~Carrot.~-- } are prepared tubes and sterilised in a manner
~Turnip.~-- } precisely similar to that described for potato.
~Parsnip.~-- }
~Hay Infusion.~--
1. Weigh out dried hay, 10 grammes, chop it up into fine particles and
place in a flask.
2. Add 1000 c.c. distilled water, heated to 70 deg. C.; close the flask
with a solid rubber stopper.
3. Macerate in a water-bath at 60 deg. C. for three hours.
4. Replace the stopper by a cotton-wool plug, and heat in the steamer at
100 deg. C. for one hour.
5. Filter through Swedish filter paper.
6. Tube, and sterilise as for nutrient bouillon.
~Haricot Bouillon.~--(_For cultivation of bacteria from tubercles of
Legumes._)
1. Measure out 1000 c.c. distilled water into a 2-litre flask.
2. Weigh out 250 grammes haricot beans and add to the water in the
flask.
3. Weigh out 10 grammes sodium chloride and add to the contents of the
flask.
4. Add 1 c.c. of a 1 per cent. solution of sodium bicarbonate.
5. Place in the steamer at 100 deg. C. for thirty minutes.
6. Filter.
7. Weigh out 20 grammes saccharose and add to the filtrate.
8. Tube, and sterilise as for nutrient bouillon.
~Haricot Agar.~--
1. Measure out 400 c.c. distilled water into a "tared" 2-litre flask.
2. Weigh out 15 grammes agar and mix into a thick paste with 100 c.c.
cold distilled water, and add to the flask.
3. Dissolve the agar by bubbling live steam through the mixture as in
making nutrient agar.
4. Weigh out 250 grammes haricot beans, place in the flask with the agar
mixture.
5. Add 1 c.c. of 1 per cent. aqueous solution sodium bicarbonate.
6. Weigh out 10 grammes sodium chloride and add to the contents of the
flask.
7. Place in the steamer at 100 deg. C. for thirty minutes.
8. Adjust the weight of the medium mass to 1030 grammes (the figure per
litre obtained experimentally) by the addition of distilled water at
100 deg. C.
9. Cool to 60 deg. C., clarify with egg and filter.
10. Weigh out 20 grammes saccharose and add to the contents of the
flask.
11. Tube, and sterilise as for nutrient agar.
~Wood Ash Agar.~--
1. Measure 400 c.c. distilled water into a tared 2-litre flask.
2. Weigh out 10 grammes agar and make into a thick paste with 100 c.c.
cold distilled water.
3. Add this agar paste to the distilled water in the flask.
4. Dissolve the agar by passing live steam through it, as in preparing
nutrient agar.
5. Weigh out 5 grammes clean wood ash and place in a second flask
containing 200 c.c. distilled water with some sterile glass beads: shake
thoroughly in a mechanical shaker for ten minutes.
6. Heat in steamer at 100 deg. C., for thirty minutes.
7. After removal from the steamer dry the outside of the flask
thoroughly, place it over a Bunsen flame and boil for one minute.
8. Filter directly into the flask containing the melted agar mixture.
9. Weigh out 4 grammes maltose. Add to the contents of the flask.
10. Adjust the weight of the medium mass to the calculated figure for
one litre (1019 grammes) by the addition of distilled water at 100
deg. C.
11. Replace the flask in the steamer for twenty minutes, cool to 60
deg. C., and clarify with egg and filter.
12. Tube, and sterilise as for nutrient agar.
_Media for the Study of Special Bacilli._
_B. Acnes._
~Oleic Acid Agar (Fleming).~--
1. Measure out into a sterile stout glass bottle which already contains
about 10 sterile glass beads
Ascitic fluid 250 c.c.
2. Weigh out
Oleic acid 25 grammes
and add it to the ascitic fluid in the bottle.
3. Emulsify evenly by shaking (either by hand or in a shaking machine)
for ten minutes.
4. Liquefy and measure out into a flask
Nutrient agar 750 c.c.
then cool to 55 deg. C.
5. Mix the oleic acid emulsion with the agar.
6. Add 10 c.c. sterile neutral red, 1 per cent. aqueous solution.
7. Tube in quantities of 10 c.c., slant, and allow to set.
8. Incubate for forty-eight hours at 37 deg. C. and reject any
contaminated tubes. Store the sterile tubes for future use.
_Coli-typhoid Group._
~Parietti's Bouillon.~--
1. Measure out pure hydrochloric acid, 4 c.c., and add to it carbolic
acid solution (5 per cent.), 100 c.c. Allow the solution to stand at
least a few days before use.
2. This solution is added in quantities of 0.1, 0.2. and 0.3 c.c.
(delivered by means of a sterile graduated pipette) to tubes each
containing 10 c.c. of previously sterilised nutrient bouillon (_vide_
page 163).
3. Incubate at 37 deg. C. for forty-eight hours to eliminate contaminated
tubes. Store the remainder for future use.
~Carbolised Bouillon.~--
1. Prepare nutrient bouillon (_vide_ page 163, sections 1 to 6). Measure
out 1000 c.c.
2. Weigh out carbolic acid, 1 gramme (2.5 or 5 grammes may be needed for
special purposes), and dissolve it in the medium.
3. Tube, and sterilise as for bouillon.
~Carbolised Gelatine.~--
1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). Measure
out 1000 c.c.
2. Weigh out carbolic acid, 5 grammes (= 0.5 per cent.), and dissolve it
in the gelatine.
3. Filter if necessary through papier Chardin.
4. Tube, and sterilise as for nutrient gelatine.
One or 2.5 grammes of carbolic acid (= 0.1 per cent. or 0.25 per cent.)
are occasionally used in place of the 5 grammes to meet special
requirements.
~Carbolised Agar.~--
1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out
1000 c.c.
2. Weigh out 1 gramme pure phenol and dissolve in the medium.
3. Filter if necessary through papier Chardin.
4. Tube, and sterilise as for nutrient agar.
~Litmus Gelatine.~--
1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 8).
2. Add sterile litmus solution, sufficient to tint the medium a deep
lavender colour.
3. Tube, and sterilise as for nutrient gelatine.
~Lactose Litmus Bouillon (Lakmus Molke).~--
1. Weigh out peptone, 4 grammes, and emulsify it with 200 c.c. meat
extract (_vide_ page 148), previously heated to 60 deg. C.
2. Weigh out salt, 2 grammes, and lactose, 20 grammes, and mix with the
emulsion.
3. Wash the mixture into a sterile litre flask with 200 c.c. meat
extract and add 600 c.c. distilled water.
4. Heat in the steamer at 100 deg. C. for thirty minutes, to completely
dissolve the peptone, etc.
5. _Neutralise carefully to litmus paper_ by the successive additions of
small quantities of decinormal soda solution.
6. Replace in the steamer for twenty minutes to precipitate phosphates,
etc.
7. Filter through two thicknesses of Swedish filter paper.
8. Add sterile litmus solution, sufficient to colour the medium a deep
purple.
9. Tube, and sterilise as for bouillon.
~Lactose Litmus Gelatine (Wurtz).~--
1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 4).
2. Render the reaction of the medium mass -5.
3. Replace in the steamer at 100 deg. C. for twenty minutes.
4. Clarify with egg as for gelatine.
5. Weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the
medium.
6. Filter through papier Chardin.
7. Add sufficient sterile litmus solution to colour the medium pale
lavender.
8. Tube, and sterilise as for nutrient gelatine.
~Lactose Litmus Agar (Wurtz).~--
1. Prepare nutrient agar (_vide_ page 167, sections 1 to 4).
2. Render the reaction of the medium mass -5.
3. Replace in the steamer at 100 deg. C. for twenty minutes.
4. Cool to 60 deg. C. and clarify with egg as for nutrient agar.
5. Weigh out lactose, 20 grammes (= 2 per cent.), and dissolve it in the
medium.
6. Filter through papier Chardin, using the hot-water funnel.
7. Add sterile litmus solution, sufficient to colour the medium a pale
lavender.
8. Tube, and sterilise as for nutrient agar.
~Glycerine Potato Bouillon.~--
1. Take 1 kilo of potatoes, wash thoroughly in water, peel, and grate
finely on a bread-grater.
2. Weigh the potato gratings, place them in a 2-litre flask, and add
distilled water in the proportion of 1 c.c. for every gramme weight of
potato. Allow the flask to stand in the ice-chest for twelve hours.
3. Strain the mixture through butter muslin and filter through Swedish
filter paper into a graduated cylinder. Note the amount of the filtrate.
4. Place the filtrate in a flask, add an equal quantity of distilled
water, and heat in the steam steriliser for sixty minutes.
5. Add glycerine, 4 per cent., mix thoroughly, and again filter.
6. Tube and sterilise as for nutrient bouillon.
~Potato Gelatine (Elsner).~--
1. Take 1 kilo of potatoes, wash thoroughly in water, peel, and finally
grate finely on a bread-grater.
2. Weigh the potato gratings, place them in a 2-litre flask, and add
distilled water in the proportion of 1 c.c. for every gramme weight of
potato. Allow the flask to stand in the ice-chest for twelve hours.
3. Strain the mixture through butter muslin, and filter through Swedish
filter paper into a graduated cylinder.
4. Add 15 per cent. gelatine to the potato decoction and bubble live
steam through the mixture for ten minutes.
5. Estimate the reaction; adjust the reaction of the medium mass to +25.
6. Cool the medium to below 60 deg. C.; clarify with egg as for nutrient
gelatine (_vide_ page 166).
7. Add 1 per cent. potassium iodide (powdered) to the medium.
8. Filter through papier Chardin.
9. Tube and sterilise as for nutrient gelatine.
~Aesculin Agar.~--(B. coli and allied organisms give black colonies
surrounded by black halo.)
1. Measure out 400 c.c. distilled water into a tared 2-litre flask.
2. Weigh out
Agar 15 grammes
Peptone 10 grammes
Sodium taurocholate 5 grammes
and make into a thick paste with 150 c.c. distilled water.
3. Add this paste to the distilled water in the flask.
4. Dissolve the ingredients by bubbling live steam through the mixture.
5. Weigh out
Aesculin 1.0 gramme
Ferric citrate 0.5 gramme
and dissolve in a second flask containing 100 c.c. distilled water.
6. Mix the contents of the two flasks--adjust the weight to the
calculated medium figure (in this case 1031.5 grammes) by the addition
of distilled water at 100 deg. C.
7. Clarify with egg and filter.
8. Tube and sterilise as for nutrient agar.
~Bile Salt Agar (MacConkey).~--
1. Weigh out powdered agar, 15 grammes (= 1.5. per cent.), and emulsify
with 200 c.c. _cold tap_ water.
2. Weigh out peptone, 20 grammes (= 2 per cent.), and emulsify with 200
c.c. _tap_ water previously warmed to 60 deg. C.
3. Mix the peptone and agar emulsions thoroughly.
4. Weigh out sodium taurocholate, 5 grammes (= 0.5 per cent.), dissolve
it in 300 c.c. _tap_ water, and use the solution to wash the
agar-peptone emulsion into a tared 2-litre flask.
5. Bubble live steam through the mixture for twenty minutes.
6. Adjust the weight of the medium mass to the calculated figure for one
litre (1040 grammes).
7. Cool to 60 deg. C. and clarify with egg as for nutrient agar (_vide_
page 168).
8. Filter through papier Chardin, using the hot-water funnel.
9. Weigh out lactose, 10 grammes (= 1 per cent.), and dissolve it in the
agar.
If desired, add 5 c.c. of a 1 per cent. (= 0.5 per cent.) aqueous
solution of neutral red.
10. Tube, and sterilise as for nutrient agar.
~Litmus Nutrose Agar (Drigalski-Conradi).~--
This medium should be prepared in precisely the same manner as the
Nutrose agar described on page 172 substituting meat extract for serum
water, and increasing the percentage of agar added per litre to 3 per
cent.
~Fuchsin Agar (Braun).~--
1. Liquefy and measure out into a sterile flask:
Nutrient agar 1000 c.c.
2. Weigh out: lactose 10 grammes and dissolve in the fluid agar.
3. Adjust the reaction to -5 and filter.
4. Measure out and mix thoroughly with agar:
Fuchsin, alcoholic solution 5 c.c.
The fuchsin solution is prepared by mixing:
Fuchsin (basic) 3 grammes.
Absolute alcohol 60 c.c.
Allow to stand twenty-four hours, then centrifugalise thoroughly and
decant the supernatant fluid into a well-stoppered bottle.
5. Measure out and add to the nutrient agar, sodium sulphite, 10 per
cent. aqueous solution, freshly prepared 25 c.c.
6. Tube and sterilise as for nutrient agar.
7. Store in a dark cupboard.
~Fuchsin Sulphite Agar (Endo).~--
1. Liquefy and measure out into a sterile flask:
Nutrient agar 1000 c.c.
2. Weigh out
Lactose 10 grammes.
and dissolve in the fluid agar.
3. Adjust the reaction to +3 and filter.
4. Measure out and mix thoroughly with the fluid agar.
Fuchsin, alcoholic solution (_vide supra_) 5 c.c.
5. Measure out and add to the medium
Sodium sulphite, 10 per cent. aqueous solution 25 c.c.
6. Tube and sterilise as for nutrient agar.
~Brilliant Green Agar (Conradi).~--
1. Liquefy and measure out into a sterile flask
Nutrient agar 1000 c.c.
2. Adjust reaction to +30 by the addition of normal phosphoric acid; and
filter.
3. Measure out and mix thoroughly with the fluid medium
Brilliant green (Hoechst) 1 per thousand aqueous solution 6.5 c.c.
4. Measure out and add to the medium
Picric acid (Gruebler), 1 per cent. aqueous solution 6.5 c.c.
5. Tube and sterilise as for nutrient agar.
~Brilliant Green Bile Salt Agar (Fawcus).~--
1. Weigh out agar 20 grammes and emulsify in 100 c.c. cold distilled
water.
2. Wash the emulsion into a "tared" 2-litre flask with 500 c.c.
distilled water.
3. Dissolve the agar by bubbling live steam through the flask.
4. Cool, clarify with egg and filter.
5. Weigh out
Sodium taurocholate 5 grammes
Peptone 20 grammes
and add to the medium in the flask.
6. Weigh out
Lactose 5 grammes
and add to the medium in the flask.
7. Adjust reaction to +15 and filter if necessary.
8. Measure out
Brilliant green, 1 per thousand aqueous solution 20 c.c.
and mix thoroughly with the fluid agar.
9. Measure out and add to the medium
Picric acid, 1 per cent. aqueous solution 20 c.c.
10. Tube and sterilise as for nutrient agar.
~China Green Agar (Werbitski).~--
1. Liquefy and measure out into a sterile flask
Nutrient agar 1000 c.c.
2. Adjust the reaction accurately to +13 and filter.
3. Measure out and mix thoroughly with the fluid agar
China green 0.2 per cent. aqueous solution 15 c.c.
4. Tube and sterilise as for nutrient agar.
~Malachite Green Agar (Loeffler).~--
1. Liquefy and measure out into a sterile flask
Nutrient agar 1000 c.c.
2. Weigh out
Dextrose 10 grammes.
and dissolve in nutrient agar.
3. Adjust the reaction to +3, and filter.
4. Measure out and mix thoroughly in the fluid agar
Malachite green, 0.1 per cent. aqueous solution 16 c.c.
for ~"weak"~ medium.
_4a._ To the filtered agar add
Malachite green, 2 per cent. aqueous solution 25 c.c.
for ~"strong"~ medium.
5. Tube and sterilise as for nutrient agar.
~Double Sugar Agar (Russell).~--
1. Liquefy and measure out into a sterile flask
Nutrient agar 1000 c.c.
2. Add 100 c.c. litmus solution to the fluid agar.
3. Weigh out and dissolve in the fluid agar.
Lactose 10 grammes
Dextrose 10 grammes.
4. Render the reaction of the medium neutral to litmus paper by the
cautious addition of normal caustic soda.
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.
6. Store for use in a cool dark place.
_B. Diphtheriae._
~Glycerine Blood-serum.~--
1. Prepare blood-serum as described, page 168, sections 1 to 4.
2. Add 5 per cent. pure glycerine.
3. Complete as described above for ordinary blood-serum, sections 5 to
7.
NOTE.--Different percentages of glycerine--from 4 per cent.
to 8 per cent.--are used for special purposes. Five per
cent. is that usually employed.
~Blood-serum (Loeffler).~--
1. Prepare nutrient bouillon (_vide_ page 163), using meat extract made
from veal instead of beef.
2. Add 1 per cent. glucose to the bouillon, and allow it to dissolve
completely.
3. Now add 300 c.c. clear blood-serum (_vide_ page 168, sections 1 to 4)
to every 100 c.c. of this bouillon.
4. Fill into sterile tubes and complete as for ordinary blood-serum.
~Blood-serum (Lorrain Smith).~--
1. Collect blood-serum (_vide_ page 168, sections 1 to 4), as free from
haemoglobin as possible.
2. Weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid
(or add 0.375 c.c. of dekanormal soda solution for every 100 c.c. of
serum).
3. Tube, and stiffen at 100 deg. C. in the serum inspissator.
4. Incubate at 37 deg. C. for forty-eight hours to eliminate any
contaminated tubes. Store the remainder for future use.
~Blood Serum (Councilman and Mallory).~--
1. Collect blood serum in slaughterhouse, coagulate, remove serum and
tube (_vide_ page 168).
Great care must be taken to avoid the inclusion of air bubbles--indeed
if only a few tubes are filled at one time, it is a good plan to stand
them upright in the receiver of an air pump and to exhaust as completely
as possible before transferring to the serum inspissator.
2. Heat the tubes in a slanting position in hot-air steriliser at 90 deg.
C. till firmly coagulated, say half an hour.
3. Sterilise in steam steriliser at 100 deg. C. for 20 minutes on each of
three successive days.
Resulting medium not translucent, but opaque and firm.
_B. Tuberculosis._
~Egg Medium (Lubenau).~--
This modification of Dorset's egg medium (_quod vide_ page 174) is
preferred by some for the growth of the tubercle bacillus of the human
type. It consists in the addition of one part of 6 per cent. glycerine
in normal saline solution, to the egg mixture between steps 4 and 5.
~Glycerine Bouillon.~--
1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
to 6).
2. Measure out glycerine, 60 c.c. (= 6 per cent.), and add to the
bouillon.
3. Tube, and sterilise as for bouillon.
~Glycerine Agar.~--
1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out
1000 c.c.
2. Measure out pure glycerine, 60 c.c. (= 6 per cent.), and add to the
agar.
3. Tube, and sterilise as for nutrient agar.
~Glycerine Blood-serum.~--
1. Prepare blood-serum as described, page 168, sections 1 to 4.
2. Add 5 per cent. pure glycerine.
3. Complete as described above for ordinary blood-serum, sections 5 to
7.
NOTE.--Different percentages of glycerine--from 4 per cent.
to 8 per cent.--are used for special purposes. Five per
cent. is that usually employed.
~Glycerinated Potato.~--
1. Prepare ordinary potato wedges (_vide_ page 174, sections 1 to 4).
2. Soak the wedges in 25 per cent. solution of glycerine for fifteen
minutes.
3. Moisten the cotton-wool pads at the bottom of the potato tubes with a
25 per cent. solution of glycerine.
4. Insert a wedge of potato in each tube and replug the tubes.
5. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
_five_ consecutive days.
~Animal Tissue Media (Frugoni).~--
1. Take a number of sterile test-tubes 16 x 3 or 4 cm., plugged with
cotton wool, and into each insert a 2 cm. length of stout glass tubing
(about 1 cm. diameter); fill in glycerine (6 per cent.) bouillon to the
upper level of the piece of glass tubing. Sterilise in the steamer at
100 deg. C. for twenty minutes on each of three successive days.
2. Kill a small rabbit by means of chloroform vapour.
3. Under strictly aseptic precautions remove the lungs, liver and other
solid organs and transfer them to a sterile double glass dish.
4. With the help of sterile scissors and forceps divide the organs into
roughly rectangular blocks 3 x 1.5 x 1 cm.
5. Pour into the dish a sufficient quantity of sterile glycerine
solution (6 per cent. in normal saline), cover, and allow to stand for
one hour.
6. Introduce a block of tissue into each tube so that it rests upon the
upper end of the piece of glass tubing. (The surface of the tissue will
now be kept moist by capillary attraction and condensation).
7. Sterilise in the autoclave at 120 deg. C. for thirty minutes.
8. Cap the tubes and store them in the ice chest for future use.
Tissues obtained at postmortems can also be used after preliminary
sterilisation by boiling or autoclaving.
_Media for the Study of Special Cocci._
_Diplococcus Gonorrhoeae._
~Ascitic Bouillon (Serum Bouillon).~--
1. Collect ascitic fluid (pleuritic fluid, hydrocele fluid, etc.), by
aspiration directly into sterile flasks, under strictly aseptic
precautions.
2. Mix the serum with twice its bulk of sterile nutrient bouillon
(_vide_ page 163).
3. If considered necessary (on account of the presence of blood,
crystals, etc.), filter the serum bouillon through porcelain filter
candle.
4. Tube, and sterilise in the water bath at 56 deg. C. for half an hour
on each of five consecutive days.
5. Incubate at 37 deg. C. for forty-eight hours and eliminate
contaminated tubes. Store the remainder for future use.
~Serum Agar (Heiman).~--
1. Prepare nutrient agar (_vide_ page 167), to following formula:
Agar 2.0 per cent.
Peptone 1.5 per cent.
Salt 0.5 per cent.
Meat extract _quantum sufficit._
2. Make reaction of medium + 10.
3. Filter; tube in quantities of 6 c.c.
4. Sterilise as for nutrient agar.
5. After the third sterilisation cool the tubes to 42 deg. C., and add
to each 3 c.c. of sterile hydrocele fluid, ascitic fluid, or pleuritic
effusion (previously sterilised, if necessary, by the fractional
method); allow the tubes to solidify in a sloping position.
6. When solid, incubate at 37 deg. C. for forty-eight hours, and eliminate
any contaminated tubes. Store the remainder for future use.
~Serum Agar (Wertheimer).~--
1. Prepare nutrient agar (_vide_ page 167), to the following formula:
Agar 2.0 per cent.
Peptone 2.0 per cent.
Salt 0.5 per cent.
Meat extract _quantum sufficit._
2. Make reaction of medium +10.
3. Filter; tube in quantities of 5 c.c.
4. Sterilise as for nutrient agar.
5. After the last sterilisation cool to 42 deg. C., then add 5 c.c.
sterile blood-serum from human placenta (sterilised, if necessary, by
the fractional method) to each tube; slope the tubes.
6. When solid, incubate at 37 deg. C. for forty-eight hours, and eliminate
any contaminated tubes. Store the remainder for future use.
~Serum Agar (Kanthack and Stevens).~--
1. Collect ascitic, pleuritic, or hydrocele fluid in sterile flasks and
allow to stand in the ice-chest for twelve hours to sediment.
2. Decant 1000 c.c. of the clear fluid into a measuring cylinder and
transfer to sterile litre flask.
3. Add 0.5 c.c. dekanormal NaOH solution for every 100 c.c. serum (_i.
e._, 5.0 c.c.), and mix thoroughly.
4. Heat in the steamer for twenty minutes.
5. Weigh out 15 grammes agar, emulsify in a separate vessel with 200
c.c. of the alkaline fluid previously cooled to about 20 deg. C., and
then add to the remainder of the fluid in the flask.
6. Bubble live steam through the mixture for twenty minutes to dissolve
the agar.
7. Filter through papier Chardin, using a hot-water funnel.
8. Weigh out glucose 10 grammes (= 1 per cent.), and dissolve it in the
clear agar.
8a. If desired, add glycerine, 5 per cent., to the clear agar.
9. Tube, and sterilise as for nutrient agar.
~Serum Agar (Libman).~--
1. Prepare nutrient agar (_vide_, page 167) using, however, 1.5 per
cent. peptone (that is 15 grammes per litre instead of 10 grammes).
2. Adjust the reaction to 0 (i. e., neutral to phenolphthalein).
3. Filter and transfer 1000 c.c. liquefied medium to a sterile flask.
4. Weigh out dextrose 20 grammes and dissolve in the fluid agar.
5. Tube in quantities of 6 c.c.; and sterilise in the steamer at 100
deg. C. for thirty minutes on each of three consecutive days.
6. After the third sterilisation cool to 42 deg. C. and add to each tube
3 c.c. of sterile hydrocele fluid, ascitic fluid or pleuritic effusion
(previously sterilised, if necessary, by the fractional method); allow
the tubes to solidify in a sloping position.
7. When solid, incubate at 37 deg. C. for forty-eight hours, and eliminate
any contaminated tubes. Store the remainder for future use.
~Egg-albumen, Inspissated.~--
1. Break several fresh eggs (hens', ducks', or turkeys' eggs), and
collect the "whites" in a graduated cylinder, taking care to avoid
admixture with the yolks.
2. Add 40 per cent. distilled water, and incorporate the mixture
thoroughly by the aid of an egg-whisk.
3. Weigh out 0.15 per cent. sodium hydrate and dissolve it in the fluid
(or add the amount of dekanormal caustic soda solution calculated to
yield the required percentage of soda in the total bulk of the
fluid--i. e., 0.375 c.c. of dekanormal NaOH solution per 100 c.c. of
the mixture).
_3a._ Glucose to the extent of 1 to 2 per cent. may now be added, if
desired.
4. Strain the mixture through butter muslin and filter through a
porcelain filter candle into a sterile filter flask.