I. Methods for Cultivation of the Protista
The culture of protista has become commonplace.
As a consequence, it is often possible to acquire either
cultures or culture methods which are "tried and true."
Many of the methods date back to Leeuwenhoek, and
when one examines the compilation of culture methods
by Provasoli (J. Protozool. 1958. 5:1-38), it is reassuring
to know that these organisms have not changed their
tastes in either 40 or 300 years.
In initial attempts of cultivation it is essential to
have a goal in mind. Just what kind of culture is
necessary for the study to be initiated, and what
success has preceded your effort? For studies of
morphology and morphogenesis, a monoxenic or even
agnotobiotic culture may be adequate. On the other
hand, the presence of another organism can confound
the results of a biochemical study. Here an axenic or
defined medium culture may be required.
To wean an organism from its natural environment
onto one of laboratory origin, it is usually advisable to
make this a gradual process. Attention to the food
source (i.e. examination of food vacuoles) will be
helpful in attempts of cultivation. On occasion, it may
be possible to isolate organisms directly to an axenic
medium. This procedure may fail for the organism is
not allowed a period of accommodation which is often
required for domestication of laboratory strains. It is
wise to take note of the temperature and pH of the
collecting site and, if possible, to bring back water
with which culturing can begin.
The goal of a culture is usually to reduce the
complex ecological environment to a series of
reproducible laboratory items that will faithfully
support the growth and development of the organism.
Further refinements may then be required to provide
for your experiments. The progressive refinement of
culture conditions includes switching from the water of
collection to defined synthetic salt solutions and, in
the case of phagotrophic organisms, from an undefined
diet to a specific one (i.e. specific strains of bacteria or
organic soup). While it is not possible to reproduce
here all successful recipes, general methods which
have experienced repeated successful use are
presented below. These fall into four general
categories:
A. Salt Solutions
B. Infusions
C. Organic Media
D. Strictly Defined Media
A. Salt Solutions
The salt solutions to be found below can be made, in
large part, from the following stock solutions:
Compound
CaCl2. 2H2O
Amount Stock
0.37gm/250ml
Final [C]
0.01 M
Ca(NO3) 2. 4H2O
2.36gm/100ml
0.1 M
KCl
0.75gm/100ml
0.1 M
K 2HPO4
4.35gm/250ml
0.1 M
MgSO4 (anhyd)
1.20gm/100ml
0.1 M
NaCl
5.84gm/100ml
1.0 M
NaNO3
8.50gm/100ml
1.0 M
Additional solutions, where required, are indicated
with each recipe and are also included in the table
found at the end of this section.
1. Modified Bristol's Medium (MBM) - This medium
will support the growth of photoautotrophic green
algae. It can be used in the liquid state or solidified
with 1.5-2% agar. For photoauxotrophic species,
MBM can be supplemented with vitamins, C-source.
Compound
NaNO3
CaCl2. 2H2O
Amount Stock
2.90 ml (1.0M)
Final [C]
2.94mM
17.00 ml (0.01M)
0.17mM
MgSO4 (anhyd)
3.00 ml (0.1M)
0.30mM
K 2HPO4
4.30 ml (0.1M)
0.43mM
K H2PO4
1.30 ml (1.0M)
1.30 mM
NaCl
0.43 ml (1.0M)
0.43mM
Microelements
see below
Distilled water to
1000 ml
Add 1.0 ml of each of the following microelement
stock solutions:
EDTA - 50.0 gm EDTA plus 31.0g KOH per liter of
distilled water
Fe - 4.98 gm FeSO4. 7H2O per liter of acidified
distilled water (acidify H2O by adding 1.0ml
conc. H2SO4 per liter)
Boron - 11.42gm H3B O3 per liter
H5 8.82gm ZnSO4. 7H2O per liter
1.44gm MnCl2. 4H2O per liter
0.71gm MoO3 per liter
1.57gm CuSO4. 5H2O per liter, and
0.49gm Co(NO3) 2. 6H2O per liter of acidified
water
Microelement stock solutions may be kept
refrigerated in polyethylene dropping bottles
fitted with droppers calibrated to deliver 1.0 ml.
Draft #2c - 4/04 - 1A
2. Beijerinck's Medium
Compound
Amount Stock
N H4NO3
6.25 ml (1.0M)
K 2HPO4
11.50 ml (0.1M)
MgSO4 (anhyd) 8.10 ml (0.1M)
CaCl2. 2H2O
68.0 ml (0.01M)
Final [C]
6.25mM
1.15 mM
0.81 mM
0.68 mM
Distilled water to
1000 ml
Note: Dilute phosphate separately in some of the
water and remaining components in remainder of the
water. Combine these two solutions after autoclaving
to produce Beijernick's medium.
Modifications: Suggested additions for growing
Chlamydomonas in dark: 0.5% yeast extract and 0.2%
sodium acetate. To obtain solid medium add 1.5%
agar. M. B. Allen: Add 0.2% glucose . (autoclaved
separately) and 0.1% beef extract.
3. Zender's Cyanophycean Medium
Compound
K 2HPO4
N a2CO3
NaNO3
Ca(NO3) 2. 4H2O
Amount Stock
1.8 ml (0.1M)
2.0 ml (0.1M)
5.5 ml (1.0M)
Final [C]
0.18mM
0.20 mM
5.5mM
2.5 ml (0.1M)
0.25mM
MgSO4 + microelements 1.0 ml (see below)
Distilled water to
1000 ml
MgSO4 + microelements (1.0 ml) is prepared as a
100ml stock solution by taking 1.2g MgSO4 (anhyd.)
and bringing the volume to 100 mls with 8 mls soln. a
below, 50 mls soln. b below, and 42 mls distilled H2O.
a.
H 3B O3
MnSO4.4H2O
N a2W O4. 3H2O
3100 mg
2230mg
33mg
( N H4) 6Mo7O24. 4H2O
KBr
KI
ZnSO4. 7H2O
Cd(NO3) 2. 4H2O
88mg
119mg
83mg
287mg
Co(NO3) 2. 6H2O
146mg
CuSO4. 5H2O
125mg
132mg
NiSO4.6H2O
Cr(NO3) 3. 7H2O
VoSO4. 5H2O
AlK(SO4) . 12H2O
Distilled water to
154mg
37mg
24mg
326mg
1000 ml
b.
10 mls 0.1M FeCl3 in 0.1N HCl
10 mls 0.1M Na2 EDTA
30 mls H2O
1.5% agar may be added to Zender's medium prior to
autoclaving.
4. Chalkley Solution
Compound
Amount Stock
Final [C]
NaCl
1.7 ml (1.0M)
1.70mM
KCl
0.54 ml (0.1M)
0.054mM
.
CaCl2 2H2O
5.4 ml (0.10M)
0.054mM
Distilled water to
1000 ml
Note: For use, add one grain of dry-sterilized,
polished rice to each 50 ml of medium. Replenish by
adding rice every two weeks.
5. Osterhout's Solution
Compound
Amount Stock
NaCl
1.8 ml (1.0M)
MgCl2. 6H2O
0.9 ml (0.1M)
KCl
0.31 ml (0.1M)
MgSO4 (anhyd) 0.33 ml (0.1M)
CaCl2. 2H2O
0.90 ml (0.01M)
Final [C]
1.80 mM
0.091 mM
0.031 mM
0.033 mM
0.009 mM
Distilled water to
1000 ml
pH may be adjusted with NaOH or Na2HPO4.
6. Prescott's Solution
Compound
Amount Stock
Final [C]
MgSO4 (anhyd) 0.11 ml (0.1M)
0.011 mM
KCl
0.22 ml (0.1M)
0.022 mM
CaCl2. 2H2O
3.0 ml (0.01M)
0.030 mM
K 2HPO4
0.3 ml (0.1M)
0.030 mM
Distilled water to
1000 ml
For use with cultures of Amoeba and Chaos: 2.0% agar,
made with Prescott or Pringsheim soln., is melted and
dispensed into finger bowls to a depth of 3-5 mm.
While still soft, 3-4 rice grains are embedded in the
agar layer. After the base layer solidifies, Prescott
soln. is added to a depth of one inch. Amoebae and
food organisms are added.
7. Modified Pringsheim's Solution
Compound
Amount Stock
Ca(NO3) 2. 4H2O 8.5 ml (0.1M)
KCl
MgSO4 (anhyd)
N a2HPO4. 7H2O
*FeSO4. 7H2O
Final [C]
3.5 ml (0.1M)
0.81 ml (0.1M)
0.85 mM
0.35 mM
0.081 mM
1.1 ml (0.1M)
0.11 mM
1.4 ml (0.01M)
0.014 mM
Distilled water to
1000 ml
Note: *If desired to include chelator, one can keep as a
Draft #2c - 4/04 - 2A
stock soln. 278 mg this salt + 0.5g EDTA-Na2 in 100 ml
dist. H2O.
8. 0.01% Knop's Solution
Compound
Amount Stock
Final [C]
Ca(NO3) 2. 4H2O
2.5 ml (0.1M)
0.25 mM
KNO3
1.4 ml (0.1M)
0.14 mM
MgSO4 (anhyd)
0.58 ml (0.1M)
0.058 mM
K 2HPO4
0.82 ml (0.1M)
0.082 mM
Distilled water to
1000 ml
Note: Add K2HPO4 drop by drop while shaking. One
drop of 0.1% FeCl2 may be added if needed. Knop's
solution is successfully used at a variety of
concentrations from 0.01% to 1%.
9. Artificial Spring Water
Compound
Amount Stock
NaCl
0.12 gm
.
N a2SiO3 9H2O
0.015 gm
N a2SO4
0.0006 gm
CaCl2. 2H2O
0.0065 gm
MgCl2. 6H2O
FeCl3. 6H2O
Final [C]
2.05 mM
0.052 mM
0.042 mM
0.044mM
0.0035 gm
0.017mM
0.005 gm
0.018mM
Distilled water to
1000 ml
Note: Adjust with NaOH or HCl to final pH of about
7.0.
Stocks: The silicate may be made up at 100X its final
concentration; the remaining compounds, exclusive of
FeCl3, may be made up together at 200X their final
concentration.
10. Ott's Artificial Seawater - Prepare stock
solutions listed below by dissolving each salt in
distilled or deionized water to the indicated
concentration. To approximately 700 ml of glassdistilled water add the indicated amounts of stock
solutions. To this mixture add 1 ml of each of the
EDTA, iron, and boron solutions listed under Modified
Bristol's Medium. Then bring the volume up to 1 liter
with glass-distilled water. The commercial products
Aquamarine or Instant Ocean are generally a more
convenient and a satisfactory source of artificial
seawater. Density approximately 1.025 and pH 7.7.
Compound
NaCl
MgSO4. 7H2O
MgCl2. 6H2O
KCl
CaCl2. 2H2O
Soln (%)
25
10
10
10
10
Amt.
(ml)
85
60
50
8
10
Final
[C]
363.0mM
24.4 mM
24.6 mM
10.7mM
6.8mM
NaHCO3
NaNO3
NaBr
H 3B O3
Sr(NO3) 2
N a2HPO4 (anhyd.)
N a2SiO3. 9H2O
1
1
1
1
1
1
1
Microelements (see above)
Distilled water to
1000
20
20
10
6
3
2
1
1 (each)
2.38mM
2.35mM
0.97mM
0.97mM
0.14mM
0.14mM
0.035mM
B. Infusions
Infusions or aqueous extracts of different types of
materials can provide the nutrients necessary either
for axenic growth of an organism or to support the food
organisms upon which the protistan of choice will
feed. There is an endless list of such infusions. We
provide here some in common use.
1. Pringsheim's Biphasic Soil-Water Medium (SW)
Probably the most useful and universally applicable
technique for maintaining unialgal cultures of a wide
range of freshwater algae is the soil-water culture
method of Pringsheim. A small quantity of calcium
carbonate (CaCO3) (approximately as much as the tip
of a scalpel full per a tube) is placed at the bottom of
the culture tube and covered with 1/4 to 1/2 inch of
moist garden soil. The tube is filled three-quarters
full with deionized or glass-distilled water. If the
soil is infertile, MBM may be used in place of tap or
distilled water. The culture vessel may be stoppered
loosely with cotton or covered with a small piece of
glass. The culture vessels are then steamed (without
pressure) for one hour at 100oC. on each of three
successive days. Sufficient calcium carbonate should
have been added so that it remains visible under the
soil when the steaming process has been completed.
Culture vessels may be inoculated as soon as they
have cooled sufficiently, but better results are usually
obtained if they are first stored under refrigeration
and allowed to clear by settling. Several
modifications of the soil before steaming will support
vigorous growth of certain auxotrophic or
heterotrophic organisms (Euglena, Trachelomonas,
etc.).
2. Soil-Extract Concentrate (SE) -- for use in other
media (e.g., no 3 below).
Compound
Amount
Soil (selected garden) 1000gm
Distilled water
1000 ml
Note: 1) Mix water and soil.
2) Add NaOH to pH 8-9.
Draft #2c - 4/04 - 3A
3) Autoclave at 15 lbs. pressure for 1 hour.
4) Allow to settle.
5) Decant and store in refrigerator; or filter
through Buchner funnel.
6) At time of use the above concentration is
diluted and may be supplemented with
organic and organic substances. One such
formulation is:
Soil-extract concentrate 50ml
0.1M KNO3
24.8ml
Distilled water to
1000ml
*Soil should be carefully selected. Avoid excessive
organic content and/or clay. Different samples may
not give comparable results. Use large container for
autoclaving soil-water mixture as it tends to boil over.
Soil-extract concentrate can be re-autoclaved.
3. Foyn's Erdschreiber Medium (FES) -for marine
protista (forams, etc.)
Compound
Amount Stock
KNO3
10.ml (0.1M)
N a2HPO4
0.56 ml (0.1M)
Soil extract (see 2)
50.0 ml
Sea water to
1000 ml
Final [C]
1.0mM
0.056 mM
5%
Final pH = 7.7 A wide variety of marine
photoauxotrophs have been grown in FES
supplemented with vitamins.
Vitamin mix:
Biotin
0.1 mg
B12
0.1 mg
Thiamine HCl
20.0mg
Distilled H2O to 100.0 ml
Prepare and then refrigerate a stock solution in
distilled water. This solution may be sterilized by
autoclaving. Use 1 ml of this stock solution per liter of
FES.
4. Fertilizer Medium from Carolina Biological
Supply - Use commercial fertilizer of the formula 410-4 or 5-10-5. Put 1 gm in a liter of spring water and
heat to 80-90 Co. Filter through filter paper while
hot and pour into finger bowls. When cool, inoculate
and place in a well-lighted window (we use a west or
north window). This medium is used for
chlorophyllous flagellates such as Chlamydomonas,
Pandorina, and Gonium.
5. Fishmeal Medium from Carolina Biological
Supply - Use commercial fishmeal which consists
entirely of the pulverized remains of dead fishes.
Add 0.2 gm of fishmeal to one liter of spring water and
heat to 80 to 90 deg. C. Filter through filter paper
while hot, add 1/2 cc. of a Vol solution of FeCl3 ?
6H2O to the filtrate and shake well.
Pour into culture containers. When cool, inoculate
and place in a well-lighted window. This medium is
especially useful for the culture of Volvox sp.
6. Hay Infusion - Hay yields nutrients and growth
substances which are suitable for development of
many protista. Timothy hay is regarded as the best
type, but dried grasses of other kinds are also suitable.
Care should be taken not to use grasses contaminated
by insecticides and herbicides. For a hay infusion, up
to 5 gm of hay to a liter of water can be used. If other
substances are to be added to the infusion (as wheat),
the amount of hay should probably be reduced. An
infusion favorable for many ciliates is prepared from
1-2 gm of hay to a liter of water. As the nutrient
properties of the medium decline, supplements may be
added. A hay infusion may be made up much more
concentrated than this (as in amount of ten gm to a
liter), autoclaved and kept sterile, and diluted before
use.
For observation of the succession of miscellaneous
protistan populations in a hay infusion the following
procedure may be used:
Boil 2 gm of hay in water and add enough water
(distilled, ionized, or natural) to make 500 cc. Put in a
dish or jar in the bottom of which is a 1/16" thin layer
of rich garden soil. The hay should be left in the
fluid. Keep covered. The cultures may be seeded with
a small amount of wild culture. Examine the infusion
at intervals by taking a sample from the surface and
another from the bottom. For a surface sample a cover
glass may be floated overnight. Examinations should
continue for at least 6 weeks without supplements.
7. Baked lettuce, Cerophyl, Duckweed, etc. - Dried
lettuce (dried to light tan but not burned from
thoroughly washed lettuce purchased in a grocery) 1.5
gm, or Cerophyl 1.5 gm, distilled water or balanced
salt solution 1 liter. Add slight excess of CaCO3.
Bring just to a boil, simmer on low heat for 5 - 10
minutes, filter, and place 10 ml amounts in test tubes.
Autoclave 15 minutes, 15 lbs. Cool and inoculate with
a bacterial food organism (e.g. Aerobacter cloacae).
Incubate at room temp. for 18 hours, then inoculate
with ciliate. Incubate 18-25 deg. C.
8. Wheat Kernel Infusion - Boil wheat grains in a
small amount of water for 2 or 3 minutes, not longer.
For the culture, it is best to use natural water or
purchased spring water. Add the boiled wheat
kernels to the water in the number desired. Let stand
several days before inoculating with protista. The
number of kernels to be used varies with the organism
to be maintained. The following numbers are
recommended:
Paramecium
60-70 per liter
Draft #2c - 4/04 - 4A
Stentor
Vorticella
Hypotrichs
20
20
40
9. The Carolina Wheat Medium Method Pasteurize spring water and pour while hot into
previously sterilized 2 oz. jars. Add one grain of
previously boiled wheat to each jar. When cool (room
temperature), inoculate.
The following organisms have been cultivated in
this medium: Amoeba proteus, Paramecium
multimicronucleatum, P. caudatum, P. aurelia, P.
bursaria, Stentor coeruleus, S. igneous, Vorticella,
Zoothamnium, Peranema, Chilomonas, Tetrahymena
pyriformis, Colpidium striatum, Colpoda,
Actinosphaerium, Euplotes, and Blepharisma.
10. The Carolina Hay-Wheat Medium
Combination - Pasteurize spring water and pour while
hot into previously sterilized 2 oz. jars. Add two
grains of wheat and two one inch stems of Timothy
hay which have been previously boiled. When cool,
inoculate. Use for Arcella vulgaris, A. discoidea,
Centropyxis aculeata, and Spirostomum.
11. Galigher Medium for Euglena
Compound
Amount
Rice Grains
30
Peas, split
5
Distilled water to
1000 ml
Note: Autoclave the rice and peas in the distilled
water for 20 min/15 p.s.i. Let medium cool.
Decant, and retain fluid. Replenish by adding
10grains of rice every month. (Rice grains are
sterilized by autoclaving dry for 30 min. at 15 p.s.i.
inscrew-cap tube.)
C. Organic Media
This type of medium comes in two types. Either
very rich and capable of supporting axenic growth of a
protozoan or quite dilute and used in the sense of an
infusion to support the growth of a food organism upon
which the protozoan feeds. In the former case there is
always the fear of contamination where uncontrolled
growth of the contaminant (usually bacterial or
fungal) will either destroy the culture or the
experiment in progress. It is essential to monitor axenic
cultures for contaminants. This is done by making an
agar from the media being utilized and incubating at
the culture temperature. Most contaminants may be
detected more rapidly by incubation at 370 on either
EPA or FA (see below).
1. Enriched Proteose Agar (EPA)
Compound
Amount Stock
Proteose Peptone
18.8gm
Glucose
1.9gm
Final [C]
1.88%
0.19%
Yeast Extract
0.9gm
N a2Fe - EDTA
0.028gm
Agar
20.0gm
Distilled water to
1000 ml
2. Fungal Agar (FA)
Compound
Amount Stock
Proteose Peptone
2.0gm
Glucose
20.0gm
Yeast Extract
0.9gm
Agar
20.0gm
Distilled water to
1000 ml
0.09%
0.0028%
2.0%
Final [C]
0.2%
2.0%
2.0%
2.0%
3. Peptone-Yeast Medium (PY) for Bacterized
Cultures of Small Free-Living Amoebae
Compound
Amount Stock
Final [C]
Distilled water
900. ml (at beginning)
Agar (for solid medium) 20.0gm
2.0%
Proteose-peptone
1.0gm
0.1%
Yeast extract
1.0gm
0.1%
CaCl2. 2H2O
20.0 ml (0.01M)
0.2mM
MgSO4 (anhyd)
10.0 ml (0.1M)
1.0mM
N a2HPO4. 7H2O
20.0 ml (0.1M)
10.0 mM
K H2PO4
8.0 ml (1.0M)
10.0 mM
Distilled water to
1000 ml
With each new batch of phosphate stocks, check ratio
until final pH of medium is approx. 6.5.
General Note: Prepare mixture in sequence shown here
to avoid precipitate. If desired, 0.20 mM EDTA ? 2Na
may be added to protect against ppt.
4. Peptone-Yeast-Glucose Medium (PYG) for Axenic
Acanthamoeba spp.
Compound
Amount Stock
Final [C]
Distilled water
800 ml (at beginning)
Agar (for solid medium) 20.0 gm
2.%
Proteose-peptone
20.0gm
2.%
Yeast extract
1.0gm
0.1%
oGlucose
50 ml (2.0M)
0.1M
MgSO4 (anhyd)
40 ml (0.1M)
4.0mM
.
CaCl2 2H2O
40ml (0.01M)
0.4mM
Na Citrate. 2H2O
1.0gm
.
Fe(NH4) 2(SO4) 2 6H2O 10.0ml (5mM)
N a2HPO4. 7H2O
25.0ml (0.1M)
0.1%
0.05mM
5.0mM
K H2PO4
2.5 ml (1.0M)*
5.0mM
Distilled water to
1000 ml
*Check ratio until final pH of medium is approx.
6.5.
Prepare mixture in order shown to avoid ppt.;
allow time for agar to melt at outset, or use
boilingwater to dissolve agar.
Draft #2c - 4/04 - 5A
oAutoclave separately, and add aseptically to rest
of medium.
5. Stock Algal Flagellate Medium (ATY Medium
of Pringsheim)
Compound
Amount Stock
Final [C]
.
Na acetate 3H2O
1.0gm
0.1%
Tryptone
2.0gm
0.2%
Yeast extract
1.0gm
0.1%
Distilled water to
1000 ml
6. Gastric Mucin Medium for Bacterized Parasitic
Amoebae
Compound
Amount Stock
Final [C]
Gastric mucin
3.0gm
0.3%
"Rock Salt"
5.0gm
0. 5 %
Distilled water to
1000 ml
Note: Heat to mix thoroughly; filter through
paper; autoclave at 15 lbs for 20 min, add rice starch
before inoculation of parasitic amoebae.
*The salt impurities accompanying NaCl appear
to be essential in this formulation. It is possible to
substitute a roughly equivalent amount of Dobell's
Ringer solution with NaCl, KCl, and CaCl2 present in
a ratio of 90:2:2.
7. Enriched Proteose Peptone (EPP) for Axenic
Tetrahymenine Ciliates
See #1 above (omit agar)
8. Crithidia oncopelti Medium
Compound
Amount Stock
Trypticase
5.0gm
Yeast extract
5.0gm
Liver extract
0.05 gm
Sucrose
10.0gm
Distilled water to
1000 ml
Final [C]
0.5%
0.5%
0.005%
1.0%
9. Podphrya Medium
Compound
Amount Stock
Final [C]
Theobromine
0.01 gm.
0.055 mM
Tap water to
1000 ml
Note: Suctoria grow in ring just beneath surface (screwcap tubes).
Food: Suspension of Tetrahymena pyriformis W in
distilled water. (See below). Equivalent amount of
liquid later removed to maintain constant surface
level.
Addition of methyl purines is distinctly beneficial
for growth of suctoria fed upon Tetrahymena. Above
medium and feeding permitted several transfers with
Tetrahymena suspensions before growth was seriously
hindered. Theophylline and caffeine may be used as
well as theobromine.
Tetrahymena medium (on agar surface to prepare
for suctorians)
Compound
Amount Stock
Final [C]
Proteose peptone
20.0gm
2.0%
Glucose
5.0gm
0.5%
Agar
14.0gm
1.4%
Distilled water to
1000ml
Make up in slants. Ciliates are easily washed off
slants with distilled water to make suspensions with
minimal organic nutrients and thus suitable for
Podophyra.
10. Ochromonas Medium
Compound
Amount Stock
Trypticase
2.0gm
Yeast autolysate
2.0gm
Liver conc. (1:20)
0.05gm
Sucrose
10.0gm
Glycerol
5.0ml
Distilled water to
1000 ml
Final [C]
0.2%
0.2%
0.005%
1.0%
"0.5%"
11. Soldo's C and M Sea Ciliate Media
Compound
Amount Stock
Final [C]
Sea water
700ml
Cerophyl
5gm
0.5%
Proteose peptone
10gm
1.0%
Trypticase
10gm
1.0%
Yeast Nucleic Acid
1gm
0.1%
Vitamin mix
1ml
Sea water to
1000ml
To 700 ml of sea water (d = 1.015 to 1.025 has given
best results) 5gm of powdered Cerophyl is added and
an extract prepared by bringing the mixture to a boil.
After filtering through glass wool while hot the
remaining components are added, and the medium is
adjusted to pH 7.2 with 1N NaOH prior to
autoclaving. Although this medium was initially
devised for small bacterial feeding marine ciliates, a
distilled water version would probably be successful
for isolation of fresh water beasts.
Vitamin Mix
Biotin
Calcium pantothenate
Folic Acid
Nicotinamide
Pyridoxal-HCl
Riboflavin
Draft #2c - 4/04 - 6A
Amount
0.01mgm
100.0mgm
50.0mgm
50.0mgm
50.0mgm
50.0mgm
Thiamine-HCl
150.0mgm
DI-Thioctic Acid
1.0mgm
Distilled water to
100 ml
M medium is identical with C medium (above)
except that the Cerophyl extract is replaced with 250
ml of a lipid mixture.
Lipid Mix
Amount
Asolectin
0.8gm
Cephalin
0.8gm
Tween 80
0.8gm
Distilled water to
1000ml
Note: In compounding the lipid mix, add components
while stirring at 80oC.
D. Strictly Defined Media
In the case of photoautotrophic organisms a
strictly defined medium may consist of only a salt
solution such as Modified Bristol's Medium or
Zender's Cyanophycean Medium. For
photoauxotrophs minimal substitutions to such "basal
media" are necessary. In the case of heterotrophically
growing organisms the range of necessary supplements
becomes vast. Indeed the fastidious nature of these
organisms can be seen by carefully examining the Holz
medium for Tetrahymena.
1. Chilomonas paramecium Medium
This organism is a non-exacting heterotroph that
grows in a balanced salt solution containing thiamine
(=basal medium) with the addition of a wide variety
of single organic compounds.
Basal Medium
Compound
Amount Stock
Final [C]
N H4Cl
460.0mgm
8.6mM
( N H4) 2SO4
100.0mgm
0.76mM
K 2HPO4
150.0mgm
8.6mM
MgCl2. 6H2O
22.5mgm
0.11 mM
CaCl2
FeCl3. 6H2O
2.0mgm
0.11 mM
1.6mgm
0.006 mM
Thiamine HCl
0.01mgm
Distilled water to
1000. ml
Note: This formula yields pH 6.75. To obtain pH 6.0
add approximately 4.8 ml 1N HCl per 1000 ml basal
medium.
CarbonSources
Compound
Final [C]
n-Butanol
0.01M
.
Na Acetate 3H2O
0.01M
Glycerol
0.01M
Glucose
0.01M
Note: In the case of compounds whose density differs
materially from that of water, a correction must be
made in order to determine the exact volume to be
added. For example, butanol has a density of 0.81, so
that 0.915 ml of n-butanol per liter of medium yields
0.01M concentration. For convenience we keep on hand
a stock soln. of 0.5M conc. (4.575 ml n-butanol per 100
ml aq. soln.); to obtain final 0.01M conc. add 20 ml
stock soln. to 980 ml adjusted basal medium. For the
same reason 0.73 ml/liter of glycerol (density 1.26)
yields 0.01M conc.
2. Holtz Tetrahymena Medium
This medium is best described as a rich, strictly
defined medium. For a minimal or "basal medium"
refer to the Kidder and Dewey medium which has
fewer components but is sufficient to support growth of
Tetrahymena. The rate of growth and titer yield in
the Holtz medium generally surpasses that of the
Kidder and Dewey medium. The compounding of a
medium such as this (with 40 components) requires
strict attention to the cookbookery provided here.
Compound
Amount Stock
Final [C]
Amino Acids
Alanine
1.5gm
150
Arginine-HCl
3.0gm
300
.
Asparagine H 2O
2.0gm
200
Glutamic Acid
4.0gm
400
Glutamine
1.0gm
100
Glycine
4.0gm
400
Histidine-HCl. H 2O
2.0gm
200
Isoleucine
2.0gm
200
Leucine
2.0gm
200
Lysine-HCl
2.0gm
200
Methionine
1.5gm
150
Phenylalanine
1.5gm
150
Proline
2.0gm
200
Serine
1.5gm
150
Threonine
1.5gm
150
Tryptophan
1.5gm
150
Valine
1.0gm
100
Nucleic Acid Derivatives
Guanosine
0.6gm
Uracil
0.4gm
Vitamin Mix
10.0ml
CarbonSource
Glucose
100. gm
10,000
Chelator and Salts
Citric Acid. H 2O
6.0gm
600
K 2HPO4
10.0gm
1,000
MgSO4 (anhyd)
2.5gm
250
CaCO3
0.75 gm
75
Metal Mix
50.0 ml
Distilled water to
10,000 ml
This medium is "conveniently!!" compounded as a
ten liter batch made up as a 4X concentrate. In this
form it is stored refrigerated, in the dark, without
glucose and in the presence of volatile preservative (1
ml for each 100 mls to be preserved). We use an equal
mix of 1,2-dichloroethane and 1-chlorobutane as a
Draft #2c - 4/04 - 7A
preservative.
The 10L-4X concentrate is prepared by taking
1250mls of distilled water and while heating and
stirring the following are added sequentially: 1) citric
acid, 2) CaCO3, 3) MgSO4, 4) 50 mls Metal Mix, 5)
amino acids (amounts shown are for L amino acids if
DL forms are used, double the amount), 6) nucleic acid
derivatives (first dissolved in 50 mls H2O w/ KOH
pellets added until soluble), 7) K2HPO4, 8) H2O to
near final volume, 9) 10 mls Vitamin Mix, 10) adjust
pH to 6.8 with KOH pellets, 11) H2O to final volume
of 2.5L.
In preparation of the final medium a small volume
is allowed for the addition of glucose which is
autoclaved separately with 1 drop of concentrated
HCl per 100 mls of glucose solution to help prevent
browning.
Vitamin Mix
Compound
Amount Stock
Final [C]
Na Riboflavin
1.0gm
5.0
Ca Pantothenate
0.2gm
1.0
Niacinamide
0.2gm
1.0
Thiamine-HCl
0.1 gm
0.5
Pyridoxal-HCl
0.01 gm
0.05
Pyridoxamine-2HCl
0.01 gm
0.05
Folic Acid
0.002 gm
0.01
DL 6-8 Thiocitic Acid 0.002gm
0.001
D-Biotin
0.0002 gm
0.001
Ethanol
100.ml
Distilled water to
200.ml
Note: Add Biotin as 2.0 ml from a stock solution made
up of 0.01 gm Biotin in 100 ml of 80% EtOH.
Compound
Metal Mix
Amount Stock
Final [C]
Fe(NH4) 2(SO4) 2. 6H2O
1.42gm
2.7
ZnSO4. 7H2O
MnSO4. 4H2O
0.45gm
1.0
0.16gm
0.4
CuSO4. 5H2O
Co(NO3) 2. 6H2O
0.03gm
0.08
0.05 gm
0.1
( N H4) 6Mo7O24. 4H2O
0.01gm
0.012
Distilled water to
500 ml
Note: Addition of KOH is usually necessary to put the
salts into solution.
Draft #2c - 4/04 - 8A
II. Laboratory Procedure in Microscopy
In order to properly use an optical microscope a
knowledge and understanding of both the optical laws
and their practical application is essential. The
majority of protozoa and their components lie near the
limits of light microscopy; it goes without saying that
successful microscopy is a prerequisite to any
protozoological study.
A. Refraction of Light
As light passes any boundary where there is a
change in the refractive index of the medium, there
will be either a refraction or a reflection of light
"rays."
Comparative Refractive Indices: (Where medium 1 is
a vacuum)
a vacuum
air
distilled water
flint glass
crown glass
fluorite glass
cedarwood oil
(immersion oil)
xylene
glycerine
Euparal
(mounting media)
Histoclad
(mounting media)
1.0000
1.0003
1.33
1.62
1.52
1.43
1.515
1.50
1.47
1.48
1.54
The image A2B 2 is the final microscope image. In
reality (figure is diagrammatic) this is formed at
250mm (10 inches) from the eye and microscope
magnifications compare what one would see with the
unaided eye at this 250 mm distance as compared with
this final image.
The objective lens determines the significant
working features of the microscope in addition to its
primary role in resolving power.
Obj. focal N.A.
Mag. Wkng Dist Focal L
16mm
0.20-0.30 10X
4-8 mm
1-2 mm
4-5mm 0.65-0.85 40-50X 0.2-0.6mm
0.25-0.5mm
2mm
1.2-1.3
90-100X 0.11-0.16mm 0.1-0.2mm
C.
Lens Aberrations:
Chromatic aberrations:
The refractive index of a substance (glass) is not
equal for all wavelengths of light. The proportion in
which the refractive indices differ is known as the
dispersion of the substance.
White light will be dispersed by a simple lens and
the colored wavelengths will each have their own
focal point. This is chromatic aberration.
Different glasses, however, show differences in
dispersion, and as a consequence, compound lenses can
be formed which will adjust for differences in
dispersion. Such "doublet" lenses (called achromats)
are corrected for apple green (550nm) and other colors
paired off (ie. red with blue, orange with green, etc.).
Apochromatic objectives are a further refinement:
they correct for three colors and require the use of
paired compensating eyepieces.
In the air space between the coverslip and the
objective lens it is possible to have refraction and
reflection if a medium is not used to prevent this. The
effect of immersion oil is to collect as much
information from the specimen as is possible with the
optics provided. It aids by minimizing refraction and
reflection.
Spherical aberrations:
Spherical aberration is due to a curvature of the
lens which refracts more strongly at the periphery
than at the center. As a consequence, pencils of light
come to focus at different points. This is corrected by
forming an aplanatic lens which combines lenses of
different shape.
B. Optical Properties of Imaging Lenses
D.
In microscopy the imaging system is composed of
three elements:
1) Objective Lens - the principal magnifying lens
(the one critical to resolution).
2) Ocular Lens - the magnifying lens which enlarges
the specimen image for visualization.
3) Corneal Lens - that provided by the observer
which allows projection onto the retina and
subsequent visual scrutiny.
Remember: If it is right side up on your retina, it is
upside down in your mind.
Numerical Aperture
The numerical aperture of an objective or
condensing lens system is an indication of the
efficiency with which the lens system can collect or
transmit light. In practice, N.A. = n sin where n is the
refractive index of the medium in the space between
the lens and the object and is the axial angle made by
the most oblique pencil of light. From this definition
there comes a theoretical limit for the N.A. of a lens
system. < 90o sin <1.0, n <1.515, therefore, N.A.
<1.515. In practice the N.A. of an oil immersion lens
will be between 1.0 and 1.4. As you will see below, the
greater the light gathering power of a lens (larger
N.A.), the greater will be its resolving power.
Draft #2c - 4/04 - 9A
E.
Resolving Power (RP)
The resolving power of any optical instrument is
the minimum distance (usually given in micrometers or
microns) between two points which can be
distinguished as separate. It must be emphasized here
that although the resolving power of a microscope can
usually be determined from the manufacturer's
specifications, this is a theoretical RP and is based on
the optical formulae of the lenses (see below).
Practical RP is what you can actually resolve in the
specimen being examined. This will depend upon:
1) the characteristics of the specimen, (i.e. refractive
index of subcomponents and its preparation), 2) the
proper illumination of the specimen, and 3) adequate
magnification for the method of observation.
In the literature one finds numerous formulae for
the calculation of resolving power. As these tend to be
calculated by optical engineers, they tend to be
theoretical. That is, they are based on ideal
specimens such as Airy Discs (self luminous objects)
under ideal conditions. Biological specimens seldom
approach these standards, and the following
formulation probably gives a realistic evaluation of
resolving power under optimal circumstances.
where: = wavelength of light in nm
0.61 = a constant representing the minimum
contrast necessary for detection green light =
550nm = 0.55µm
N.A. cond. < N.A. obj.
An example of the RP of a typical student
microscope with a 0.9 NA condenser and a 1.25 NA oil
immersion objective is as follows.
RP = 0.61 x 0.55 µm
1/2 (0.9 + 1.25)
= 0.3 µm
General Precautions
It has been observed that the treatment of
microscopes varies as widely as does the treatment of
cattle. The following notes are intended as an
initiation into local practices.
The microscope should always be put away or
covered when not in use.
The microscope should be kept clean and free of
dust and dried immersion oil. Get in the habit of
thoroughly cleaning the instrument when you have
completed working with it.
For wiping immersion oil off slides and cleaning
the metal parts of the microscope, either Kimwipes or
Kleenex is very useful. Keep a box of it at the
microscope table.
Use only lens paper or Kimwipes for cleaning the
lenses and mirror (if it has one). Brush the lenses
lightly at first, to remove particles of dust and dirt.
These particles may result in scratches if rubbed
against the glass. To dissolve grease and oil on
objectives the lens paper may be moistened very
slightly with xylene. Grease on the exposed lenssurface of the eyepiece should also be removed with
lens paper.
The lens paper container should be kept covered at
all times to protect the paper from dust.
For protozoological work, use only No. 0, No. 1, or
No. 1.5 coverslips. Thicker ones will not generally
permit use of the high-power oil immersion lens on
whole-mounted specimens.
Kohler Illumination - the key to success
In order to approach the theoretical resolution of a
compound light microscope it is essential that the
specimen be adequately and properly illuminated. In
practice this means that a low voltage, high intensity
research type illuminator is used in the fashion first
described by Kohler in 1894.
The essential feature of Kohler illumination is to
provide parallel pencils of light at the specimen
plane.
where: S1
L
D
S2
I
C
PP
O
S3
= point source on lamp filament
= lamp condenser lens
= lamp diaphragm (field diaphragm)
= image of lamp source on condenser
diaphragm
= condenser iris diaphragm (= back
focal plane of condenser)
= condenser lens
= specimen plane
= objective lens
= rear focal plane of the objective lens
In practice, Kohler illumination is accomplished
by following the procedure outlined below.
Procedure for Proper Microscope Illumination
1. Alignment of optical axis (orienting the light
beam) (10 x objective).
a. Set up lamp and microscope with a T square
about six inches apart so that the primary optical
axis of the lamp bulb, mirror, and scope are exactly
aligned.
b. Remove frosted filter and close the lamp
diaphragm almost completely.
c. Adjust the angle of the lamp and the plane
surface of the mirror so that the light beam is
reflected onto the center of the stopped down substage
diaphragm. Carry out this operation and the
following one while looking into the mirror along the
Draft #2c - 4/04 - 10A
path of the light beam.
d. Focus the image of the lamp filament upon the
stopped-down substage diaphragm, by turning the
knob controlling the lamp condenser. If the image of
the filament does not fill the substage diaphragm,
your lamp is too close.
e. Replace the frosted filter and add a piece of
white paper to further reduce the light intensity.
Open the diaphragm of lamp partially.
f. Focus the low power (10X) objective upon a slide
preparation. At this time direct the reflected light
beam into the field of view by tilting the mirror
while looking through the microscope.
2.
Focusing upon light source [opening of lamp
(field) diaphragm].
a. While looking through the microscope focus
upon the edge of the lamp diaphragm by adjusting the
vertical position of the substage condenser. Open the
lamp diaphragm until its image maximally fills the
field of view.
b. The full numerical aperture of the microscope
objective can now be achieved by adjusting the
substage condenser diaphragm. Proceed as follows:
remove ocular and look down the body tube at the rear
lens of the objective; you should be able to see the
outline of the substage diaphragm. Slowly open the
substage diaphragm until its edge is just visible at the
periphery of the objective field.
3. Adjustments for Study Under Higher
Magnifications (43X & 97X objectives)
Caution: With a parfocal microscope and suitable
thin-slide preparations, other objectives can be
rotated into place without first raising the microscope
barrel. Until you are familiar with a given
microscope, however, always check this by watching
the clearance above the slide as the nosepiece is being
rotated. If the instrument is truly parfocal, objects
will remain almost in focus when objectives are
changed. It should be realized that individual
physiological differences restrict accurate
parfocalization to the average "normal" observer.
Each student must empirically determine the relative
behavior of his microscope.
a. Change to higher power objective, according to
size of object, and focus upon object.
b. Close down the lamp diaphragm and repeat #2
above.
Kohler illumination embodies three basic
principles which ensure optimal resolution (but see
below) in the light microscope. They are:
1. Axial alignment.
2. Parallel pencils of light at the specimen
plane.
3. Reduction of extraneous light which will
otherwise degrade the image.
Non-Kohler Illumination
Although Kohler illumination will provide
optimal resolving power for a specimen, it may not be
possible to observe some details of the specimen.
Staining methods increase contrast of the subject
material and allow for brightfield study with Kohler
illumination. However, it is not always possible or
expedient to utilize specific staining methods. At this
point we get to a compromise of light microscopy. In
order to increase the contrast of an object (especially
necessary for living material) several alternatives
are available. Each of those has a detrimental effect
on the resolving capabilities of the microscope but
may allow you to visualize structures otherwise
invisible by the standard methods of brightfield
microscopy described above.
Contrast achieved by:
1) Stopping down the condenser diaphragm,
2) oblique illumination or
3) phase contrast microscopy.
The first two methods can be achieved with
standard compound microscopes and in some cases
substantial loss of resolution in experienced at the
expense of increased specimen contrast.
In phase contrast microscopy a percentage of light
gathering power of the objective is given up for the
ability to differentially analyze this light. As the
light passes through parts of the specimen it is either
1) undeviated or 2) deviated due to diffraction or
retardation. By matching a phase ring in the objective
with an annular ring of illumination light, Zernike
found that it was possible to separate the undeviated
pencils from those that were deviated (diffracted).
The undeviated pencils will pass through the
objective annular ring and can be treated and
manipulated at will. In standard positive contrast
phase they are reduced in intensity by a special
aluminum coating while the deviated pencils are
retarded 1/4 wavelength by a magnesium fluoride
coating outside of the objective ring. As the pencils are
reunited at the eyepiece diaphragm we find that:
1) the undeviated pencils (background) have been
reduced in intensity, and
2) the deviated pencils have been phase shifted
1/2 wavelength. Thus the image is a destructive one
at specimen points of highly refractile material. The
total phase difference has been enhanced, and
structures previously difficult to see become evident.
Draft #2c - 4/04 - 11A
Measurement in the Microscope
Ocular micrometers are used to determine the
length of objects seen in the microscope. The ocular
micrometer consists of a scale on a small glass disc
inserted into an ocular with the scale exactly at the
level where the primary image from the objective is
formed. These devices are not interchangeable, and
furthermore, they must be calibrated separately for
each objective.
In order to measure objects with this scale it is
necessary to calibrate it for each objective with a
stage micrometer. Refer to Fig. 124 on pg. 49 of the
Figures. Place the stage micrometer on the microscope
stage and bring its scale into focus. The stage
micrometer has a scale which is usually 2 mm long
divided by 20 or 200 lines. Rotate the ocular and move
the stage micrometer until the two scales are
superimposed. Now find two places as far apart as
possible at which the lines from the two scales
exactly coincide. Determine the number of spaces on
each scale between these two points. For example,
suppose 100 divisions of the ocular micrometer
correspond to 70 divisions of the stage micrometer.
Each division of the stage micrometer represents
0.01mm and 70 divisions equal 0.7mm or 700
micrometers (microns). 700/100=7 micrometers
(microns). With this ocular and this objective each
ocular micrometer division represents 7 microns.
In the same way, calibrate the ocular micrometer
for each objective. With the higher objectives the
stage micrometer divisions appear quite thick, and
greater accuracy is obtained by reading from one edge
of the beginning line to the corresponding edge of the
line where the scales coincide.
These calibrations are accurate only when the
same instrument and lens- combinations are used. A
calibrated ocular micrometer may then be used
directly in measurement. Referring to the example
given above, an object measuring 42 divisions in length
would be 42 x 7 = 294 micrometers.
Draft #2c - 4/04 - 12A
III. Staining Methods for the Cytological Examination of the Protozoa
It is nearly impossible to put together a
comprehensive list of staining methods that are useful
for study of the protozoa. The reason for this is
twofold: 1) All staining methods have had or could
have some utility in the examination of protozoa and
2) it is impossible to predict what structures or
organisms will be examined during the course of
various studies. As a compromise we offer in this
section a variety of "tried-and-true" methods that
have been used successfully in this and other
laboratories. The words of E. A. Minchin (see preface)
should, however, be remembered in this regard.
A. Vital Stains
These stains are applied to living organisms and
stain specific structures, organelles or molecules in
living beasts. In most cases the stain is prepare as a
stable stock solution in 100% ethanol. The stock is
diluted further with ETOH; a small amount is placed
on a slide; the ETOH is allowed to evaporate, and a
drop of cells is added to the film of stain. Stain
concentration is critical to the result and can be toxic to
the organisms. Indeed, structures revealed and colors
of stained structures will depend on concentration, pH
of stain-organism solution and the organism itself.
Essentially, these are "try and determine" stains that
are quickly performed and may be revealing.
1.
2.
3.
4.
5.
6.
Acridine Orange for nucleic acids.
1% stock solution in ETOH.
Use diluted 1/10 or 1/100.
Blue fluorescence microscopy reveals green
staining of nucleus while RNP particles stain
cytoplasm red.
Bismark Brown for nuclei and cytoplasm.
1% stock solution in ETOH.
Dilute stock 1/10 to 1/1500 for use.
Brilliant Cresyl Blue for structural protein.
1% stock solution in ETOH.
Dilute stock 1/10 - 1/1000 for use.
Janus Green B for mitochondria.
1% stock solution in ETOH.
Dilute stock 1/10 to 1/1800 for use.
Neutral Red for lysosomes and food vacuoles.
1% stock solution in ETOH.
Dilute stock 1/10 to 1/1000 for use.
Tetracycline for mitochondria.
Staining solution 0.002 - 0.010%
The antibiotic tetracycline appears to have
binding affinity for bacterial and
mitochondrial ribosomes. When bound it can
be excited by near ultraviolet and fluorescent
yellow.
B. Temporary Preparations
Each of the stains listed above can be used in
higher concentrations than those recommended for
vital staining. Under these conditions the dyes are
toxic and will give intense staining although
specificities no longer hold. Organisms may be fixed
prior to staining. Again, desirable results must be
empirically determined. Here are a few other
recommended methods.
1. Dippell's All Purpose Stain
0.5% Acetocarmine
58.3 ml
45% HAc
25.0 ml
1N HCl
11.1 ml
1% Fast Green FCF (in 95% ETOH) 5.6 ml
This is a combination fixative-stain. Equal volumes
of stain and organisms are mixed. Nuclei stain - red,
cytoplasm - green and nuclear analagen - blue-green.
This method is especially useful for the identification
of cultures undergoing nuclear reorganization.
2. Lugol's Iodine
Iodine
Potassium iodide
ETOH
Distilled water
6gm
4gm
10 ml
100 ml
When added to a preparation, flagella and cilia
are stained - light brown, glycogen - reddish brown,
starch -black and nuclei become generally darkened. A
few drops of Lugol's Iodine can also be added to 70%
alcohol to remove Hg++ from organisms fixed with
mercuric chloride-containing fixatives.
3. 1% Methyl Green for nuclei
This stain will color nuclei intensely green. By prior
fixation with OsO4 vapor and 1 hour hydrolysis in 1N
HCl at 60oC, this stain may differentiate
chromosomes.
Note: During preparation of the stain it is
desirable to extract a methyl blue impurity by
repeated chloroform extraction. To the remaining
aqueous stain a 0.5% volume of glacial HAc is added.
4. Parducz's Stain for cilia and flagella
Parducz Fixative (see section C)
10% Ferric ammonium sulfate
0.5% Heidenhain’s hematoxylin (see sec. D)
Basically a hematoxylin stain, the Parducz
method provides an elegant demonstration of
Draft #2c - 4/04 - 13A
metachrony in cilia and flagella.
a. Wash cells in distilled water when possible.
b. Add 4 parts fixative to 1 part cells and fix for 15
minutes.
c. Wash repeatedly with distilled water.
d. Add FeNH4(SO)2 - 4 parts for each part of cells
and sit for 2 minutes total time.
e. Wash with distilled water.
f. Add 2-3 parts hematoxylin for each part of cells
and watch the staining develop.
5. Duboscq and Brasil's Fixative
80% isopropyl alcohol
Picric acid
Formalin
Glacial acetic acid
150 ml
1 gm
60 ml
15 ml
6. Hollande's Fixative
5. Sudan IV for fats and lipids
Sudan IV
Potassium hydroxide
70% alcohol
to saturation
2gm
100 ml
This stain is apparently only soluble in true fats,
certain lipids, and alcohol. It has a greater affinity
for fats and lipids than it does for its alcohol solvent.
Large fat globules appear red while smaller ones
appear in shades of orange. It may be preferable to
filter the stain just prior to use.
C. Fixatives
The recipes which follow are commonly used in
protozoological work.
1. Bouin's Fixative
Picric acid, saturated soln
Formalin
Glacial acetic acid
75 ml
25 ml
5 ml
2. Cajal's Fixative
Neutralized formalin
Ammonium bromide
Distilled water
15 ml
2 gm
85 ml
Note: Leave undisturbed 2-5 days before use.
Picric acid
Cupric acetate
Distilled water
Formalin
Glacial acetic acid
4.0 gm
2.5 gm
100.0 ml
10.0 ml
2.0 ml
7. Parducz's Fixative (must be freshly prepared)
Mercuric chloride, saturated soln
2% Osmium tetroxide
1 part
6 parts
8. Schaudinn's Fixative
Mercuric chloride, saturated soln
95% isopropyl alcohol
Glacial acetic acid
66 ml
33 ml
5 ml
D. Adhering Organisms
Success in the preparation of permanent slides of
protozoa is guaranteed by securing the organisms to
either glass microscope slides or coverslips. After the
beasts are adhered to these substrates they can
handled with ease and without damage. In the case of
organisms contained in high protein environments (i.e.
blood, feces) sufficient adhesive properties are
present and further technique is unnecessary. For freeliving organisms there are a variety of methods for
adhesion; form these we offer three.
3. Champy's Fixative (must be freshly prepared)
1. Mayer's Egg Albumin Affixative (MEA)
3% Potassium dichromate
1% Chromic acid
2% Osmium tetroxide
Fresh egg white
Glycerine
Sodium salicylate
7 parts
7 parts
4 parts
4. DaFano's Fixative (salinated)
Cobalt nitrate
Sodium chloride
Formalin
Distilled water
1 gm
1 gm
10 ml
90 ml
50 pts
50 pts
1 pt
Note: Egg white is whipped briefly until
homogeneous and allowed to stand until bubbles have
surfaced. Bubbles are skimmed off and glycerine and
sodium salicylate added.
Mayer's affixative is spread in a very thin film on
the coverglass or slide, allowed to dry, and
concentrated organisms are added in one drop of 85%
alcohol. After a brief interval, and without allowing
Draft #2c - 4/04 - 14A
the beasts to dry out, a drop of acid-alcohol (100 mls
95% alcohol and 1 ml conc. HCl is added to further
harden the albumin. From this point the material is
rehydrated and staining may proceed.
2. McArdle's Fried Egg
This is a simple method which has had
outstanding results with the feulgen nucleal reaction.
To one drop of concentrated organisms, one drop of 2%
OsO4 is added. Dilute Mayer's Egg Albumin (1 pt
MEA: 4 pts water) is added, 1-2 drops. The uncovered
preparation is mixed and incubated at 60oC for 1 hour.
(In variations of this method, overnight in a
dessicator at room temperature may replace the 60oC
oven.) At the end of this period the cells will be
embedded in a viscous soln. which can be acid
hydrolyzed directly for the feulgen reaction or
congealed in 95% alcohol and rehydrated for other
staining procedures.
3. Parlodion Blanket
In certain procedures (notably the protargol stain)
the reagents used have a sufficiently detergent effect
to make MEA alone insufficient to retain beasts. In
this case it is desirable to use the parlodion blanket
technique. After affixing organisms to a cover glass or
slide substrate as in #1 above, they are further
dehydrated through changes of absolute alcohol and
then methanol. They are then placed in 0.5%
parlodion (in absolute methanol) for 10 to 20 seconds.
Substrate is quickly drained and alcohol is allowed to
evaporate slightly (but not to dryness). As a haze
begins to appear on the surface the substrate is quickly
immersed in 70% isopropyl alcohol. (The use of
isopropyl alcohol is essential for success as parlodion
is insoluble in this alcohol while it is soluble to some
extent in ethanol and completely in methanol.) From
70% isopropyl alcohol specimens are rehydrated,
stained, dehydrated and mounted.
E. Generalized Staining Procedures
1. Nigrosin Relief Stain
10% Nigrosin is prepared by boiling for 30 minutes
and suction filtering upon cooling.
0.1% Formalin is added to prevent decomposition.
a. Equal amounts of cells (they may be fixed by
OsO4 vapors) and stain are mixed on a slide.
b. Allow preparation to air dry.
c. Mount coverslip if desired.
2. Gelei's Osmium-Toluidine Blue Method for cilia
and flagella.
Fixative (10 pts 2% OsO4: 1 pt formalin)
Alum-Potassium Dichromate (1 gm AlK(SO4) 2)
1% Ammonium molybdate
0.3% Toluidine blue
a. Fix cells for 1-12 hours in the refrigerator in
equal parts: organisms and freshly prepared
fixative.
b. Mount on substrate as in D above.
c. Place in alum-potassium dichromate for 1-12
hours at room temperature.
d. Rinse in distilled water.
e. Place in ammonium molybdate for 1-12 hrs.
f. Rinse 2X in distilled water.
g. Stain in toluidine blue for 2-5 minutes at 50oC. If
a red-violet ppt. appears, rinse was insufficient.
h. Place in 95% alcohol, dehydrate, clear and
mount. As toluidine blue is quickly extracted in
alcohols, these steps must be rapid.
3. Giemsa Stain for blood smears
Giemsa stain purchased in liquid from (certified)
Giemsa buffer (7 parts Na2HPO4 + 4 parts
K H2PO4)
2 gms of mixture ground in mortar are added to I
liter of distilled water)
a. Blood smears are fixed from 1/2 to 3 minutes in
methanol.
b. Rinse with distilled water.
c. Overlay smear with stain freshly prepared
from one drop of stain and 1 ml of buffer at pH 7.07.2. Stain for 30-45 minutes. (For overnight
staining add stain to buffer at 1:60 ratio).
d. Wash off stain with a jet of distilled water.
e. Air dry.
f. Mount if desired.
4. Masson's Modified Trichrome Stain
Chromotrope 2R
Fast Green FCF
Light Green SF
Phosphotungstic Acid
Glacial Acetic Acid
Distilled water
0.6 gm
0.15 gm
0.15 gm
0.7 gm
1.0 ml
100.0 ml
Note: Add 1.0 ml glacial acetic acid to dry
components; allow to "ripen" for 15 to 30 minutes
and then add water.
a. Fix in Bouin's, Hollande's or other suitable
fixatives.
b. Wash 2X in 70% alcohol.
c. Mount on substrate.
d. Stain for 2 to 8 minutes.
e. Place into acid alcohol (10ml-90% alcohol + 1
drop glacial acetic acid) for 5-10 seconds or until
stain barely runs from slide.
Draft #2c - 4/04 - 15A
f. Dehydrate, clear and mount.
5. Heidenhain's Hematoxylin for general purpose
staining.
Bouin's or Hollande's Fixative 2% Ferric
ammonium sulfate (prepared fresh from pale
violet crystals without heating)
0.5% Hematoxylin (prepared as an aqueous soln.
from a 10% "ripened" stock in 95% alcohol)
Saturated picric acid
a. Organisms are fixed in a suitable fixative.
b. Mount on substrate.
c. Rehydrate.
d. Mordant for 10 minutes or more in ferric
ammonium sulfate.
e. Wash repeatedly in distilled water.
f. Stain for 10 minutes or more in hematoxylin.
Staining time usually equals time of mordant.
g. Wash repeatedly in distilled water.
h. Differentiate in picric acid while observing
under the microscope. Stop destaining at first sign
of removal of stain from the nucleus.
i. Wash thoroughly in water, dehydrate, clear
and mount.
6. Klein's Dry Silver Stain
1 - 3% Silver nitrate
a. Spread out fluid containing the specimens in a
thin layer on a grease-free slide.
b. Air dry, neither too rapidly nor too slow
c. Cover dried film with silver nitrate. Leave 6 to
8 minutes for small ciliates.
d. Wash with distilled water. Place in clean
white dish and cover with distilled water.
e. Leave in diffuse daylight at a bright window 3
to 6 hours or under an ultraviolet lamp for 2-30
minutes. When reduction is sufficient the film
will appear rusty.
f. Rinse in distilled water, air dry and mount
7. Chatton-Lwoff Silver Impregnation for basal
bodies, parasomal sacs, cytoproct and CVP
Champy's fixative
DaFano's fixative, salinated
3% silver nitrate
salinated gelatin (see below)
a. Fix concentrated beasts for 1-5 minutes in
Champy's fixative. A truly hemisphericalbottomed embryological watch-glass, used
directly on the stage of dissecting microscope,
makes a very convenient receptacle for carrying
out early steps.
b. Replace Champy's quickly with salinated
DaFano's fixative. Change twice. Ciliates may be
left in this fluid for weeks.
c. Wash out the DaFano's with distilled water.
d. Place small concentrated drop of organisms on
very clean, grease free slide; add somewhat
smaller drops of warm (35o-40oC) salinated
gelatin in solated condition (powdered gelatin, 10
gm, sodium chloride, 0.05 gm; distilled water, 100
ml.). Mix with clean warmed needle. Quickly
withdraw excess fluid until specimens remain just
embedded in thin gelatin layer.
e. Transfer slide immediately from warm stage to
cold chamber (covered dish with moist filter
paper in bottom, temp. about 5oC). Leave until
gelatin has jelled sufficiently.
f. Place preparation in cold (5oC) solution of silver
nitrate for 10-20 minutes. Keep it cold.
g. Flush slide thoroughly with cold distilled
water and immediately submerge it to depth of 34 cm in cold distilled water in a whitebottomed
dish under a source of ultraviolet light.Good
distance of preparation from a 253nm lamp is 2030 cm. Change water over slides if it becomes
warm or cloudy. Expose to light for 10-30 minutes.
h. Remove to cold 30,50, 70% alcohol; complete
the dehydration 85% 95%, 2 changes 100%
alcohol at room temperature; several minutes in
each). Clear in xylene and mount.
8. Protargol Silver Impregnation
Hollande's or Bouin's Fixative
0.5% Potassium Permanganate
4% Oxalic Acid
1% Protargol (freshly prepared by allowing
Protargol sprinkled on surface of water to
dissolved without agitation)
1% Hydroquinone in 5% sodium sulfite
1% Gold chloride
2% Oxalic Acid
5% Sodium Thiosulfate
a. Fix in suitable fixative.
b. Wash in 70% alcohol.
c. Mount on substrate.
d. Carry the preparations to water.
e. Put into KMnO4 for 5 minutes.
f. Wash in distilled water.
g. Put into 4% oxalic acid for 5 minutes.
h. Wash well in distilled water.
i. Stain in freshly prepared Protargol solution
with copper metal added (approximately 0.5 gm
for each 10 ml of stain). With incubation at 37oC
for 24 hours as a desirable time and temperature.
j. Wash briefly in distilled water and quickly go
Draft #2c - 4/04 - 16A
to the next step.
k. Reduce in hydroquinone for 5-10 minutes, or
until cytoplasmic staining becomes apparent under
the microscope.
l. Wash several times in distilled water.
m. Place in gold chloride, 10-30 seconds.
n. Wash once in distilled water.
o. Place in 2% oxalic acid until a purplish color
appears. This should take about 3 minutes.
p. Wash well several times.
q. Place in sodium thiosulfate, 3 to 10 minutes, or
until destaining becomes apparent.
r. Wash well several times in distilled water.
s. Dehydrate, clear and mount.
9. Wessenberg's Double Silver Stain
Cajal's Fixative
1% Ammonium hydroxide (by volume)
3% Silver nitrate
Hortega Silver ammonium carbonate stock
solution:
10% AgNO3
25 ml
5% Na2CO3
75 ml
N H4OH
add drop by drop until
ppt. dissolves
Stock Reducer: 4% pyridine in formalin. 5%
Sodium thiosulfate.
a. Kill organisms by exposing to fumes of 2%OsO4
for 20 seconds.
b. Add a drop of 2% soln. of gelatin (previously
melted and cooled), mix with specimens, remove
excess and chill.
c. Fix in Cajal's fixative for 2-5 days.
d. Wash in ammonium hydroxide for 3 minutes.
e. Rinse in distilled water several minutes.
f. Stain 10-15 minutes in 3% AgNO3 plus 5 drops of
pyridine per 10 ml.
g. Rinse quickly in distilled water plus 2-3 drops
of pyridine per 10 ml.
h. Stain 10-15 minutes in dilute Hortega silver
ammonium carbonate: made by adding 2 parts of
water to 1 part of the stock solution plus 5 drops of
pyridine per 10 ml stain.
i. Rinse quickly in distilled water plus 2-3 drops of
pyridine per 10 ml.
j. Reduce 10-20 minutes in a solution made by
adding 9 parts of water to I part of the stock
reducer.
k. Rinse briefly in distilled water.
l. Place in sodium thiosulfate for 5 minutes.
m. Wash in running tapwater 5-10 minutes.
n. Dehydrate, clear and mount.
Note: It is best to stain not more than 2 cover glasses at
a time. Handle them with glass or Teflon forceps.
The fixative and the reducing solution can be used
only once, the others may be used as long as they
remain clear. Wash glassware with nitric acid.
STRUCTURES REVEALED WITH THE COMMON
METHODS OF SILVER IMPREGNATION
Klein
Macronucleus
Micronucleus
CV
*CV Pore
+
*Cytoproct
+
Trichocyst
Mucocyst
+
Basal Bodies
+
Cilia
+/Cirri
+
Membranelles
+
Undul.membrane +
Caudal Cilium
+
Parasomal sacs
+
*Ribbed wall
+
Trichite apparatus Kinetorhize
*Argyrome
+
*Used in Taxonomy
Ch-Lw Protgl Wessenbg
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+
+
+
+
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+
+
+
+/+
+
+
+
+
+
+
+
+
+
+
+
+/maybelost+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
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+
+
+
+
+
+
+
F. Specific Staining Procedures
In addition to generalized methods for staining
cellular structure, there is an array of cytochemical
methods for localizing specific macromolecular
components within cells. In some cases these methods
also allow quantitation by techniques of microspectrophotometry. We offer four such methods here.
1. Bauer's Glycogen Stain
1.5% Chromic acid in Bouin's Fixative
4% Chromic acid
Schiff's reagent (see 3 below)
Sulfurous acid (see 3 below)
1% Orange G in 90% alcohol
a. Fix in Bouin's + chromic acid.
b. Wash in 70% alcohol.
c. Mount on substrate.
d. Transfer to distilled water.
e. Transfer to chromic acid for 1 hour.
f. Wash repeatedly.
g. Stain for 15-30 minutes in Schiff's reagent.
h. Pass through 3 changes of dilute sulfurous acid,
1-2 minutes per change.
i. Wash several times.
j. Dehydrate to 85% alcohol.
k. Counterstain if desired in Orange G in 90%
alcohol.
l. Dehydrate, clear and mount.
Draft #2c - 4/04 - 17A
2. Bromphenol Blue for structural proteins
Bouin's or other non-OsO4 containing fixative
Mercury-Bromphenol Blue Reagent (Hg-BPB)
Mercuric chloride 10 gm
Bromphenol blue 0.1 gm
Distilled water
100 ml
Distilled water
10% sodium bisulfite
1N HCl
200 ml
10 ml
10 ml
1% Orange G in 90% alcohol
0.5% Acetic acid
a. Fix in Bouin's.
b. Wash in 70% alcohol.
c. Mount organisms on substrate
d. Carry to water.
e. Stain for 15 minutes in Hg-BPB.
f. Wash for 20 minutes in 0.5% acetic acid.
g. Rapidly dehydrate, clear and mount.
3. Feulgen Nucleal Reaction for DNA
Bouin's Fixative
5N Hydrochloric acid
Schiff's reagent
Distilled water
Basic Fuchsin
1N HCl
Sodium bisuIfite
Sulphurous Acid
200 ml
1 gm
20 ml
1 gm
Note: Prepare by bringing water to boil, add basic
fuchsin, stir, cool to 50oC and filter. Add HCl,
cool to room temperature and add sodium
bisulfite. Allow to stand in the dark for 12-24
hours before use. It should be straw-yellow. If red,
pink or with precipitate, discard. Keep
refrigerated in the dark and well-stoppered.
a. Fix in Bouin's or see V-D-2 above.
b. Mount on substrate if necessary.
c. Carry to distilled water.
d. Hydrolyze for 20 minutes in 5N HCl. (Time of
hydrolysis is critically dependent upon fixative.
20 minutes is for Bouin's material. If OsO4
fixatives are used this time may be as much as 5X
as long.)
e. Rinse in distilled water.
f. Stain with Schiff's reagent for 1-2 hours.
g. Wash in 3-1 minute changes of sulphurous acid.
h . Wash well in water.
i. Dehydrate to 85% alcohol.
j. Counterstain in 1% Orange G in 90% alcohol if
desired.
k. Dehydrate, clear and mount.
4. Sudan IV for Lipids.
See V-B-5 above, transfer organisms to glycerine
and mount in warm glycerine jelly.
Glycerine Jelly
Gelatin
Distilled water
Glycerine
Phenol crystals
(preservative)
8 gms
52 ml
50 ml
0.1 gm
Note: Soak gelatin in water for 2 hours, add
glycerine, stir and heat to 70oC for 30 minutes.
Draft #2c - 4/04 - 18A