unaltered.)
~Urine Gelatine.~--
1. Collect freshly passed urine in sterile flask.
2. Take the specific gravity, and, if above 1010, dilute with sterile
water until that gravity is reached.
3. Estimate (with control) at the boiling-point, and note the reaction
of the urine.
4. Weigh out gelatine, 10 per cent., and add to the urine in the flask.
5. Heat in the steamer at 100 deg. C. for one hour to dissolve the
gelatine.
6. Estimate the reaction and add sufficient caustic soda solution to
restore the reaction of the medium mass to the equivalent of the
original urine.
7. Cool to 60 deg. C. and clarify with egg as for nutrient gelatine
(_vide_ page 166).
8. Filter through papier Chardin.
9. Tube, and sterilise as for nutrient gelatine.
~Urine Gelatine (Heller).~--
1. Collect freshly passed urine in sterile flask.
2. Filter through animal charcoal to remove part of the colouring
matter.
3. Take the specific gravity, and if above 1010, dilute with sterile
water till this gravity is reached.
4. Add Witte's peptone, 1 per cent.; salt, 0.5 per cent.; gelatine, 10
per cent.
5. Heat in the steamer at 100 deg. C. for one hour, to dissolve the
gelatine, etc.
6. Add normal caustic soda solution in successive small quantities, and
test the reaction from time to time with litmus paper, until the fluid
reacts faintly alkaline.
7. Cool to 60 deg. C. and clarify with egg as for nutrient gelatine
(_vide_ page 166).
8. Filter through papier Chardin.
9. Tube, and sterilise as for nutrient gelatine.
~Urine Agar.~--
1. Collect freshly passed urine in sterile flask.
2. Take the specific gravity and if above 1010, dilute with sterile
water till this gravity is reached.
3. Weigh out 1.5 per cent. or 2 per cent. powdered agar, and add it to
the urine.
4. Heat in the steamer at 100 deg. C. for ninety minutes to dissolve the
agar.
5. Cool to 60 deg. C. and clarify with egg as for nutrient agar (_vide_
page 168).
6. Filter through papier Chardin, using the hot-water funnel.
7. Tube, and sterilise as for nutrient agar.
(Leave the reaction unaltered.)
~Serum Sugar Media (Hiss).~--
In these media the fermentation of carbohydrate substance by bacterial
action is indicated by the coagulation of the serum proteids in addition
to the production of an acid reaction.
~Serum Dextrose Water (Hiss).~--
1. Measure out into a litre flask
Serum water (See page 170) 1000 c.c.
2. Weigh out
Dextrose 10 grammes
and dissolve in the serum water.
3. Filter through Swedish filter paper.
4. Measure out and add to the medium
Litmus solution (Kahlbaum) 50 c.c.
5. Tube in quantities of 10 c.c. and sterilise in the steamer at 100
deg. C. for twenty minutes on each of three successive days.
Laevulose, galactose, maltose, lactose, etc., can be substituted in
similar amounts for dextrose and the medium completed as above.
~Omeliansky's Nutrient Fluid~ (_For Cellulose Fermenters_).--
1. Weigh out and mix
Potassium phosphate 4.0 grammes
Magnesium sulphate 2.0 grammes
Ammonium sulphate 4.0 grammes
Sodium chloride 0.25 gramme
2. Dissolve in distilled water 4000 c.c.
3. Flask in quantities of 250 c.c.
4. Weigh out and add 5 grammes precipitated chalk to each flask.
5. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
three successive days.
_Media for the Study of Chromogenic Bacteria._
~Milk Rice (Eisenberg).~--
1. Measure out nutrient bouillon, 70 c.c., and milk, 210 c.c., and mix
thoroughly.
2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with
the milk and broth mixture.
3. Fill the paste into sterile capsules, spreading it out so as to form
a layer about 0.5 cm. thick, over the bottom of each.
4. Heat over a water-bath at 100 deg. C. until the mixture solidifies.
5. Replace the lids of the capsules. Sterilise in the steamer at 100
deg. C. for thirty minutes on each of three consecutive days.
(A solid medium of the colour of _cafe au lait_ is thus produced.)
~Milk Rice (Soyka).~--
1. Measure out nutrient bouillon, 50 c.c., and milk, 150 c.c., and mix
thoroughly.
2. Weigh out rice powder, 100 grammes, and rub it up in a mortar with
the milk and broth mixture.
3. Fill the paste into sterile capsules, to form a layer over the bottom
of each.
4. Replace the lids of the capsules.
5. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
three consecutive days.
(A pure white, opaque medium is thus formed.)
_Media for the Study of Phosphorescent and Photogenic Bacteria._
~Fish Bouillon.~--
1. Weigh out herring, mackerel, or cod, 500 grammes, and place in a
large porcelain beaker (or enamelled iron pot).
2. Weigh out sodium chloride, 26.5 grammes; potassium chloride, 0.75
gramme; magnesium chloride, 3.25 grammes; and dissolve in 500 c.c.
distilled water. Add the solution to the fish in the beaker.
3. Place the beaker in a water-bath and proceed as in preparing meat
extract--i. e., heat gently at 40 deg. C. for twenty minutes, then rapidly
raise the temperature to, and maintain at, the boiling-point for ten
minutes.
4. Strain the mixture through butter muslin into a clean flask.
5. Weigh out peptone, 5 grammes, and emulsify with about 200 c.c. of the
hot fish water; incorporate thoroughly with the remainder of the fish
water in the flask.
6. Heat in the steamer at 100 deg. C. for twenty minutes to complete the
solution of the peptone.
7. Filter through Swedish filter paper.
8. When the fish bouillon is cold, if it is to be used as fluid medium,
make up to 1000 c.c. by the addition of distilled water. If, however, it
is to be used as the basis for agar or gelatine media store it in the
"Double Strength" condition.
9. Tube and sterilise as for nutrient bouillon.
As an alternative method "Marvis" fish food (16 grammes) may be
substituted for the 500 grammes of fresh fish.
~Fish Gelatine.~--
1. Measure out double strength fish bouillon, 500 c.c., into a "tared"
2-litre flask.
2. Add sheet gelatine, 100 grammes, cut into small pieces.
3. Bubble live steam through the mixture for fifteen minutes to dissolve
the gelatine.
4. Weigh the flask and its contents; adjust the weight to the calculated
figure for one litre of medium (1135.5 grammes) by the addition of
distilled water at 100 deg. C. (_vide_ page 166).
5. Cool to below 60 deg. C., and clarify with egg.
6. Filter through papier Chardin.
7. Tube, and sterilise as for nutrient gelatine.
Shake well after the final sterilisation, to aerate the medium.
~Fish Gelatine-Agar.~--
1. Weigh out powdered agar, 5 grammes, and emulsify it with 200 c.c.
double strength fish bouillon.
2. Wash the emulsion into a "tared" 2-litre flask with 300 c.c. fish
bouillon.
3. Weigh out sheet gelatine, 70 grammes, cut it into small pieces and
add it to the contents of the flask.
4. Bubble live steam through the mixture to dissolve the gelatine and
agar.
5. Weigh the flask and contents. Adjust the weight to the calculated
figure for one litre of medium (1110.5 grammes) by the addition of
distilled water at 100 deg. C. (_vide_ page 166).
6. Cool to below 60 deg. C. and clarify with egg.
7. Filter through papier Chardin.
8. Tube, and sterilise as for nutrient gelatine.
Shake well after the final sterilisation, to aerate the medium.
_Media for the Study of Yeasts and Moulds._
~Pasteur's Solution.~--
(Reaction alkaline).
1. Weigh out and mix the ash from 10 grammes of yeast; ammonium
tartrate, 10 grammes; cane sugar, 100 grammes.
2. Dissolve the mixture in distilled water, 1000 c.c.
3. Tube or flask, and sterilise as for nutrient bouillon.
~Yeast Water (Pasteur).~--
1. Weigh out pressed yeast, 75 grammes; place in a 2-litre flask and add
1000 c.c. distilled water.
2. Heat in the steamer at 100 deg. C. for thirty minutes.
3. Filter through papier Chardin.
4. Tube or flask, and sterilise as for nutrient bouillon.
~Cohn's Solution.~--
1. Weigh out and mix
Acid potassium phosphate (KH_{2}PO_{4}) 5.0 grammes
Calcium phosphate 0.5 gramme
Magnesium sulphate 5.0 grammes
Ammonium tartrate 10.0 grammes
and dissolve in
Distilled water 1000 c.c.
2. Tube, or flask and sterilise as for nutrient bouillon.
~Naegeli's Solution.~--
1. Weigh out and mix
Dibasic potassium phosphate (K_{2}HPO_{4}) 1.0 gramme
Magnesium sulphate 0.2 gramme
Calcium chloride 0.1 gramme
Ammonium tartrate 10.0 grammes
and dissolve in
Distilled water 1000 c.c.
2. Tube or flask; sterilise as for nutrient bouillon.
~Plaster-of-Paris Discs.~--
1. Take large corks, 2.5 cm. diameter, and roll a piece of stiff
note-paper round each, so that about a centimetre projects as a ridge
above the upper surface of the cork, and secure in position with a pin
(Fig. 112).
2. Mix plaster-of-Paris into a stiff paste with distilled water, and
fill each of the cork moulds with the paste.
3. When the plaster has set, remove the paper from the corks, and raise
the plaster discs.
4. Place the plaster discs on a piece of asbestos board and sterilise by
exposing in the hot-air oven to 150 deg. C. for half an hour.
[Illustration: Fig. 112.--Cork and paper mould for plaster-of-Paris
disc.]
5. Remove the sterile discs from the oven by means of sterile forceps,
place each inside a sterile capsule, and moisten with a little sterile
water.
6. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
three consecutive days.
~Gypsum Blocks (Engel and Hansen).~--
These are in the form of truncated cones and for their preparation small
tin moulds are required, each having a diameter of 5.5 cm. at the base
and 4 cm. at the truncated apex. The height (or depth) of a mould is 4.5
to 5 cm.
1. Mix powdered calcined gypsum into a stiff paste with distilled water.
2. Fill the paste into the moulds and allow it to set and dry by
exposure to air.
3. Remove the block from the mould and transfer it to a double glass
dish of adequate size (7 cm. diameter x 7 cm. high).
4. Sterilise block in its dish for one hour in the hot-air oven at
115 deg. C.
5. Carefully open the dish and add sterile distilled water to moisten
the block and form a layer in the bottom of the dish 1 cm. deep.
~Wine Must.~--(Wine must is obtained from Sicily, in hermetically sealed
tins, in a highly concentrated form--as a thick syrup--but not
sterilised.)
1. Weigh out "wine must," 200 grammes, place in a 2-litre flask and add
distilled water, 800 c.c.
2. Weigh out ammonium tartrate, 5 grammes, and add to the dilute must.
3. Place the flask in a water-bath regulated to 60 deg. C. for one hour
and incorporate the mixture thoroughly by frequent shaking.
4. Filter through papier Chardin.
5. Tube, and sterilise as for nutrient bouillon.
~Wheat Bouillon (Gasperini).~--
1. Weigh out and mix wheat flour, 150 grammes; magnesium sulphate, 0.5
gramme; potassium nitrate, 1 gramme; glucose, 15 grammes.
2. Dissolve the mixture in 1000 c.c. of water heated to 100 deg. C.
3. Filter through papier Chardin.
4. Tube, and sterilise as for nutrient bouillon.
~Bread Paste.~--
1. Grate stale bread finely on a bread-grater.
2. Distribute the crumbs in sterile Erlenmeyer flasks, sufficient to
form a layer about one centimetre thick over the bottom of each.
3. Add as much distilled water as the crumbs will soak up, but not
enough to cover the bread.
4. Plug the flasks and sterilise in the steamer at 100 deg. C. for
thirty minutes on each of _four_ consecutive days.
_Media for the Study of Parasitic Moulds._
~French Proof Agar (Sabouraud).~--
1. Weigh out Chassaing's peptone, 10 grammes, and emulsify it with 200
c.c. distilled water previously heated to 60 deg. C.
2. Weigh out powdered agar, 13 grammes, and emulsify with 200 c.c. cold
distilled water.
3. Mix the two emulsions and wash into a tared 2-litre flask with 600
c.c. distilled water.
4. Bubble live steam through the mixture for twenty minutes, to dissolve
the agar.
5. Cool to 60 deg. C. and clarify with egg as for nutrient agar (_vide_
page 168).
6. Filter through Papier Chardin, using the hot-water funnel.
7. Weigh out _French_ maltose, 40 grammes, and dissolve in the agar.
8. Tube, and sterilise as for nutrient agar.
~English Proof Agar (Blaxall).~--Substitute Witte's peptone for that of
Chassaing, and proceed as for French proof agar.
~French Mannite Agar, Sabouraud.~--(_For cultivation of Favus._)
Proceed exactly as in preparing French Proof agar _vide supra_
substituting Mannite (38 grammes) for maltose.
_Media for the Study of Milk Bacteria._
~Gelatine Agar.~--This medium is prepared by adding to nutrient gelatine
sufficient agar to ensure the solidity of the medium when incubated at
temperatures above 22 deg. C. If it is intended to employ an incubating
temperature of 30 deg. C., 10 per cent. gelatine and 0.5 per cent. agar
must be dissolved in the meat extract before the addition of the peptone
and salt; while for incubating at 37 deg. C., 12 per cent. gelatine and
0.75 per cent. agar must be used. Avoid the addition of more agar than is
absolutely necessary, otherwise the action upon the medium of such
organisms as elaborate a liquefying ferment may be retarded or
completely absent.
1. Measure out 400 c.c. double strength meat extract into a "tared"
2-litre flask, and add to it gelatine, 100 grammes.
2. Weigh out powdered agar, 5 grammes, emulsify with 100 c.c., cold
distilled water and add to the contents of the flask.
3. Dissolve the agar and gelatine by bubbling live steam through the
flask for twenty minutes.
4. Weigh out peptone, 10 grammes; salt, 5 grammes; emulsify with 100
c.c. double strength meat extract previously heated to 60 deg. C., and
add to the contents of the flask.
5. Replace in the steamer for fifteen minutes. Then adjust the weight to
the calculated figure for one litre (in this instance 1120 grammes) by
the addition of distilled water at 100 deg. C.
6. Estimate the reaction; control the result. Then add sufficient
caustic soda solution to render the reaction +10.
7. Replace in the steamer at 100 deg. C. for twenty minutes.
8. Cool to 60 deg. C. Clarify with egg as for nutrient agar.
9. Filter through papier Chardin, using the hot-water funnel.
10. Tube, and sterilise as for nutrient agar.
~Agar Gelatine (Guarniari).~--
1. Measure out double strength meat extract, 400 c.c., into a "tared"
2-litre flask, and add to it gelatine, 50 grammes.
2. Weigh out powdered agar, 3 grammes; emulsify with cold distilled
water, 50 c.c., and add to the contents of the flask.
3. Dissolve the agar and gelatine by bubbling live steam through the
flask for twenty minutes.
4. Weigh out Witte's peptone, 25 grammes; salt, 5 grammes, and emulsify
with 100 c.c. double strength meat extract previously heated to 60 deg.
C., and add to the contents of the flask.
5. Replace in the steamer for fifteen minutes.
6. Weigh the flask and make up the medium mass to the calculated figure
for one litre (1083 grammes) by the addition of distilled water at
100 deg. C.
7. Neutralise carefully to litmus paper by the successive additions of
small quantities of normal soda solution.
8. Replace in the steamer at 100 deg. C. for twenty minutes.
9. Cool to 60 deg. C. Clarify with egg as for nutrient agar.
10. Filter through papier Chardin, using the hot-water funnel.
11. Tube, and sterilise as for nutrient agar.
~Whey Gelatine.~--
1. Curdle fresh milk by warming to 60 deg. C., and adding rennet; filter
off the whey into a sterile "tared" flask.
2. Estimate and note the reaction of the whey.
3. Weigh out gelatine, 10 per cent., and add it to the whey in the
flask.
4. Bubble live steam through the mixture fifteen minutes to dissolve the
gelatine; and weigh.
5. Estimate the reaction of the medium mass; then add sufficient caustic
soda solution to restore the reaction of the medium mass (i. e., total
weight minus weight of flask) to the equivalent of the original whey.
6. Cool to 60 deg. C. and clarify with egg as for nutrient gelatine
(_vide_ page 166).
7. Filter through papier Chardin.
8. Tube, and sterilise as for nutrient gelatine.
~Whey Agar.~--
1. Curdle fresh milk by warming to 60 deg. C., and adding rennet; filter
off the whey into a sterile flask.
2. Weigh out agar, 1.5 or 2 per cent., and add it to the whey in the
flask.
3. Bubble live steam through the mixture for twenty minutes, to dissolve
the agar.
4. Cool to 60 deg. C.; clarify with egg as for nutrient agar (_vide_ page
168).
5. Filter through papier Chardin, using the hot-water funnel.
6. Tube, and sterilise as for nutrient agar.
~Litmus Whey.~--
1. Curdle fresh milk by warming to 60 deg. C. and adding rennet.
2. Filter off the whey through butter muslin into a sterile flask.
3. Neutralise to litmus by the cautious addition of citric acid solution
4 per cent. (Do not neutralise with _mineral_ acid.)
4. Heat in the steamer at 100 deg. C. for one hour to coagulate all the
proteid.
(If the whey is cloudy when removed from the steamer allow it to stand
for forty-eight hours in the ice chest and then decant off the clear
fluid--or filter through a Berkefeld filter candle.)
5. Filter into a sterile flask.
6. Tint the whey with litmus solution to a deep purple red.
7. Tube, and sterilise as for milk.
~Litmus Whey (Petruschky).~--
1. Measure out into a flask
Fresh milk 1000 c.c.
2. Add
Hydrochloric acid (or glacial acetic acid) 1.5 c.c.
and boil.
3. Filter off coagulated casein.
4. Neutralise to litmus by the addition of n/1 caustic soda solution and
boil. Whey now cloudy and acid again.
5. Again neutralise to litmus by addition of n/10 caustic soda solution.
6. Filter.
7. Tint the whey with neutral litmus solution to a deep purple colour.
8. Tube and sterilise as for milk.
~Litmus Whey Gelatine.~--
1. Measure out milk 1000 c.c. into a tared 2-litre flask.
2. Add hydrochloric acid (or glacial acetic acid) 1.5 c.c. and boil for
five minutes.
3. Filter off the casein, and make the whey faintly alkaline to litmus.
4. Weigh out
Peptone 10 grammes
and emulsify in a few cubic centimeters of the whey and return to the
flask.
5. Weigh out
Gelatine 50 grammes
add it to the whey in the flask and incorporate the mixture by bubbling
through live steam.
6. Clear with egg and filter.
7. Make the weight of the medium mass to the calculated figure for one
litre (1060 grammes) by the addition of distilled water.
8. Weigh out
Dextrose 15 grammes
and dissolve in the fluid whey gelatine.
9. Add sterile litmus solution to the required tint.
10. Tube and sterilise for twenty minutes in steamer at 100 deg. C. on
each of five successive days.
This medium will remain semi-fluid at the room temperature, and may be
used for cultures in the cool or hot incubator.
~Litmus Whey Agar~ is prepared in a similar manner to Whey Gelatine, with
the substitution of 15 grammes of agar for the gelatine.
~Malt Extract Solution (Herschell).~--
1. Measure into a flask distilled water 1000 c.c.
2. Weigh out
Extractum malti (malt extract) 25 grammes
and add to distilled water in flask.
3. Boil for five minutes, allow to stand, and decant off clear fluid
from sediment.
4. Tube and sterilise as for nutrient bouillon.
_Media for the Study of Earth Bacteria, Nitrogen Fixers._
~Earthy Salts Agar (Lipman and Brown).~--(_For the enumeration of soil
organisms._)
1. Measure out
Agar 20 grammes.
Emulsify in 200 c.c. distilled water.
2. Wash the agar emulsion into a tared 2-litre flask with 400 c.c.
distilled water.
3. Weigh out
Peptone 0.5 gramme.
Emulsify in 50 c.c. distilled water and add to the contents of the
flask.
4. Bubble live steam through the mixture for twenty minutes to dissolve
the agar.
5. Weigh out and mix
Dextrose 10.0 grammes.
Potassium phosphate 0.5 gramme.
Magnesium sulphate 0.2 gramme.
Potassium nitrate 0.06 gramme.
and add to the contents of the flask.
6. Adjust the weight of the medium mass to the calculated figure for one
litre (1025 grammes) by the addition of distilled water at 100 deg. C.
7. Titrate the medium mass and adjust the reaction to +5.
8. Cool to 60 deg. C. Clarify with egg and filter.
9. Tube in quantities of 10 c.c. and sterilise as for nutrient agar.
~Beyrinck's Solution. I.~--(_For the cultivation of nitrogen fixing
organisms._)
1. Weigh out and mix 1 gramme potassium hydrogen phosphate, 0.2 gramme
magnesium sulphate, and 0.02 gramme sodium chloride.
2. Dissolve in water 1000 c.c., in a 2-litre flask.
3. Add 1 c.c. of a one per thousand aqueous solution of ferrous
sulphate.
4. Add 1 c.c. of a one per thousand solution manganese sulphate.
5. Weigh out 20 grammes dextrose and add to the contents of the flask
(dextrose up to 40 grammes may be used for the different organisms).
6. Steam for twenty minutes, filter.
7. Tube, and sterilise as for nutrient bouillon.
~Beyrinck's Solution. II.~--(_For growth of Azobacter._)
Proceed as in preparing solution No. I, substituting mannite for
dextrose in step 5.
~Winogradsky's Solution (for Nitric Organisms).~--
1. Weigh out and mix.
Potassium phosphate 1.0 gramme
Magnesium sulphate 0.5 gramme
Calcium chloride 0.01 gramme
Sodium chloride 2.0 grammes
and dissolve in
Distilled water 1000 c.c.
2. Fill into flasks, in quantities of 20 c.c. and add to each a small
quantity of freshly washed magnesium carbonate.
3. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
three consecutive days.
4. Add to each flask containing 20 c.c. solution, 2 c.c. of a sterile 2
per cent. solution of ammonium sulphate.
5. Incubate at 37 deg. C. for forty-eight hours and eliminate any
contaminated culture flasks. Store the remainder for future use.
~Winogradsky's Solution (for Nitrous Organisms).~--
1. Weigh out and mix
Ammonium sulphate 1 gramme
Potassium sulphate 1 gramme
and dissolve in
Distilled water 1000 c.c.
2. Add 5 to 10 grammes basic magnesium carbonate, previously sterilised
by boiling.
3. Fill into flasks and sterilise, etc., as for previous solution.
~Silicate Jelly (Winogradsky).~--
1. Weigh out and mix
Ammonium sulphate 0.40 gramme
Magnesium sulphate 0.05 gramme
Calcium chloride 0.01 gramme
and dissolve in
Distilled water 50 c.c.
Label--Solution A.
2. Weigh out and mix
Potassium phosphate 0.10 gramme
Sodium carbonate 0.60 gramme
and dissolve in
Distilled water 50 c.c.
Label--Solution B.
3. Weigh out
Silicic acid 3.4 grammes
and dissolve in
Distilled water 100 c.c.
4. Pour the silicic acid solution into a large porcelain basin.
5. Mix equal quantities of the solutions A and B; then add successive
small quantities of the mixed salts to the silicic acid solution,
stirring continuously with a glass rod, until a jelly of sufficiently
firm consistence has been formed.
6. Spread a layer of this jelly over the bottom of each of several large
capsules or "plates."
7. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
three consecutive days.
_Media for the Study of Water Bacteria._
~Naehrstoff Agar (Hesse and Niedner).~--(_For enumeration of water
organisms._)
1. Weigh out: agar, 12.5 grammes and emulsify in 250 c.c. distilled
water.
2. Wash the agar emulsion into a tared 2-litre flask with a further 250
c.c. distilled water.
3. Dissolve by bubbling live steam through the mixture.
4. Emulsify Naehrstoff-Heyden (albumose) 7.5 grammes in 200 c.c. cold
distilled water and add to melted agar.
5. Adjust weight of medium mass to the calculated figure for one litre
(1020 grammes) by addition of distilled water at 100 deg. C.
6. Clarify with white of egg and filter.
7. Tube in quantities of 10 c.c. and sterilise in the steamer at 100
deg. C. for twenty minutes on each of three successive days.
~Bile Salt Broth--Double Strength.~--
1. Weigh out Witte's peptone, 40 grammes, and emulsify with 300 c.c.
distilled water previously warmed to 60 deg. C.
2. Wash the peptone emulsion into a litre flask with 600 c.c. distilled
water.
3. Weigh out sodium taurocholate, 10 grammes, and glucose, 10 grammes;
dissolve in 100 c.c. distilled water and add to the peptone emulsion in
the flask.
4. Heat in the steamer at 100 deg. C. for twenty minutes.
5. Filter through Swedish filter paper into a sterile flask.
6. Add sterile neutral litmus solution sufficient to colour the medium
to a deep purple.
7. Fill into small Erlenmeyer flasks in quantities of 25 c.c.
8. Sterilise as for nutrient bouillon.
_Media for the Study of Plant Bacteria._