dry. Treat with solution a for 3-5 min.
2. Treat the smear with solution b for 1-2 min.
3. Dilute with an equal amount of solution c.
4. Allow it react for 3 -5 min.
5. Wash and differentiate with solution c.
6. Air dry and examine under oil immersion lens.
Result: Same as in Leishman staining method.
4
Reagents
Fixatives
1. Bouin's fixative
Saturated aqueous solutions of picric acid
75.0 ml
Formalin
25.0 ml
Glacial acetic acid
5.0 ml
Use:
1. This fixative is useful for the investigation of virus inclusion bodies.
2. Used in cell wall staining (method of Bouin)
2. Chrome-acetic acid fixative
10% aqueous chromic acid
2.5 ml
10 % aqueous acetic acid
5.0 ml
Distilled water to
100 ml
Use: For fixation of chromosomes, nucleoli and centrioles of fungi.
3. Flemming fluid
Osmic acid
0.1 g
Chromic acid
0.2 g
Glacial acetic acid
01 ml
Distilled water to
100 ml
The osmic and chromic acids when mixed will keep for only3-4 weeks, The acetic acid
should be added immediately before use.
Use: Used as a fixative for tissue section.
4. Fontan's fixative (Ruge's solution)
Glacial acetic acid
1.0 ml
Formalin (37-40% formaldehyde)
2.0 ml
Distilled water to
100 ml
Use: Used in spirochete staining.
5. Fomalin-acetic acid alcohol (FAA) mixture
60% Alcohol
90.0 ml
Glacial acetic acid
5.0 ml
Formalin
5.0 ml
Use: For fixation of plant material before staining.
6. Mercuric chloride-formalin fixative
Saturated aqueous
9.0 g
Solution of mercuric chloride
280.0 ml
Commercial formalin
20.0 ml
Use: The minimum amount of destoration and fairly good cytological details are
obtained, particularly in staining of various inclusion bodies if above fixative is used.
7. Moller's fixation
Lead acetate
9.0 g
Distilled water to
2800 ml
Formalin
20.0 ml
Precaution: Store in tightly stoppered bottle.
Use: Used in capsule (method of Moller) staining.
8. Newcomer's fixative.
Dioxane
10.0 ml
Acetone
10.0 ml
Petroleum ether
10.0 ml
Propionic acid
30.0 ml
Isopropyl alcohol
60.0 ml
Use: Most acetic and alcohol mixtures result in to loss of nuclei staining property and
dissolution of mitochondria. This fixative overcomes this difficulty during fixation of
smear.
9. Schaudinn's fixative
Absolute ethyl alcohol
33.0 ml
Glacial acetic acid
1.0 ml
Saturated solution of HgC12
(6.9g/100 ml at 20°C) in water
66.0 ml
Use:
1. Used to fur the smear in staining of protozoa.
2. Used in nuclear staining (method of Robinow)
10. Susa's Fixative
Mercuric chloride
45.0 g
Distilled water to
800 ml
Sodium chloride
5.0 g
Trichloroacetic acid
20.0 g
Acetic acid (Glacial)
40.0 ml
Formalin (40% formaldehyde)
200 ml
Use: Fixative for normal and pathological specimens before staining. This fixative give
better result.
11. Zenker's fluid
Mercuric chloride
5.0 g
Potassium bicarbonate
2.5 g
Sodium sulphate
1.0 g
Distilled water to
100 ml
Immediately before use, add 5 ml of glacial acetic acid per 100 ml of above mixture.
Use: It is a fixative for animal tissue staining.
12. Zenker-formal fixative
This is similar to Zenker's fluid except that the 5 ml of formalin is added instead of acetic
acid.
Salines
1. Azide saline
Sodium azide
0.8 g
Physiological saline OR buffered saline
100 ml
Use: Azide prevents microbial decomposition. It is used as a diluting fluid.
2. Borate-calcium saline
NaCl
8.0 g
CaCl2
1.0 g
H3BO3
1.2 g
Na2B4O7.10H2O
0.052 g
Distilled water to
1000 ml
Use: It is used as a cell suspension and diluting fluid tor haemagglutination experiments,
where calcium is required and phosphate should be absent.
3. Buffered saline
NaCl
8.0 g
K2HPO4
1 : 21 g
KH2PO4
0.34 g
Distilled water to
1000 ml
Use: This solution gives a pH of about 7.3 and also provides potassium and phosphate
ions. It is general diluent and suspending fluid. NaCl can be diluted in different buffer
solution giving desired pH.
4. Formal saline
Sodium chloride (0.9%) solution
90.0 ml
40% formaldehyde
10.0 ml
The pH is adjusted at 7.0 by addition of CaCO3 granules.
Use: In staining of virus inclusion bodies.
5. Phenolized saline
Sodium chloride
8.5 g
Phenol
5.0 g
Distilled water to
1000 ml
6. Physiological saline
Sodium chloride
8.5 g
Distilled water to
1000 ml
Use: Used for preparation of bacterial suspensions. It prevents osmolysis of bacteria.
7. VDRL, buffered saline solution (pH 6.0)
Formaldehyde (neutral, c.p.)
0.5 ml
Na2HPO4.12H2O
0.093 g
KH2PO4
0.170 g
NaCl
10.0 g
Distilled water to
1000 ml
Dissolve all the constituents in distilled water. Check the pH of the solution. Store the
reagent in screw capped bottles.
Use: Used for preparation of VDRL antigen.
8. Vernol-NaCI diluent
NaCl
8.5 g
Barbitone (diethyl-barbituric acid)
0.575 g
Sodium barbitone
0.2 g
MgCl2.6H2O
0.168 g
CaCl2
0.028 g
Distilled water to
1000 ml
Preparation: stock solution concentrated x 5 is made by dissolving 5.75 g barbitone in
500 ml hot distilled water. Add 85 gm, NaCl and make up the volume to about 1400 ml.
Dissolve 2.0 gm sodium barbitone in 500 ml distilled water and add it to the NaCl
barbitone solution. Make up to 2000 ml. Add 1.68 g MgC12.6H20 and 0.28 g CaCl2. For
use dilute 1 in 5 with distilled water.
Use: This saline may be used for complement fixation tests and gives more reproducible
results.
IMViC Test Reagents
1. Kovac's reagent
n-amyl alcohol
75.0 ml
Hydrochloride acid (conc.)
25.0 ml
P-dimethylamino-benzaldehyde
5.0 g
Add aldehyde to a flask containing alcohol and dissolve by gently warming to 5S°C in a
water bath. Cool and add HCI. Store the reagent in a dark glass bottle in a refrigerator.
Use: Kovac's reagent develop red colour in presence of indole.
2. Methyle red solution
Methyl red
0.04 g
Ethyl alcohol (absolute)
40.0 ml
Distilled water to
60.0 ml
Dissolve methyl red in ethyl alcohol and add water.
Use: Used in methyl red test. Add about 5 drops of the methyl red reagent in to
inoculated and incubated liquid medium. Mix. Positive tests are bright red and negative
are yellow.
3. Barritt's reagent (Voges Proskauer test)
Solution A: 5g alpha-naphthol in 100 ml of 95% ethyl alcohol. Dissolve the alpha
naphthol in the ethyl alcohol with constant stirring.
Solution B: 40g potassium hydroxide in 100 ml water.
Use: Used in Voges Proskauer test. Add 1 ml of Potassium hydroxide and 3 ml of alpha
naphthol in the inoculated and incubated liquid medium. A positive reaction is detected
by the development of an eosin-pink colour usually in 2-5 minutes.
Caution: Avoid all contact with human tissues, alpha-naphthol is considered to be
carcinogenic.
Other Reagents
Andrade's indicator
Preparation: Prepare 0.5% solution of acid fuchsin. Add 1 N NaOH to it, slowly, till
colour turns slight yellow.
Use: Used as an indicator in sugar fermentation tube. It becomes dark red in colour at or
below pH 5.5.
Gelatin Hydrolysis
1. Mercuric chloride solution (Frazier's solution)
Mercuric chloride
15.0 g
Hydrochloric acid (conc.)
20.0 ml
Distilled water to
100.0 ml
Use: Used for detection of gelatin breakdown. After flooding the plate with mercuric
chloride solution gelatin liquifying microorganisms show clear zone around colony
against opaque background.
2. Tannic acid solution
Tannic acid
1.0 g
Distilled water to
100.0 ml
Use: To test gelatin hydrolysis. Tannic acid causes relative opacity around gelatin
liquifying colonies, quick to develop but fading as the medium also becomes opaque.
3. Trichloroacetic acid
Trichloroacetic acid (C.P.)
5.0 g
Distilled water to
100.0 ml
Dissolve the acid in the water with constant stirring.
Use: Used to test gelatin hydrolysis by microorganisms on the gelatin agar.
Pectin Hydrolysis
Hexadecyltrimethyl ammonium
bromde
Distilled water to
Use: To detect pectin hydrolysis. It precipitates intact pectin.
1.0 g
100.0 ml
Starch Hydrolysis
Lugol's Iodine (Gram's Iodine)
Use: After flooding the plate of starch agar with iodine solution amylolytic
microorganisms show clear zone around the colony against purple background.
Oxidase test reagent
Dimethyl-p-phenylene diamine hydrochloride
1.0 g
Distilled water to
100.0 ml
The reagent should be made fresh daily. It should not be stored longer than one
week in the refrigerator.
If the preparation becomes darkened, discard. Tetramethyl-p-phelylenediamine
dihydrochloride (1%) is even more sensitive but it is more expensive and difficult to
obtain.
Use: To test presence of certain oxidises in bacteria, the dye is reduced to deep purple
colour if the test is positive.
Methylene blue (1:25000) solution
Methylene blue dye
0.04 g
Distilled water to
1000 ml
Preparation: Dissolve the methylene blue in the distilled water and dispense into regular
staining bottles.
Use: Used in methylene blue reduction test (for determining the quality of milk.) Dilute
10 times before use.
Motility studies reagent
Carboxy-methyl-cellulose
2.0 g
Sucrose (0.2 M)
98.0 ml
Distilled water to
1000 ml
Preparation: Prepare 0.2 M sucrose solution by adding 68.4 g. of sucrose in 1000 ml of
distilled water.
Use: If a bacterial culture growing on a solid medium is to be examined for motility, a
loopful of culture should be mixed with a drop of 2% CMC at the centre of coverslip.
Methyl cellulose reagent
Carboxy methyl cellulose
10.0 g
Distilled water to
90.0 ml
Reparation: Dissolve the methyl cellulose in warm distilled water.
Use: For microscopic observation of protozoa.
Significance: Methyl cellulose slows down the movement of protozoa.
Resazurin (1:20,000) solution
Resazurin
Distilled water to
Preparation: Dissolve the resazurin in distilled water.
Use: Used in resazurin reduction test (for determining the quality of milk.)
Cleaning solution for glassware
(Sulphuric acid dichromate solution)
Sodium dichromate
25.0 g
Sulphuric acid (conc.)
1000 ml
Distilled water to
50 ml
Preparation: Dissolve the dichromate crystals in 50 ml of warm water. Cool to, room
temperature and add acid slowly to the preparation. Use: This preparation is used for the
removal of residual organic matter from laboratory glassware. Glassware should be
soaked in this solution for a number of days. Then rinsed in running tap water at least 10
times, then rinsed twice in single distilled water and finally rinsed once in double glass
distilled water.
Ferric chloride reagent
FeCl36H2O
12.0 g
2% Aqueous HCl
100.0 ml
Made up the 2% aq. HCl by adding 5.4 ml of concentrated HCl (37%) to 94.6 ml H2O.
Use: Used in phehyyalanine deaminase test. Development of green colour indicate
positive phenylalanine deaminase test.
Diphenylamine reagent (nitrate test)
Diphenylamine
0.7 g
Sulphuric acid (conc.)
60 ml
Distilled water
28.8 ml
Preparation: Dissolve diphenylamine in a mixture of sulphuric acid and water. Cool and
add slowly 11.3 ml of concentrated hydrochloric acid. After the solution has stood for 12
hours some of the base separates, showing that the reagent is saturated.
Use: Used in detection of nitrate in the medium. This reagent produces a blue black
colour in the presence of either nitrites or nitrates. It is necessary to make sure that no
nitrites are present by Trommsdorf -regent when it is used as a test for nitrates.
Trommsdorf's reagent
Zinc, chloride solution (20%)
100.0 ml
Starch
4.0 g
potassium iodide
2.0 g
Distilled water
100.0 ml
Preparation: Prepare 100 ml of 20% aqueous ZnC12 solution and add slowly with
constant stirring to a mixture of 4.0 g of starch in water. Dissolve completely by heating.
Dilute with water and add the potassium iodide. Made the volume to 1 litre by additions
of more water, filter and store in brown-stoppered bottle.
Use: For detection of nitrites. This reagent produce intense blue-black colour in present
of nitrite.
Nitrite test reagents
Solution A:
Sulfanilic acid
8.0 g
5 N acetic acid (1 part glacial acetic acid to
2.5 parts water)
1000 ml
Solution B:
Dimethyl-alpha-napthylamine
5.0 g
5 N acetic acid
1000 ml
Immediately before use, mix equal volumes of solution A and solution B.
Use: Add 0.1 nil of test reagent to 5 ml of broth. A development of red colour indicate
presence of nitrite.
Caution: Although at this time it is not know for sure, there is a possibility that
dimethyl-alphanapthylamine in solution B may be carcinogenic. For reasons of safety,
avoid all contact with tissues.
Nessler's reagent (Ammonia test)
Potassium iodide
50.0 g
Ammonia free distilled water (cold)
35.0 ml
Mercuric chloride solution (saturated)
35.0 ml
Potassium hydroxide (50% aqueous solution)
400 ml
Distilled water to
530 ml
Dissolve potassium iodide in ammonia free distilled water. Add a saturated solution of
mercuric chloride to it. (slight precipitate persists.) Add solution of potassium hydroxide.
Dilute to 1 litre, allow to settle for one week and decant the supernatant for use. Store in
brown bottle.
Use: Used for detection of ammonia. Positive reaction is shown by a colour ranging from
a pale but distinct yellow to a dark brown precipitate.
Buffers
1. Acetic buffer
Solution required:
A: 0.2 M solution of acetic acid (11.55 m1 in 1000 ml).
B: 0.2 M solution of sodium acetate (16.4 g of C2H3O2Na or 27.2 g of C2H3O2Na.3H2O
in 1000 ml). X ml of A + Y ml of B, diluted to a total of 400 ml
X
Y
pH
185.2
14.8
3.6
176.0
24.0
3.8
164.0
36.0
4.0
147.2
52.8
4.2
122.0
78.0
4.4
102.0
98.0
4.6
80.0
120.0
4.8
59.2
140.8
5.0
42.0
158.0
5.2
35.2
164.8
5.4
19.2
180.8
5.6
2. Barbitone (veronal) buffer
Solutions required:
A: 0.2M solution of sodium barbitone (sodium diethyl barbiturate) (7.37 g in 1000 ml).
B: 0.2M HCl 100 ml of A+ X of B, diluted to a total of 400 ml
X
pH
3.0
9.2
5.0
8.0
12.0
18.0
25.4
35.0
45.0
55.0
65.0
78.0
86.0
90.0
3. Bicarbobnate-CO2 buffer
9.0
8.8
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
6.8
Concentration of CO2 in gaseous phase
5%
10%
20%
Concentration
0.02 M
7.4
7.1
6.8
Of NaHCO3
0.05 M
7.8
7.5
7.2
0
Above solution give desired pH only at 37 C.
4. Borax-NaOX buffer
A: 0.05 M solution of borax (19.05 g in 1000 ml, 0.02 M in terms of sodium borate)
B: 0.2 M NaOH 100 ml of A + X ml B, diluted to a total of 400 ml
X
pH
0.0
9.28
14.0
9.35
22.0
9.4
35.2
9.5
46.0
9.6
58.0
9.7
68.0
9.8
77.2
9.9
86.0
10.0
92.0
10.1
5. Boric acid-Borax buffer
Solution required:
A: 0.2 M solution of boric acid (12.4 g in 1000 ml).
B: 0.2 M solution of borax (19.05 g in 1000 ml : 0.2 M in terms of sodium borate).
100 mi of A + X ml of B, diluted to a total of 400 ml.
X
pH
4.0
7.6
6.2
9.8
14.6
23.0
35.0
60.0
118.0
230
7.8
8.0
8.2
8.4
8.6
8.8
9.0
9.2
6. Cacodylate buffer
Solutions required:
A: 0.2 M solution of sodium cacodylate [42.8 g of Na (CH3)2 AsO2.3H2O in 1000 ml]/.
B: 0.2 M NaOH 100 ml of A + X ml of B diluted to a total of 400 ml
X
pH
5.4
7.4
8.4
7.2
12.6
7.0
18.6
6.8
26.6
6.6
36.6
6.4
47.6
6.2
59.2
6.0
69.6
5.8
78.4
5.6
86.0
5.4
90.0
5.2
94.0
5.0
7. Carbonate-Bicarbonate buffer
Solutions required:
A: 0.2 M solution of anhydrous sodium carbonate. (2.12 g in 100 ml).
B: 0.2 M solution of sodium bicarbonate. (1-68 g in 100 ml).
X ml of A + Y ml of B, diluted to a total of 400 ml
X
Y
pH
8.0
92.0
9.2
19.0
81.0
9.4
32.0
68.0
9.6
44.0
56.0
9.8
55.0
45.0
10.0
66.0
34.0
10.2
77.0
23.0
10.4
85.0
15.0
10.6
8. Citrate buffer
Solutions required:
A: 0.1 M solution of citric acid (19.21 g in 1000 ml).
B: 0.1 M solution of sodium citrate (29.41 g C6H5O7Na3.2H2O in 1000 ml).
X ml of A + Y ml of B, diluted to a total of 400 ml.
X
Y
pH
186.0
14.0
3.0
174.8
25.2
3.2
160.0
40.0
3.4
148.0
52.0
3.6
140.0
60.0
3.8
132.0
68.0
4.0
126.0
74.0
4.2
112.0
88.0
4.4
102.0
98.0
4.6
92.0
108.0
5.0
82.0
118.0
5.0
72.0
128.0
5.2
64.0
136.0
5.4
54.8
145.2
5.6
47.2
152.8
5.8
38.0
162.0
6.0
28.8
171.2
6.2
9. Citrate-Phosphate buffer
Solutions required:
A: 0.1 M solution of citric acid (19.21 g in 1000 ml).
B: 0.2 M solution of dibasic sodium phosphate (28.39 g of Na2HPO4 or 71.7 g of
Na2HPO4.12H2O in 1000 ml). X ml of A + Y Ml of B, diluted to a total of 100 ml.
X
Y
pH
44.6
5.4
2.6
42.2
7.8
2.8
39.8
10.2
3.0
37.7
12.3
3.2
35.9
14.1
3.4
33.9
16.1
3.6
32.3
17.7
3.8
30.7
19.3
4.0
29.4
20.6
4.2
27.8
22.2
4.4
26.7
23.3
4.6
25.2
24.8
4.8
24.3
26.7
5.2
23.3
26.7
5.2
22.2
27.8
5.4
21.0
29.0
5.6
19.7
30.3
5.8
17.9
32.1
6.0
16.9
33.1
6.2
15.4
34.6
6.4
13.6
36.4
6.6
9.1
40.9
6.8
6.4
43.6
7.0
10. Glycine-NaOH buffer
Solutions required:
A: 0.2 M solutions of &cine (15.01 g in 100 ml).
B: 0.2M NaOH 100 ml of A + X ml of B, diluted to a total of 400 ml.
X
pH
8.0
8.6
12.0
8.8
17.6
9.0
24.0
9.2
33.6
9.4
44.8
9.6
54.4
9.8
64.0
10.0
77.2
10.4
91.0
10.6
11. Maleate buffer
Solutions required:
A: 0.2M solutions of acid sodium maleate (8 g of NaOH+23.2 g of maleic acid of 19.6 g
of maleic anhydride in 1000 ml).
B: 0.2MNaOH 100 ml of A + X pf B, diluted to a total of 400 ml.
X
pH
14.4
5.2
21.0
5.4
30.6
5.6
41.6
5.8
53.8
66.0
76.0
83.2
88.8
6.0
6.2
6.4
6.6
6.8
12. Phosphate buffer
Solutions required:
A: 0.2 M solution of monobasic sodium phosphate. (31.2 g NaH2PO4.2H2O in 1000 ml).
B: 0.2 M solution of dibasic sodium phosphate. (28.39 g of Na 2HPO4 or 7 1.7 g of
N3HP0,. 12H20 in 1000 ml). X ml of A + Y of B, diluted to a total of 200 ml.
13. Succinate buffer
X
Y
pH
92.0
8.0
5.8
87.7
12.3
6.0
81.5
18.5
6.2
73.5
26.5
6.4
62.5
37.5
6.6
51.0
49.0
6.8
39.0
61.0
7.0
28.0
72.0
7.2
19.0
81.0
7.4
13.0
87.0
7.6
8.5
91.5
7.8
5.3
94.7
8.0
Solutions required:
A: 0.2 M solution of succinic acid (23.6 g in 1000 ml)
B: 0.2MNaOH 100 ml of A + X ml of B, diluted to a total of 400 ml.
X
pH
30.0
3.8
40.0
4.0
53.0
4.2
66.8
4.4
80.0
4.6
94.0
4.8
106.8
5.0
121.2
5.2
136.8
5.4
150.0
5.6
162.8
5.8
174.0
6.0
14. Tris (hydroxymethyl) aminomethane HCL (Tris HCL buffer)
Solution required:
A: 0.2 M solution of tris (hydroxymethyl) aminomethane (24.2 g in 1000 ml).
B: 0.2 M HCL 100 ml of A + X ml of B, diluted to a total of 400 ml.
X
pH
10.0
9.0
16.2
8.8
24.4
8.6
33.0
8.4
43.8
8.2
53.6
8.0
65.0
7.8
76.8
7.6
82.8
7.4
88.4
7.2
5
The Standard Solutions
A standard solution is one of known concentration. Following standard solutions are
frequently required in
microbiology laboratory:
(1) Molar (2) Molal (3) Nomal
(1) Molarity (M) = Molarity is defined as the number of moles
of solute contained in one litre.
A 1 molar (1.0 M) solution of sodium hydroxide contain 1.0
mole or 40 g of sodium hydroxide in one litre of solution because,
NaOH (aq.)

Na++OH
1 mole of Na+
=
23 g (from table no. 2)
1 mole of oxygen
=
16 g (from table no. 2)
1 mole of hydrogen
=
1 g (from table no. 2)
Thus,
1 mole of NaOH = Total 40g
Thus,
When 40g (I mole) of NaOH is dissolved in 1 litre of water it gives 1 molar solution.
(2) Molality (M) = Molality expresses the concentration of a solution in which the
quantity of solute is given in moles, and the quantity of solvent in kilograms. Molality is
defined as the number of moles of solute dissolved in 1000 g (1 kg) of solvent. A one
molal (1.0 m) solution of sodium hydroxide contain 1.0 mole of 40 g of NaOH dissolved
in 1 kilogram of water, that is
(3) Normality (N) = Normality expresses the concentration of a solution in which the
quantity of solute is given in gram equivalent weights and the volume of solution in
litres.
Normality therefore represents the number of gram equivalent weights of solute
dissolved in one litre of solution. That is,
Gram equivalent weight of solutes are dependent on the total positive or negative charges
in the formula of acid, base or salt.
PPM (Parts Per Million Solution)
Gram of solution per million grams of solution or one g of solute1 million ml of solution.
OR
therefore 1 ppm = 1 mg NaCl/100 ml or 1 mg NaCl/ml.
Table 1
The gram equivalent weights of few substances
Compound
Lons
Lonic
1.00 mol.
1.00 gram
change
(in g.)
equivalent
weight (in g)
+
HCl
H +Cl
1
36.47
36.47
+
H2SO4
2H +SO4-2
2
98.08
49.04
+
NaOH
Na +OH1
40.00
40.00
+2
Ca(OH)2
Ca +2OH
2
74.10
37.05
K3PO4
3K++PO4-3
3
212.28
70.76
Table 2
International atomic weights of the element
Element
Aluminium
Antimony
Arsenic
Barium
Beryllium
Bismuth
Boron
Bromine
Cadmium
Calcium
Carbon
Chlorine
Chromium
Cobalt
Copper
Fluorine
Gold
Hydrogen
Iodine
Iron
Lead
Magnesium
Mercury
Nickel
Nitrogen
Oxygen
Palladium
Phosphorus
Platinum
Potassium
Radium
Selenium
Silicon
Symbol
Al
Sb
As
Ba
Be
Bi
B
Br
Cd
Ca
C
Cl
Cr
Co
Cu
F
Au
H
P
Fe
Pb
Mg
Hg
Ni
N
O
Pd
P
Pt
K
Ra
Se
Si
Atomic weight
26.98
121.75
74.92
137.34
9.01
208.98
10.82
79.90
112.40
40.08
12.01
35.45
52.00
58.93
63.55
19.00
196.97
1.01
126.90
55.85
207.20
24.31
200.59
58.69
14.008
16.0000
106.7
30.98
195.23
39.096
22605
78.96
28.06
Element
Symbol
Ag
Na
Sr
S
Sn
Ti
W
U
V
Zn
Zr
Atomic weight
107.880
22.997
87.63
32.066
118.70
47.90
183.92
238.07
50.95
65.38
91.2
Silver
Sodium
Strontium
Sulfur
Tin
Titanium
Tungsten
Uranium
Vanadium
Zinc
Zirconium
Solution required:
A. 1% aqueous barium chloride solution
B. 1% aqueous sulphuric acid solution.
Add the amount given in the following table to clean, dry ampoules. Ampoules
should have same diameter as the test tube to be used in subsequent density determinates.
Seal the ampoules and label them.
Table 3
McFarland nephelometer barium sulphate standards
Tube No.
Barium chloride
Sulphuric Acid
Corresponding Approx. Density of
1% (ml)
1% (ml)
bacteria (million/ml)
1
0.1
9.9
300
2
0.2
9.8
600
3
0.3
9.7
900
4
0.4
9.6
1200
5
0.5
9.5
1500
6
0.6
9.4
1800
7
0.7
9.3
2100
8
0.8
9.2
2400
9
0.9
9.1
2700
10
1.0
9.0
3000
Use: used to determine density of bacteria in suspension. (specially for preparation of
vaccines.)
Table 4
Antimicrobic zone of inhibition evaluation (Kirby Bauermethod).
Significance of zone diameters with disk potency.
Antimicrobial agent
Amikacin
Ampicillin
Gram negative organisms and
enterococci.
Staphylococci
Bacitracin
Carbenicillin
Proteus sp. & E. coli.
Pseudomonas aeruginosa
Cefadroxil
Cefotaxime
Ceftriaxone
Cephalothin
Chroramphernicol
Ciprofloxacin
Clindamycin
Colistin
Cotrimoxazole
Erythromycin
Gentamicin
Ps. aeruginosa
Kanamycin
Lincomycin
Methicillin
Nafcillin
Nalidixic aci
Neomycin
Metilmycin
Mitroflurantoin
Norfloxacin
Oleandomycin
Oxolinic acid
Penicillin G
Staphylococci
Other organisms
Disk
potency
10 mcg
Resistant
mm
<12
Intermidat Sensitive
e mm
mm
12-13
>13
10 mcg
<12
12-13
>13
10 mcg
10 units
<21
<9
21-13
9-12
>28
>12
50 mcg
50 mcg
30 mcg
30 mcg
30 mcg
30 mcg
30 mcg
5 mcg
2 mcg
10 mcg
30 mcg
15 mcg
<13
<15
<15
<15
<14
<15
<13
<16
<15
<9
<14
<14
18-22
13-14
15-17
15-22
14-20
>22
>14
>17
>22
>20
>14
>17
>20
>16
>10
>20
>17
10 mcg
30 mcg
2 mcg
5 mcg
1 mcg
30 mcg
30 mcg
30 mcg
30 mcg
5 mcg
15 mcg
2 mcg
<13
<14
<17
<10
<11
<14
<13
<15
<15
<16
<21
<11
14-17
17-20
10-13
11-12
14-18
13-16
15-16
15-16
16-20
12-16
>12
>17
>20
>13
>12
>18
>16
>16
>16
>20
>16
>10
10 units
10 units
<21
<12
21-28
12-21
>28
>21
13-17
16-20
15-16
9-10
14-20
14-17
Antimicrobial agent
Polymyxin B
Rifampicin
Neisseria
Meningitides
Streptomycin
Tetracycline
Tobramycin
Triple sulfa
Vancomycin
Disk
potency
300 units
Resistant
mm
<9
5 mcg
10 mcg
30 mcg
10 mcg
250 mcg
30 mcg
<25
<12
<15
<12
<12
<10
Intermidat Sensitive
e mm
mm
9-11
>11
>24
>14
>18
>13
>16
>11
12-14
15-18
12-13
13-16
10-11
Table 5
Relation between trasmittancy (T %) & optical density (O.S.)
T%
100
99
98
97
96
95
94
93
92
91
90
89
88
87
86
85
84
83
82
81
80
79
78
O.D.
0.000
0.04
0.009
0.013
0.018
0.022
0.027
0.032
0.036
0.041
0.046
0.051
0.056
0.061
0.066
0.071
0.076
0.081
0.086
0.092
0.097
0.102
0.108
T%
50
49
48
47
46
45
44
43
42
41
40
39
38
37
36
35
34
33
32
31
30
29
28
O.D.
0.301
0.310
0.319
0.328
0.339
0.347
0.357
0367
0.377
0.387
0.398
0.409
0.120
0.432
0.444
0.456
0.459
0.482
0.495
0.509
0523
0.538
0552
T%
O.D.
T%
O.D.
77
1.114
27
0.569
76
0.119
26
0.585
75
0.125
25
0.602
74
0.131
24
0.620
73
0.137
23
0638
72
0.143
22
0.658
71
0149
21
0.678
70
0155
20
0.699
69
0.161
19
0721
68
0.168
18
0.745
67
0.174
17
0.770
66
0.181
16
0.796
65
0.187
15
0.824
64
0194
14
0.854
63
0.201
13
0.886
62
0.208
12
0.921
61
0.215
11
0.950
60
0.222
10
1.000
59
0.229
9
1.046
58
0.237
8
1.097
57
0.244
7
1.155
56
0.252
6
1.222
55
0260
5
1.301
54
0.268
4
1.398
53
0.276
3
1.523
52
0.248
2
1.699
51
0.292
1
2.000
Optical density of given transmittance can be determined by using following formula:
O.D. = 2 – Log % Transmittance
Table 6
Temperature and Pressure Relationship
Steam pressure (psi)
1
0
1
2
Temperature (0C)
2
100.0
101.9
103.6
Steam pressure (psi)
Temperature (0C)
3
105.3
4
106.9
5
108.4
6
109.8
7
111.3
8
112.6
9
113.9
10
115.2
11
116.4
12
117.6
13
118.8
14
119.9
15
121.0
16
122.0
17
123.0
18
124.1
19
125.0
20
126.0
21
126.9
22
127.8
23
128.9
24
129.6
25
130.4
26
131.3
27
132.1
28
132.9
29
133.7
30
134.5
Around the working range of 15 psi, each raises the autoclave temperature by
approximately 10C.
Table 7
MPN/100 ml using one tube of 50 ml & five tubes of 10 ml
50 ml tube positive
10 ml tubes positive
MNP / 100 ml
0
0
0
0
1
1
0
2
2
0
3
4
0
4
5
0
1
1
1
1
1
1
5
7
0
2
1
3
2
6
3
9
4
16
5
18+
Table 8
MPN determination from multiple tubes test (McCrady’s table) for set of nine tubes
Number of tubes giving positive reaction
out of
3 of 10 ml
3 of 1 ml
3 of 0.1 ml
each
each
each
0
0
0
0
0
1
0
1
0
1
0
0
1
0
1
1
1
0
1
1
1
1
2
0
2
0
0
2
0
1
2
1
0
2
1
1
2
2
0
2
2
1
3
0
0
3
0
1
3
0
2
3
1
0
3
1
1
3
1
2
3
2
0
3
2
1
3
2
2
3
3
0
3
3
1
MPN Index
/ 100 ml
<3
3
3
4
7
7
11
11
9
14
15
20
21
28
23
39
64
43
75
120
93
150
210
240
460
95 % confidence limits
Lower
Upper
<0.5
<0.5
<0.5
1
1
3
3
1
3
3
7
4
10
4
7
15
7
14
30
15
30
35
36
71
9
13
20
21
23
36
36
36
37
44
89
47
150
120
130
380
210
230
380
380
440
470
1300
2400
3
3
3
3
2
3
1100
>2400
150
-
4800
-
Table 9
MPN determination from multiple tube test (McCrady’s table) for set fifteen tubes.
Number of tubes giving positive reaction
out of
3 of 10 ml
3 of 1 ml
3 of 0.1 ml
each
each
each
0
0
0
0
0
1
0
1
0
0
2
0
1
0
0
1
0
1
1
1
0
1
1
1
1
2
0
2
0
0
2
1
0
2
1
0
2
1
1
2
2
0
2
3
0
3
0
0
3
0
1
3
1
0
3
1
1
3
2
0
3
2
1
4
0
0
4
0
1
4
1
0
4
1
1
4
1
1
4
1
2
4
2
0
MPN Index
/ 100 ml
<2
2
2
4
2
4
4
6
6
5
7
7
9
9
12
8
11
11
14
14
17
13
17
17
17
21
26
22
95 % confidence limits
Lower
Upper
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
<0.5
1.0
1.0
2.0
2.0
3.0
1.0
2.0
2.0
4.0
4.0
5.0
3.0
5.0
5.0
7.0
7.0
9.0
7.0
7
7
11
7
11
11
15
15
13
17
17
21
21
28
19
25
25
34
34
46
31
46
46
63
63
78
67
Number of tubes giving positive reaction
out of
3 of 10 ml
3 of 1 ml
3 of 0.1 ml
each
each
each
4
2
1
4
3
0
4
3
1
4
4
0
5
0
0
5
0
1
5
0
2
5
1
0
5
1
1
5
1
2
5
2
0
5
2
1
5
2
2
5
3
0
5
3
1
5
3
2
5
3
3
5
4
0
5
4
1
5
4
2
5
4
3
5
4
4
5
5
0
5
5
1
5
5
2
5
5
3
5
5
4
5
5
5
MPN Index
/ 100 ml
26
27
33
34
23
31
43
33
46
63
49
70
94
779
110
140
180
130
170
220
280
350
240
350
450
920
1600
>2400
MPN / 100 ml can be calculated by using following formula:
=
√
Table 10
95 % confidence limits
Lower
Upper
9.0
9.0
11
12
7
11
15
11
16
21
17
23
28
25
25
37
44
35
43
57
90
120
68
120
180
300
640
-
78
80
93
93
70
89
110
93
120
150
130
170
220
190
250
340
500
300
490
700
850
1000
750
1000
1400
3200
5800
-
Preparation of indicators
Indicator solution
Indicator (g)
965 % Ethanol (ml) Distilled water (ml)
Bromocresol green
0.4
500
500
Bromocresol purple
0.4
500
500
Bromocresol blue
0.4
500
500
Cresol red
0.2
500
500
Methyl red
0.2
500
500
Phenolphthalein
1.0
50
50
Phenol red
0.2
500
500
Thymol blue
0.4
500
500
Preparation: Dissolve the indicator in 95 % ethanol. Add water, Filter before use.
Table 11
Power of units – prefixes
Multiple
Prefix
Symbol
12
10
Tera
T
9
10
Giga
G
6
10
Mega
M
3
10
Kilo
K
2
10
Hector
H
10
Deca
Da
-1
10
Deci
D
-2
10
Centi
C
-3
10
Milli
M
-6
10
Micro
µ
-9
10
Nano
N
-12
10
Pico
P
-15
10
femto
F
Indicators
1
Cresol red (acid range)
Meta cresol purple (acid range)
Thymol blue (acid range)
Bromo phenol blue
Bromo cresol green
Table 12
Indicators with pH range
Full acid Colour
Full alkaline Colour
2
3
Red
Yellow
Red
Yellow
Red
Yellow
Yellow
Blue
Yellow
Blue
pH range.
4
0.2-1.8
1.2-2.8
1.2-2.8
3.0-4.6
3.8-5.4
Indicators
1
Chlor cresol green
Methyl red
Chlor phenol red
Bromo cresol purple
Bromo Thymol blue
Neutral red
Phenol red
Cresol red (alkaline range)
Meta cresol purple (alkaline
range)
Thymol blue (alkaline range)
Cresolphthalein
Phenolphthalein
Acyl blue
Parazo orange
Toly red
Full acid Colour
2
Yellow
Red
Yellow
Yellow
Yellow
Red
Yellow
Yellow
Yellow
Full alkaline Colour
3
Blue
Yellow
Red
Purple
Blue
Amber
Yellow
Yellow
Purple
pH range.
4
4.0-5.6
4.4-6.4
4.8-6.4
5.2-6.8
6.0-7.6
6.8-8.0
6.8-8.4
7.2-8.8
7.4-9.0
Yellow
Colourless
Colourless
Red
Yellow
red
Blue
Red
Red
Blue
Orange
yellow
8.0-96
8.2-9.8
8.3-10.0
12.0-13.6
11.0-12.6
10.0-11.6
Table 13
Test organisms for microbiological assay of antibiotics
Antibiotic
1
Amikacin
Antifimial
Bacitracin
Bleomycin
Carbenicillin
Cephalosporin
Chloranphencol
Clindamycin
Doxycycline
Erythromycin
Framycetin
Gentamicin
Test organism
2
Staphylococcus aureus
Sacchromyces cerevisiae
Candida albicans
Micrococcus luteus
Mycobacterium smegmatis
Pseudomonas aeruginosa
Bacillus subtilis
Staphylococcus aureus
S. lutea
Bacillus subtilis
Staphylococcus aureus
Staphylococcus aureus
Micrococcus luteus
Bacillus pumilus
Bacillus subtilis
Staphylococus epidermidis
ATC-1 No. NTTC2 No. (NCIB3 No.)
3
4
29737
7447
9763
10716
10240
7743
607
25619
6633
8236
29737
6571
8340
6633
8236
29737
6571
29737
7447
9341
(8553)
14884
8241
6633
8236, 10400
12228
(8853)
Antibiotic
1
ATC-1 No. NTTC2 No. (NCIB3 No.)
3
4
6633
8236
10896
14884
8241
29737
7447
6633
8236
Bacillus subtilis
12228
(8853)
Staphylococcus epidermidis
12228
(8853)
Staphylococcus epidermidis
2601
10716
Saccharomyces cfrevisie
11778
10320
Bacillus cereus var. mycodies
29737
7447
Staphylococcus aureus
8236
Bacillus subtilis
6571
Staphylococcus aureus
4617
8344
BordeteNa bronchiseptica
6633
8236
Bacillus subtilis
6633
8236
Bacillus subtilis
10031
(9111)
Klebsiella pneumoniae
14884
8241
Bacillus pumilis
14884
8241
Bacillus pumilus
10490
Pseudomonas aeruginosa
11778
10320
Bacillus cereus
29737
7447
Staphyoccoccus aureus
6633
82368236
Bacillus subtilis
10896
Klebsiella edwardsii
Test organism
2
Bacillus subtilis
Klebsiella edwardsii
Kanamycin sulphate Baccillus pumilus
Staphylococcus aureus
Kanamycin B
Neomycin
Novobiocin
Nystatin
Oxytetracycline
Penicillin
Polymyxin B
Rifampicin
Streptomycin
Sulphonamides
Trimethoprim
Ticarcillin
Tetracycline
Tobramycin
1. American Type culture Collection, 2 1301 Park Lawn Drive, Rockville, MD
20852m USA.
2 National Collections of Type Culture, Central Public Health Laboratory, Colindale
Avenue, London NW9 5HT, England.
2. National Collection of Industrial Bacteria, toryy Research Station P.O. Box 31,
135 Abbey Road, Aberdeen 98 DC Scotland.
Table 14
Temperature Celsius-Fehrenheit Relation
Fehrenheit Celsius Fehrenheit Celsius Fehrenheit Celsius Fehrenheit Celsius
1
2
3
4
5
6
7
8
0.0
32.0
26.0
78.8
51.0
123.8
76.0
168.8
1.0
33.8
27.0
80.6
52.0
125.6
77.0
170.6
2.0
35.6
28.0
82.4
53.0
127.4
78.0
1724
3.0
37.4
29.0
842
54.0
129.2
79.0
174.2
4.0
39.2
30.0
86.
55.0
131.0
80.0
176.0
5.0
41.0
31.0
87.8
56.0
132.8
81.0
177.8
6.0
42.8
32.0
89.6
57.0
134.6
82.0
179.6
7.0
44.6
33.0
91.4
58.0
136.4
83.0
181.4
8.0
46.4
34.0
93.2
59.0
138.2
84.0
183.2
9.0
48.2
35.0
95.0
60.0
140.0
85.0
185.0
10.0
50.0
36.0
96.8
61.0
141.8
86.0
186.8
11.0
51.8
37.0
98.6
62.0
143.6
87.0
1886
12.0
53.6
38.0
100.4
63.0
145.4
88.0
190.4
13.0
55.4
39.0
102.2
64.0
147.2
89.0
192.2
14.0
57.2
40.0
104.0
65.0
149.0
90.0
194.0
15.0
59.0
41.0
105.8
66.0
150.8
91.0
195.8
16.0
60.8
42.0
107.6
67.0
152.6
92.0
197.6
17.0
62.6
43.0
109.4
68.0
154.4
93.0
199.0
18.0
64.4
44.0
111.2
69.0
156.2
94.0
201.2
19.0
66.2
45.0
113.0
70.0
158.0
95.0
203.0
20.0
68.0
46.0
114.8
71.0
159.8
96.0
204.8
21.0
69.8
47.0
116.6
72.0
161.6
97.0
206.6
22.0
71.6
48.0
118.4
73.0
163.4
98.0
208.4
23.0
73.4
49.0
120.2
74.0
165.2
99.0
210.2
24.0
75.2
50.0
122.0
75.0
167.0
100.0
212.0
25.0
77.07
0
C=
Table 15
Percentages by volumes at 15.60 (60 F) of ethyl alcohol corresponding to apparent
specific gravity at various temperatures*
Apparent 15.56/ 20/
22/ 24/ 25/ 26/ 28/ 30/ 32/ 34/ 35/ 36/
Specific 15.56
20
22
24
25
26
28
30
32
34
35
36
Gravity
1
2
3
4
5
6
7
8
9
10
11
12
13
0
1.000
0.999
0.998
0.997
0.996
0.995
0.994
0.993
0.992
0.991
0.990
0.989
0.988
0.987
0.986
0.985
0.984
0.983
0.982
0.981
0.979
0.978
0.977
0.976
0.975
0.974
0.973
0.972
0.971
0.970
0.969
0.968
0.967
0.966
0.965
0.964
0.963
0.962
0.00
0.67
1.34
2.02
2.71
3.41
4.13
4.86
5.61
6.38
7.17
7.98
8.82
9.66
10.50
11.37
12.25
13.16
14.08
15.02
16.92
17.91
18.91
19.93
20.93
21.92
22.90
23.87
24.81
25.85
26.69
27.60
28.50
29.39
30.26
31.11
31.93
32.72
0.00
0.66
1.34
2.2
2.70
3.40
4.11
4.84
5.58
6.34
7.12
7.90
8.71
9.54
10.36
11.19
12.04
12.90
13.77
14.66
16.46
17.38
18.31
19.26
20.20
21.14
22.05
22.96
23.87
24.84
25.62
26.50
27.36
28.19
29.03
29.85
30.66
31.44
0.00
0.66
1.34
2.01
2.70
3.40
4.10
4.83
5.56
6.32
7.09
7.87
8.67
9.49
10.30
11.12
11.95
12.79
13.64
14.51
16.27
17.17
18.08
18.99
19.91
20.82
21.72
22.61
23.49
24.44
25.21
2606
26.89
27.72
28.53
29.34
30.12
30.91
0.00
0.66
1.34
2.01
2.70
3.39
4.10
4.82
5.55
6.30
7.06
7.84
8.63
9.43
10.24
11.04
11.86
12.68
13.52
14.37
16.09
16.97
17.85
18.74
19.64
20.53
21.41
22.27
23.13
24.06
24.82
25.65
2646
27.27
28.07
28.86
29.64
30.41
0
0.00
0.66
1.34
201
2.70
3.39
4.09
4.81
5.54
6.29
7.05
7.83
8.61
9.41
10.21
11.00
11.81
11.81
13.46
14.60
1600
16.87
17.74
18.62
19.50
20.38
21.25
22.11
22.96
23.88
24.63
25.45
26.26
27.06
27.85
28.63
29.40
30.17
0.00
0.66
1.33
2.01
2.70
3.39
4.09
4.80
5.53
6.28
7.03
7.81
859
9.39
10.18
10.96
11.77
11.77
13.40
14.24
15.92
16.78
17.63
18.50
19.38
20.25
21.11
21.96
22.88
23.72
24.45
25.26
2606
26.85
27.6
28.41
29.18
29.94
0.00
0.66
1.33
2.00
2.70
3.38
4.07
4.79
5.51
6.25
7.00
7.78
855
9.33
10.11
10.89
11.68
11.68
13.29
14.11
15.75
16.59
17.43
19.28
19.13
19.99
20.83
21.66
22.49
23.38
24.10
45.89
25.68
26.46
27.23
27.99
28.74
29.50
0.00
0.66
1.32
2.00
2.69
3.37
4.06
4.77
5.49
6.23
6.98
7.74
8.50
9.27
10.05
10.82
11.60
11.60
13.18
13.98
15.59
16.41
17.24
18.07
18.90
19.74
20.56
21.38
22.19
23.06
23.77
24.54
25.32
26.09
26.85
27.59
28.33
29.07
0.00
0.66
1.32
2.00
2.68
3.36
4.05
4.75
5.47
6.20
6.94
7.70
8.46
9.22
9.99
10.74
11.51
11.51
13.06
13.85
15.44
16.24
17.05
17.87
18.68
1950
20.31
20.11
21.91
22.77
23.46
24.21
2498
25.74
26.49
27.2
27.95
28.67
0.00
0.66
1.32
1.99
2.67
3.35
4.04
4.74
5.45
6.17
6.91
7.66
8.41
9.17
9.92
10.67
11.42
11.42
1295
13.73
15.29
16.08
16.88
17.67
18.47
19.28
20.07
20.86
21.64
22.48
23.16
23.90
24.66
25.40
26.14
26.86
27.58
28.29
0.00
0.66
1.32
1.99
2.66
3.34
4.03
4.73
5.44
6.16
6.90
7.64
8.39
9.14
9.89
10.63
11.38
11.38
12.90
13.67
15.22
16.00
16.79
17.58
18.37
19.17
19.95
20.73
21.50
22.34
23.01
23.76
24.50
25.24
25.97
26.69
27.39
28.10
0.00
0.66
1.32
1.99
2.66
3.34
4.03
4.72
543
6.15
6.88
7.62
8.37
9.12
9.86
10.59
11.34
11.34
12.85
13.61
15.15
15.92
16.71
17.49
18.27
19.06
19.84
20.61
21.37
22.21
22.88
23.61
24.35
25.08
25.81
36.52
27.92
27.92
0.961
0.960
0.959
0.958
0.957
0.956
0.955
0.954
0.953
0.952
0.951
0.950
0.949
0.948
0.947
0.946
0.945
0.944
0.943
0.942
0.941
0.940
0.939
0.938
0.937
0.936
0.935
0.934
0.933
0.932
0.931
0.930
0.929
0.928
0.927
0.926
0.925
0.924
0.923
0.922
0.921
0.920
0.919
0.918
0.917
0.916
33.50
34.27
35.02
35.75
36.46
37.16
37.84
38.51
39.17
39.82
40.46
41.09
41.70
42.31
42.92
43.51
43.10
44.68
45.25
45.81
46.37
46.92
47.46
48.00
48.53
49.06
49.58
50.09
50.60
51.11
51.61
52.11
52.61
53.10
53.59
54.08
54.57
55.05
55.52
5600
56.47
56.95
57.41
57.88
58.34
58.79
32.21
32.96
33.69
34.41
35.12
35.82
36.49
37.16
37.82
38.46
39.10
39.73
40.35
40.96
41.56
4215
42.74
43.33
43.90
44.46
45.03
45.58
46.12
46.67
47.20
47.73
48.26
48.78
49.80
49.30
50.31
50.81
51.31
51.80
52.29
52.79
53.27
53.76
54.24
54.72
55.19
55.67
56.14
56.60
56.07
57.53
-
-
30.92
31.65
32.37
33.08
33.78
34.46
35.13
35.79
36.44
37.09
37.71
38.33
38.95
39.56
40.17
40.76
41.34
41.92
42.49
43.06
43.62
44.18
44.73
45.27
45.81
46.34
46.87
47.40
47.92
48.43
48.94
49.44
49.94
50.44
50.93
51.42
51.91
52.40
52.88
53.36
53.84
54.31
54.78
55.25
55.72
56.18
-
-
29.80
30.51
31.22
31.91
32.58
33.25
33.92
34.57
35.22
35.85
36.47
37.09
37.7
38.30
38.90
39.49
40.07
40.64
41.21
41.77
42.33
42.89
43.44
43.98
44.52
45.06
45.58
46.11
4663
47.15
47.65
48.16
48.66
49.16
49.66
50.15
50.64
51.13
51.61
52.09
52.57
53.05
53.51
53.98
54.45
54.92
-
-
28.80
29.50
30.18
30.86
31.53
32.18
32.83
33.46
34.10
34.72
35.34
35.95
3655
3715
37.74
38.32
38.90
39.47
40.04
40.59
41.15
41.70
42.24
42.79
43.33
43.86
44.39
44.91
45.43
45.95
46.46
46.97
47.47
47.97
48.47
48.96
49.44
49.93
50.41
50.89
51.37
51.84
52.32
52.79
53.26
53.72
-
0.915
0.914
0.913
0.912
0.911
0.910
0.909
0.908
0.907
0.906
0.905
0.904
0.903
0.902
0.901
0.900
0.899
0.898
0.897
0.896
0.895
0.894
0.893
0.892
0.891
0.890
0.888
0.887
0.886
0.885
0.884
0.883
0.882
0.881
0.880
0.879
0.878
0.877
0.876
0.875
0.874
0.873
0.872
0.871
0.870
0.869
59.25
59.70
60.15
60.59
6104
61.48
61.92
62.36
62.79
63.23
63.66
64.09
64.53
64.96
65.38
65.81
66.23
66.65
67.07
67.48
67.90
68.31
68.72
69.13
69.54
69.94
70.75
71.15
71.54
71.94
72.33
72.73
73.12
73.50
73.89
74.28
74.66
75.05
75.43
75.81
76.19
76.56
76.94
77.31
77.68
78.05
57.99
58.44
59.89
59.34
59.79
60.24
60.68
61.12
61.56
62.00
62.43
62.86
63.30
63.73
64.16
64.58
65.01
65.43
65.85
66.27
66.69
67.10
67.52
67.93
68.34
68.75
69.56
69.96
70.36
70.76
71.16
71.55
71.95
72.34
72.73
73.12
73.50
73.89
74.28
74.66
75.04
75.42
75.80
76.18
76.55
76.92
-
-
56.65
57.10
57.56
58.01
58.46
58.91
59.36
59.80
60.25
50.68
61.12
61.55
61.99
62.42
62.85
63.27
63.70
64.13
64.55
64.97
65.39
65.81
66.23
66.64
67.06
67.47
68.28
68.69
69.10
69.50
69.90
70.29
70.69
71.09
71.48
72.87
78.26
72.65
73.04
73.42
73.81
73.19
73.57
73.95
75.33
75.70
-
-
55.38
55.84
56.30
56.76
57.21
57.66
58.11
58.55
59.00
59.43
59.87
60.31
60.75
61.18
61.61
62.04
62.47
62.89
63.32
63.74
64.16
64.58
65.00
65.42
65.83
66.25
67.07
67.48
67.49
68.29
68.69
69.09
69.49
69.89
70.29
71.68
71.07
71.46
71.85
72.24
72.63
73.01
73.39
73.77
74.16
74.54
-
-
54.18
54.65
55.11
55.56
56.02
56.47
56.93
59.39
57.81
58.26
58.70
59.13
59.57
60.00
60.43
60.86
61.29
61.72
62.14
62.57
62.99
63.41
63.83
64.32
64.67
65.08
65.91
66.32
66.73
68.13
68.54
68.94
69.34
68.74
69.14
70.53
70.93
72.32
70.71
71.10
71.49
71.88
71.27
71.65
73.03
73.41
-
0.868
0.867
0.866
0.865
0.864
0.863
0.862
0.861
0.860
0.859
0.858
0.857
0.856
0.855
0.854
0.853
0.852
0.851
0.850
0.849
0.848
0.847
0.846
0.845
0.844
0.843
0.842
0.841
0.840
0.839
0.838
0.837
0.836
0.835
0.834
0.833
0.832
0.831
0.830
0.829
0.828
0.827
0.826
0.825
0.824
0.823
78.41
78.78
79.14
79.51
79.87
80.22
80.58
80.94
81.29
81.65
82.00
82.35
82.70
83.04
83.39
83.73
84.07
84.41
84.75
85.09
85.42
85.75
86.08
86.40
86.73
87.05
87.38
87.70
88.02
88.33
88.65
88.96
89.27
89.58
89.58
90.19
90.49
90.78
91.08
91.37
91.66
91.95
92.24
92.53
92.80
93.08
77.29
77.66
78.03
78.39
78.76
79.12
79.48
79.84
80.20
80.55
80.91
81.26
81.61
81.96
82.31
82.66
83.01
83.35
83.69
84.03
84.37
84.71
85.04
85.38
85.71
86.03
86.36
86.68
87.01
87.33
87.65
87.97
88.29
88.60
88.91
89.22
89.53
89.83
90.14
90.44
90.73
91.03
91.32
91.62
91.90
92.19
-
-
76.08
76.45
76.82
77.19
77.56
77.93
78.29
78.65
79.01
79.37
79.73
80.09
80.44
80.80
81.15
81.50
81.85
82.20
82.54
82.89
83.23
83.57
83.91
83.25
83.59
84.92
85.25
85.59
85.92
86.24
87.57
87.89
87.21
87.53
87.85
88.16
88.48
88.79
89.10
89.41
89.71
90.02
90.32
90.61
90.91
91.21
-
-
74.92
75.29
75.66
76.04
76.41
76.78
77.15
77.51
77.88
78.24
78.60
78.96
79.32
79.67
80.03
80.38
80.73
81.09
81.44
81.78
82.13
82.48
82.82
82.17
82.51
82.85
84.18
84.52
84.85
85.18
86.51
86.84
86.16
86.49
86.81
88.13
87.45
87.77
88.08
88.40
88.71
89.02
89.33
89.64
89.94
90.24
-
-
73.79
74.17
74.55
74.92
75.29
75.67
76.04
76.41
76.78
77.14
77.51
77.87
78.23
78.59
78.95
79.30
79.66
80.01
80.37
80.72
81.07
81.42
81.77
81.11
81.46
81.80
83.14
83.48
83.82
84.16
85.49
85.82
85.15
85.48
85.80
86.13
86.45
86.77
87.10
87.41
87.73
88.05
88.36
88.67
88.98
89.29
-
0.822
93.36
92.48
91.50
92.54
89.59
0.821
93.63
92.76
91.79
92.84
89.90
0.820
93.90
93.04
92.07
91.13
90.20
0.819
94.16
93.31
92.36
91.42
90.49
0.818
94.43
93.58
92.64
91.71
90.79
0.817
94.69
93.85
92.92
92.00
91.09
0.816
94.95
94.12
93.20
92.28
91.38
0.815
95.20
94.38
93.47
92.56
91.66
0.814
95.46
94.64
93.74
92.84
91.95
0.813
95.71
94.90
94.01
93.12
92.24
0.812
95.96
95.16
94.27
93.40
92.53
0.811
96.20
95.42
94.53
93.67
92.80
0.810
96.45
95.67
94.79
93.94
93.08
0.809
96.69
95.92
95.05
94.20
93.36
0.808
96.93
96.16
95.31
94.47
93.63
0.807
97.16
96.41
95.56
94.73
93.90
0.806
97.39
96.65
95.81
94.99
94.16
0.805
97.62
96.89
96.06
95.24
94.43
0.804
97.85
97.12
96.31
95.50
94.69
0.803
98.07
97.36
96.55
95.75
94.95
0.802
98.29
97.59
96.79
96.00
95.21
0.801
98.50
97.81
97.03
96.25
95.46
0.800
98.72
98.03
97.26
96.49
95.72
0.799
98.92
98.26
97.50
96.73
95.97
0.798
99.13
98.47
97.72
96.97
96.21
0.797
99.33
98.68
97.95
97.21
96.46
0.796
99.54
98.89
98.17
97.44
96.70
0.795
99.73
99.10
98.39
97.67
96.94
0.794
99.93
99.30
98.60
97.89
97.18
0.793
99.50
98.81
98.12
97.41
0.792
99.70
99.02
98.33
97.64
0.791
99.90
99.22
98.55
97.86
0.790
99.43
98.76
98.09
0.789
99.63
98.97
98.31
0.788
99.83
99.18
98.53
0.787
99.38
98.74
0.786
99.58
98.95
0.785
99.78
99.16
0.784
99.98
99.36
0.783
99.56
0.782
99.76
0.781
99.96
0.7809
99.98
0.7808
100.0
*Specific gravity is determined by taking weight of water & sample at the same temp. e.g. 25/250C
-
Table 16
Basic SI units and their abbreviaions
Physical quantity
Lenght
Mass
Time
Amount of substance
Thermodynamics temp.
Electric current
Luminous intensity
Name
Meter
Kilogram
Second
Mole
Kelvin
Ampere
Candela
Symbol
M
kg
s
mol
K
A
cd
Table 17
The special names & symbols of some derived SI units
Physical quantity
Frequency
Force
Presure
Energy or work
Power
Electrical charge
Electric capacitance
Potential diference
Resistance
Conductance
Radioactivity
Absorbed dose of radiation
Customary temp.
Name
Hertz
Newton
Pascal
Joule
Watt
Coloumb
Farad
Volt
Ohm
Siemens
Becquerel
Gray
Degree Celcius
Symbol
Hz
N
Pa
J
W
C
F
V
Ω
S
Bq
Gy
0
C
Units
S-1
Kg ms-2
N m-2
Nm
Js-1
As
AsV-1
WA-1
VA-1
Ω
S-1
J kg-1
0
C=K-273.15
Table 18
Notations
10-15
10-12
10-9
10-6
10-3
10-2
10-1
102
103
106
109
=
=
=
=
=
=
=
=
=
=
=
quardillionth
trillionth
billionth
millionth
thousandth
hundredth
tenth
hundred
thousand
million
billion
Table 19
Refractive indicates of diferent substances used as mounting medium
Substance
Glass
Distilled water
Cedar wood oil (immersion oil)
CCL4
Sandal wod oil
Olive oil
Glycerol
Canada balsam
Eupapa 1
Polysterene
Air
Naphrax
Refractive index
1.56
1.33
1.51
1.46
1.51
1.47
1.47
1.54
1.48
1.59
1.00
1.74
Appendies
Appendix-I
List of minimum equipment required for good microbiology laboratory.
1. Microscope with oil immersion lens
One for every student in the batch
2. Hot air oven
one
3. Autoclave
one
4. Bacteriological incubator
Two
5. Rotary shaker
one
6. Incubator shaker
one
7. Calorimeter
one
8. Stirror
one
9. Laminar air flow unit
one
10. Refrigemtor
one
11. Monopan balance (digital)
one
12. Unit for glass distilled water
one
13. W chamber for genetics experiments
one
14. Heating mantle
one
15. pH meter
one
16. Medical centrihge
one
17. Laboratory hot plate
one
18. Chemical balance
one
19. Antibiotic zone diameter reader
one
20. Colony counter
one
21. Phase contrast microscope
one
22. Laboratory fermenter
one
Appendix-2
Addresses for getting chemicals, instruments, readyrnade media and cultures required for
microbiology practicals
1. Readymade media
* Himedia Laborat0riePs vt. Ltd.
A-406, Bhaveshwar Plaza,
235 Marg, Mumbai-400 086, India
* LOBA Chemie Pvt. Ltd.
78/80, Babu Genu Road,
P.B. No. 2042
Mumbai-400 002.
2. Chemicals
Poona Chemical Laboratory
207, Magalwar Peth,
Near Gadital
Behind Modi Petrol pump,
Pune - 411011.
S.D. Fine Chen Ltd.
3 15 H.O., T.V. Ind. Estate,
248 Worli Road, Post Box No. 19160
Mumbai-400 030.
Glaxo India Limited
Dr. Annie Besant Road,
Mumbai-400025.
Ranbaxy Fine Chemical Division
12th Floor, Derika Towers 6, Nehru Place
New Delhi-110019.
Borosil Glass works Ltd.
4031404, Kaliandas Udyog Bhavan.
Near Century Bazar, Worli Murnbai-400025
Superfit Continental Private Ltd.
5-6, Old sitaram Building 1st floor.
204 Princess Street, Mumbai-400 002.
Qualigens Fine Chemicals Division.
Glaxo SmithKline Pharmaceuticals Ltd.
Dr.Annie Besant Road, Worli.
Mumbai-400030.
Loba Chemie Pvt. Ltd.
Jehangir Villa, 107 Wode House Road,
Colaba, Mumbai - 400005 (India).
Asgi Enterprises.
10, Dadi Santook Lane, Mumbai-2
Thomas Baker. (Chemicals) Pvt. Ltd,
Head Office.
4/86, Bharat Mahal, Marine Drive, Mumbai 400002 (India).
Universal Laboratories Pvt. Ltd.
Corporate office-507, Raheja Centre,
214, Nariman point, Mumbai-400021. (India).
Biotech Pvt Ltd.
18/1, Madhukunj Society, Panchavati,
Off Pashan Road, Pune-411008.
Bangalore Geni.
No. 6, 6th Main, BDA. Industrial Suburb,
Near SRS Road Peenya,
Bangalore-560058. (India).
3. Instruments
M/S Dynarnicro [Bacteriology Equipment]
M/S Toshniwal Brothers Pvt. Ltd.
198, Jarnshedji Tata Road,
Mumbai-400020.
M/S MICLAB Instruments
Gulmohar 'B', Flat No. 5
428-43013, Gultekadi,
Pune-411009.
M/s TEMPO Industrial Corporation
I, lamington Chambers
394, Dr. Bhadkamkar Marg,
Mumbai-400004.
M/S Ope1 Instruments Pvt. Ltd.
562, Sadashiv Peth, Laxi Road,
Bhanuvilas Chowk,
Pune-411030.
Biotron Healthcare (India) P. Ltd.
30 1, Coral classic, 20h Road.
Chembur, Mumbai400071.
Micro Devices Metrohm Ltd.
Millenium Business park,Mahape,
Navi. Mumiotrobai-400710.
Remi Equipments Ltd.
14, Shah Industrial Estate, Veera Desai Road.
Andheri (W), Mumbai 400053.
SAKSHAM Technologies Pvt.,Ltd.
502, Niti Apartments, Underai Road,
Near New Era.
Cinema, Malad(W), Mumbai-400064.
Thermo Electron Cotpration,
Laboratory Consumables & pipetting
Ratatie 2, P. 0. Box 100.
Fin-o1621 Vantaa.
Finland.
Elico Limited.
B-90, A.P.I.E. Sanathnagar,
Hyderabad-500018, A.P. India.
Millipore (India) F'vt. Ltd.
50-A. I1 Phase, Ring Road, Peenya,
Bangalore-560058, India.
Labindia Instruments Pvt. Ltd.
201, Nand chambers, L.B.S. Marg,
Near Vandana cinema, Thane-400602.
4. Microbial Cultures
Fermentation Technology Laboratory
Indian Institute of Science
Bangalore-3 (Karnataka)
National Collection of Industrial Microorganisms
National Chemical Laboratory,
Council of Scientific and Industrial Research;
Pashan Road,
Pune (Maharashtra)
5. Fungal Cultures
Herbarium Cryptogame India Orientaiks
Division of Mycology and Plant Pathology
Indian Agricultur Raels earch Institute,
New Delhi- 12, India
Microbial Type Culture Collections
I Gene Bank (MTCO) Institute of Microbial Technology
Post box No. 1304, Sector 39-A,
Chandigarh-160014, India.
Agharkar Research Institute
Bhandarkar Road,
Pune.
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Microbiology Concepts and Application, Pelczar, Chan and Krieg. McGreaw
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Microbiological Applications, Sixth edition, Harold J. Benson w.m.c. Brown
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Practical Zoology, K.C. Ghose B. Manna New Central Book Agency,
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Pollution Microbiology A Laboratory Manual, Melvin S. Finstein Marcel Dekkar
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Index
Acetate buffer, 254
1991 Acetobacter medium,
Ws Acholeplasma medium, 45-46
Acid fast staining, 196-99
t and Actinomycetes medium, 39-43
Agrobactarium isolation medium,
46-47
Alcaligenes medium, 46
Algal medium, 173-76
Alternasia medium, 145
Aminoacid decarbexylation test,
1-2
Moeller's medium, 1-2
Falkow's medium, 2
Ammonification medium, 47
Ammonium medium, 17
Amylase production test, 2-3
Anaerobic bacteria medium,
Antibiotic assay medium, 2 1-22
Ascomycetes medium, 145-46
Bordetella medium, 64-65
Brycella medium, 65-67
Burk's medium, 59
Campylobacter medium, 68
Candida medium, 149-50
Capsule staining, 199-204
Carbon utilization medium, 4 1-43
Carry-Blair medium, 138-39
Casein hydrolysis, 3
Catalose test, 35
Cellulolytic fungal medium,
150-5 1
Cellulolytic medium, 68-69
Aspergillus medium, 146-47
44-46 Azospirillum semisolid medium,
60-61
Azotobacter medium, 5740
Bacteriophage, 185-90
Bagnra, 50
Balch, 55-56
Basal medium, 25
Basdiomycets medium, 147-48
Bateroides medium, 62
Bdellovibrio medium, 62-63
Beadle and Tatum, 165-66
Becking's medium, 63
Bennet's agar, 39
Bile escutin medium, 5
Bile sensitivity test, 3-6
Bioluminescent bacteria medium,
Blood agar, 82
Blue green algal medium, 176-78
Dextrose tripone a g e 40
Dine's shining, 194-95
DNzse agar, 4
DNase test, 4-5
Durham's peptone water, 9- 10
Egg yolk agar, 11-12, 13
Eijkman text, 5
Eimhjellen medium, 87
Endo's agar, 73
Entamoeba medium, 18 1
Enteric bacteria medium, 78-79
Equipments required in
microbial lab, 295
Cell wall stairling, 214-16
Chaetomium medium, 15 1-52
Chitin hydrolyse, 3
Christensen's medium, 33-34
Citrate utilization test, 3-4
Clostridium medium, 70-72
Coaglucose test, 36-37
Coliform medium, 72-73
Corder, 54
Corynebacterium medium, 74-76
Cultivation medium, 39-190
algae, 173-80
bacteria, 39- 144
bacteriophage, 185-90
fungi, 145-72
protozoa, 1 8 1-84
Cytopha medium, 76
Deaminase test, 15- 16
Dhedrogenase activity, 38
Denitrifying bacteria medium,
77-78
Dermatophyte test medium, 152
Derxia medium, 76-77
Gram-negative broth, 85
Gross's agar, 197-99
Haemophilus medium, 86
Halophiles medium, 86-88
Hankin's medium, 20-21
Hartley's broth, 131
Hayflick medium, 106-07
Hippurate hydrolysis, 7
Homo and heterofermentation
differentiation, 6-7
Hoyer's medium, 44
Hugh and Leifson medium, 20
Hungate, 51
Hydrogen sulphide production:
Erwinia selective medium,
79-80
Esculin hydrolysis, 5
Falkow's medium, 2
Fat agar, 12-13
Filde's extract, 66-67
Flagella staining, 207-1 1
Flat sour bacteria medium,
80-8 1
Fontana's staining, 204-05
Fungal medium, 153-61
Fusarium medium, 16 1-64
Fuibacterium medium, 8 1
Garlic abr, 169
Gelatin agar, 5-6
Gelatin hydrolysis, 5-6
General medium, 82-85
Gluconate test, 6
Glucose peptone agar, 114
Glucose phosphate broth, 34
Glycerol yeast extract agar, 40
Gram's staining, 224-29
Lactate agar, 11
Lactate fermentation, 11
Lactic acid bacteria medium,
88-89
Lactobacilli medium, 89-90
Lapage, 49
Lecithinase production, 11 - 12
Leishmania medium, 183-84
Leptospira medium, 9 1-93
Lignolytic fungi medium, 164-65
Lipid staining, 213- 14
Lipolysis test, 12-13
Litmus milk medium, 13-14
Litmus milk test, 13-14
Lysozyme broth, 10
Kligler's iron agar, 8-9
lysine iron agar, 9
pepton water, 8
SIM agar, 7-8
Hydrolysis of fats, 38
India ink staining method, 204
Indole test, 9-10
International atomic weights of
elements, 265-66
Iron haematomylin staining, 239
Jensen's medium, 60
Katznelson and base medium, 24
Kauffmann-Muller broth, 120
Kelton medium, 105
King, Ward and Raney's medium,
112-13 Motility agar,
Kligler's iron agar, 8-9
Knop's solution, 151
Korthof's medium, 92
Koser's citrate broth, 3-4
Mycobacterium medium, 96-98
Mycoplasma medium, 10 1-07
Myxobacterium medium,
98-101
Nagler test, 37
Negative staining, 199; 200-04
Neisseria transport medium,
107-08
Neurospora medium, 165-66
Nitrate medium, 18- 19
Nitrate reduction, 18- 19
Nitrification test, 17- 18
Nitrosomonasm edium, 108
Noren's medium, 100
Normality, 264
Lysozyme sensitivity test, 10
MacConkey's broth, 5, 79
Malonate utilization and
phenylalanine, 15- 16
Malonate utilization test, 14-1 5
Mandel and Reese medium, 69
Media for biochemical test, 1-38
Melanine production, 14
Metachromatic granules staining,
211-12
Methyl red test, 16
Milk agar, 3
Minimal medium, 94-95
Moeller's medium, 1-2
Molality, 263-64
Molarity, 263
Monsur's medium, 139 i
16
Motility and nitrate reduction test,
17
MR-VP broth, 16
Mucor-synthetic medium, 155
Pikovskaya's medium, 24-25
Plant tissue culture medium,
142-43
Pneumococcal medium, 110
Positive stainingonethods,
199-200
Potassium cyanide test, 25
Potato-carrot agar, 41
PPM (Parts Per Minute), 264
Pseudomonas medium, 111 - 13
Reagents, 244-62
buffers, 254-62
fixatives, 244-47
IMVIC test, 249
other, 249-5 1
Nucluear staining, 222-23
salines, 247-49
Oatmeal agar, 40-41
ONPG test, 19-20
Optochin test, 37
Oxidase negative, 35-36
Oxidation fermentation test, 20
Rhizobium medium, 113-14
Rhizoctonia agar, 169
Robertson's broth, 47-48
Rue's staining, 206
Rumen bacteria medium, 1 15- 16
Salmonella medium, 116-24
Seedlings medium, 143-44
Sijpesteijn, 52
Simmon's citrate medium, 4
Simple staining, 191-96
Sloppy agar medium, 103
Snyder test, 30-31
Sodium chloride tolerance test,
25-26
Sodium hippurate broth, 7
Soil microorganisms medium,
124-25
Solenite broth, 121
Sowers, 53
Spirochete medium, 125-26
6-7
Transmittaney and optical density:.
relation between, 268-70
Transport medium, 104
Treponema medium, 136
Trichomonas medium, 181-83
Triple sugar-iron agar, 32-33
Tryptone nitrate broth, 18
TSI agar test, 32-33
Tween pliospliate buffered
substrate medium, 11
Tyrosine agar, 14
Pathogen preservation medium,
108-09
Pectin hydrolysis, 20-21
Penassay seed agar, 22
Penicillin assay, 2 1-22
Penicillin medium, 167-68
Phenolphthalein phosphate agar,
23
Phenylalanine deaminase test, 23
Phormidium agar, 179-80
Photosynthetic bacteria medium,
109-10
Phsphate solubilization, 24-25
Phsphotase production, 23
Spore staining, 216- 19
Sprirochete staining, 204-07
Staining methods, 191-243
bacteria, 191-235
fungi, 235-38
protozoa, 239-43
Staining virus elementary bodies.
229-33
Staining virus inclusion bodies,
233-35
Standard solution, 263-94
Staphylococcus medium, 126-30
Starch agar, 2-3
Starcll hydrolysis. 250-54
Streptococcus medium, 130 - 33
Sucrose gelatin agar, 90
Sugar fermentation medium,
Urea hydrolysis. 33-34
Viability staining, 223-24
Vibrio medium, 137-42
26-29
Sugar fermentation, 26-29
Sulfatase test, 29-30
Sulphate reducing bacteria
medium, 133-35
Sulphur oxidizing bacteria
medium, 135-36
Synthetic agar, 42
Temperature and pressure:
relation, 270-71
Terathionate broth, 122
Thiocyanate utilization test,
31-32
Todol-Hewit broth, 133
Tomato juice agar, 90
Tomato juice-gelatin medium,
Vogel-Johnson agar, 129
Voges Proskauer test, 34
Wayne's sulfatase medium,
29-30
Wilson and Blair's medium,
123-24
Xanthine decomposition, 34-35
Xanthomonas medium, 142
Xanthine agar, 34
Yeast extract-malt extract agar,
43
Yeast glucose agar, 42-43
Yeast medium, 17 1-72
Ziehl-Neelsen staining, 176-97