Append.
APPENDIX
COMPOSITION
1. Carbon Utilization Medium
*Pridham and Gottlieb trace Salts
Solution
Ingredient
(g/l)
.2.64
CuS04,5H20
0.64
KH2PO4
2.38
FeS04,7H20
O.ll
K2HPO4
5.69
MnCl2,4H20
0.79
MgS04,7H20
1.00
ZnS04.7H20
0.15
Pridham and Gottlieb
trace salt solution*
pH
1.0 ml
3. Egg Yolk Agar
Ingredient
(%)
Ingredient
(NH4)2S04
(g/l)
4.5
10.0 g carbon source added, sterilized
separately by filtration
2. Czapeck's-Dox-Thom agar
Peptone
1.0
Glucose
0.1
Ingredient
(g/l)
NaCl
1.0
Sucrose
30.0
Yeast Extract
0.5
NaNOj
2.0
Agar
0.5
K2HPO4
I.O
pH
4.5
MgS04
0.01
Agar
20
Autoclaved at 121° C for 15 min.
Cooled to 50°C and 5.0% egg yolk
was added.
pH
4.5
208
Append.
5. Glycerol Asparagine Agar
4. Gelatin Agar
Ingredient
(g/1)
Nutrient broth
100 ml
Gelatine
5.0
Agar
2.0
Autoclaved at 121° C for 15 minutes
pH
4.5
L-Asparagine
Glycerol
K2HPO4
Trace salt's solution
Agar
pH
1.0
10.0
1.0
1.0 ml
20.0
4.5
6. Hugh-Leifson's medium (H-L test)
7. Inorganic Salt-Starch Agar
Ingredient
Ingredient
(g/1)
(g/1)
Solution I: Paste of the starch was made
with a small amount of cold distilled water
and brought to a volume of 500 ml.
Solution II:
Peptone
2.0
Ingredient
(g/500ml)
NaCl
5.0
K2HPO4 (anhydrous)
1.0
K2HP04
0.3
Agar
3.0
pH
7.2
MgS04,7H20
NaCl
(NH4)2S04
CaCOs
Trace salt solution
pH
The medium was heated to dissolve,
the components pH was adjusted to 4.5. 15 ml of
0.2% bromothymol blue and sterile glucose was
added to get a final 1 % concentration.
LO
1.0
2.0
• 2.0
1.0 ml
4.5
Starch suspension (I) and salt solution (II)
were mixed. 20.0 g of agar was added and
sterilized at 121° C for 15 minutes.
209
Append.
8. Luria -Bertani Medium (LB)
9. M9 medium
450 ml of distilled water was added to a 1
lit beaker, following amount of each
component was weighed and dissolved
them in water
Ingredient
(g/1)
Ingredient
Tryptone
NaCl
pH
10.0
5.0
7.5
(g/1)
NH4C1
1.0
€.0
NA2HP04
NaCl
0.5
3.0
KH2PO4
pH was adjusted to 4.5 with 1 N HCl.
autoclaved at 151 lbs for 20 min. and the
volume was brought to 988 ml. 1 M
MgS04, 20% glucose and IM CaClj
solutions were prepared separately in
screw cap bottles, autoclaved and
aseptically added in upper solution in
following quaintity
1 M MgS04
2.0 ml
20% glucose
10 ml
IMCaCb
100|il
10. MM9 Medium (Chrome Azurol S Assay)
Ingredients
g/1
10xMM9
KH2PO4
3
NaCl
5
NH4CI
10
Autoclaved at 15 lbs for 20 min.
CAS-HDTMA
Chrome Azurol S Assay
Water
605 mg
500 ml
210
Append.
Hexadecyltrimethylammonium bromide (HDTMA) solution (729 mg
HDTMA in 400 ml water) was added slowly.
Autoclaved
Deferrated casamino acids; 10 g casamino acids dissolved in 100 ml water. Extract
the casamino acids solution with an equal volume of SVo 8-hydroxyquinoline in
chloroform to remove contaminating iron. Extract an equal volume chloroform to
remove traces of 8-hydroxyquinoline.
Chrome Azurol S (CAS) agar
Water
750 ml
NaOH
6.0g (Dissolved in water)
Pipes
30.24
10 X MM9 medium
100 ml
Agar
15g
Autoclaved and cooled to SO^^C.
Addition of the following chemicals :
Deferrated casamino acids
20% Glucose
lMMgC12
lOOmMCaCb
Vitamin and amino acid supplemented if required
Mixed. 100 ml CAS- HDTMA solution was added. Mixed gently to avoid foaming &
Plates were poured. Colonies of siderophore producing bacteria grown on this media will
be surrounded by yellow or orange halo.
11. Medium for detection of lAA
12. Medium for detection of
pectinolytic activity
Ingredient
K2HP04
KH2P04
(NH4)2S04
MgSd4
NaCl
CaCb
Tryptophan
Glucose
0.1
0.1
0.3
0.05
0.015
Trace
0.1
2.0
Ingredient
KH2PO4
Na2HP04
Pectin
(NH4)2S04
Yeast Extract
MgS04,7H20
FeS04,7H20
CaCl2
(%)
0.4
0.6
0.5
0.2
0.1
0.2
0.000
0.000
211
Append.
Agar
1.0
Hydrolysis
zones detected
by
flooding the plates with aqueous
solution of hexadecyl trimethyl
ammonium bromide (1% w/v).
13, Medium for Nitrate Reduction Test
Ingredient
(g/1)
14. Medium for utilization of
Nitrogen Source Basal
Medium
(%)
Ingredient
Peptone
5.0
Meat Extract
3.0
1.0
KNO3
pH
4.5
Autoclaved at 121° C for 15 minutes
1.0
D-glucose
0.05
MgS04,7H20
NaCl
0.05
0.001
FeS04,7H20
0.1
K2HPO4
Agar
12
Source of Nitrogen under test
0.1 %(w/v)
15. NaCl Tolerance Medium
16. Nutrient Agar
Ingredient
Ingredient
(g/l)
100 ml
1.0
1.5-12% (w/v)
Peptone
Yeast Extract
NaCl
10.0
3.0
5.0
15 minutes
Agar
pH
20.0
7.0
Nutrient broth
Glucose
NaCl at different
concentrations
Autoclaved at 121° C for
17. Oat Meal Agar
Oat meal
Agar
20.0
18.0
20 g oat meal was cooked in 1000 ml distilled water for 20 minutes, filtered through cheese
cloth. Distilled water was added to restore volume of filtrate to 1000 ml. Trace salt solution
1.0 ml was added, pH was adjusted to 4.5 with NaOH. Agar 18 gm was added and liquefied
by steaming at 100r C for 15 to 20 minutes. Sterilized at 121° C for 15 mir
mmutes.
212
Append.
18. Peptone-Yeast Extract Iron Agar
Peptone Iron Agar
Yeast Extract
pH
36.0
2.0
4.5
Agar was liquefied by steaming at 100° C
for 15 to 20 min. Autoclaved at 121° C for 15 min.
19. Pikovaskaya's medium
20. SI agar
Ingredient
(g/1)
Ingredient
(g/1)
Glucose
Ca3(P04)2
KCl
(NH4)2S04
MgS04 7H2O
MnS04
FeS04
Yeast extract
lO.Og
5.0
0.2
0.5
0.1
trace
trace
0.5
Sucrose
Glycerol
Casamino acid
10.0
10.0
5.0
LO
1-0
2.3
1.2
Agar
pH
15
7.0 or 4.5
NaHCOa
MgS04. 7H2O
K2HPO4
Sodium lauroyl
sarcosine
Trimethoprim
Agar
pH
21. Sabouraud's Agar
20.0 mg
15.0
7.4-7.6
22. Starch Agar
Ingredient
(g/1)
Ingredient
D-Glucose
Peptone
pH
Agar
40.0
10.0
5.6
20.0
Nutrient Agar
100 ml
Starch
1.0
Soluble starch solution (10%) was
prepared in water and steam for 1
h. 20 ml of this solution was added
to 100 ml of melted nutrient agar
(%)
213
Append.
. Starch Casein Agar
Ingredient
25. Tyrosine Agar
(g/1)
Soluble Starch
10
Casein
1.0
K2HPO4
0.5
MgS04,7H20
0.5
pH
4.5
Agar
20.0
Ingredients are dissolved in
order of PO4, SO4, Starch and
Casein
24 Tributyrin agar
Ingredient
(g/1)
Glycerol
L-Tyrosine
L-Asparagine
K2HPO4 (anhydrous)
MgS04,7H20
NaCl
FeS04,7H20
15.0
0.5
1.0
0.5
0.5
0.5
0.01
Trace Salts solution*
pH
Agar
1.0 ml
4.5
20.0
Beef extract
0.1
*Trace Salts Solution water
Peptone
0.5
FeS04,7H20
0.1
2.75
1ml
MnCl2,4H20
0.1
NaCl
Tributyrin
26 Yeast Extract - Malt Extract Agar
Ingredient
(g/1)
Yeast Extract
Malt Extract
Dextrose
Agar
pH
4.0
10.0
4.0
20.0
4.5
214
Append.
REAGENTS
1.
Anmmonium molybdate and 1-amino 2-Napthol 4-SuIphonic acid (ANSA)
30g sodium metabisulphite, 6 g sodium sulphite and 500 mg ANSA dissolved
separately in small quantities of water. Combined all the solutions and made
upto 250 ml of water. Allow to stand overnight and filtered. Stored in
refrigerator in an amber colour bottle. Prepare fresh reagent after every fortnight.
2*
Barton's reagent
(A) 25 g of ammonium molybdate to be dissolved in 400 ml distilled
(B) 1.25 g of ammonium metavalanadate dissolved in 300 ml of boiling water,
cooled to room temperature and the 250 ml of concentrated HNO3 added.
Afterwords (A) and (B) solutions were mixed and made upto 1 lit.
3*
Reagents for Nitrate Reduction Test (Griess-Illosvay reagent)
Solution I: Suphanilic acid reagent
Dissolve 0.5 g in 30 ml glacial acetic acid and make upto 100 ml with
distilled water , filter through Whatman filter paper No. I.
Solution II : a-Naphthylamine Solution
0.1 gof a- Naphthylamine dissolved in 100 ml of boiling distilled water, cooled
and 30 ml of glacial acetic acid was added. For use \solution 1 and 2 were mixed
in equal amounts.
4.
Salkowaski reagent
0.05 M FeCb in 35% (v/v) perchloric acid.
215