Chapter VIII: Appendix: 492 (6)

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._

~Beetroot.~-- }
~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.

5. Tube, and stiffen at 100 deg. C. in the serum inspissator.

6. Incubate at 37 deg. C. for forty-eight hours and eliminate any contaminated tubes; store the remainder for future use.

~Egg-albumen (Tarchanoff and Kolesnikoff).~--

1. Place unbroken hens' eggs in dekanormal caustic soda solution for ten days. (After this time the white becomes firm like gelatine.)

2. Carefully remove the shell and cut the egg into fine slices.

3. Wash for two hours in running water.

4. Place the egg slices in a large beaker and sterilise in the steamer at 100 deg. C. for one hour.

5. Transfer each slice of egg by means of a pair of sterilised forceps to a Petri dish or large capsule.

6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of three consecutive days.

~Egg Albumin Broth (Lipschuetz).~--

1. Weigh out

Egg albumin (extra fine powder, Merck). 4 grammes

and place in a 2-litre flask with a number of sterile glass beads.

2. Measure out distilled water 200 c.c. into a half-litre flask and warm to 37 deg. C. in the incubator.

3. Add the water to the flask containing the albumin and beads and dissolve by shaking.

4. Add n/10-NaOH, 40 c.c. Allow the mixture to stand for thirty minutes with frequent shaking.

5. Filter through Swedish filter paper.

6. Sterilise by boiling two or three times at intervals of two hours.

7. Add ordinary nutrient bouillon 600 c.c.

8. Fill into small Erlenmeyer flasks in quantities of 50 c.c.

9. Incubate for forty-eight hours at 37 deg. C.--discard any contaminated flasks and store the remainder for future use.

~Egg Albumin Agar.~--

1. Prepare egg albumin solution as above 1-6.

2. Liquefy and measure out ordinary nutrient agar 600 c.c. and add to the egg albumin solution (in place of the nutrient broth).

3. Complete as above 8-9.

_Diplococcus Meningitidis Intracellularis._

~Ascitic Fluid Agar (Wassermann)~ _Synonym_ ~N-as-gar (Mervyn Gordon).~

1. Liquefy and measure out into a sterile flask:

Nutrient agar 600 c.c.

2. Measure out into a half litre flask

Distilled water 210 c.c.

and add to it

Ascitic fluid 90 c.c.
Nutrose 6 grammes

3. Heat over a bunsen flame, shaking constantly until the fluid boils, and the nutrose is dissolved.

4. Add the nutrose ascitic solution to the fluid agar.

5. Heat in the steamer for thirty minutes, then filter.

6. Tube and sterilise as for nutrient agar.

NOTE.--The finished medium in this case measures 900 c.c.
only since inconvenient fractions would be introduced in
making up to one litre exactly.

_Diplococcus Pneumoniae._

~Blood Agar (Washbourn).~--

1. Melt up several tubes of nutrient agar (_vide_ page 167) and allow them to solidify in the oblique position.

2. Place the tubes, in the horizontal position, in the "hot" incubator for forty-eight hours, to evaporate off some of the condensation water.

3. Kill a small rabbit with chloroform and nail it out on a board (as for a necropsy). Moisten the hair thoroughly with 2 per cent. solution of lysol.

4. Sterilise several pairs of forceps, scissors, etc., by boiling.

5. Reflect the skin over the thorax with sterile instruments.

6. Open the thoracic cavity by the aid of a fresh set of sterile instruments.

7. Open the pericardium with another set of sterile instruments.

8. Sear the surface of the left ventricle with a red-hot iron and remove fluid blood from the heart by means of sterile pipettes (e. g., those shown in Fig. 13, c).

9. Deliver a small quantity of the blood on the slanted surface of the agar in each of the tubes, and allow it to run over the entire surface of the medium.

10. Place the tubes in the slanting position and allow the blood to coagulate.

11. Return the "blood agar" to the hot incubator for forty-eight hours and eliminate any contaminated tubes. Store the remainder for future use.

_Media for the Study of Mouth Bacteria Generally._

~Potato Gelatine (Goadby).~--

1. Prepare glycerine potato broth (see page 203, sections 1 to 5).

2. Add 10 per cent. gelatine to the potato decoction and bubble live steam through the mixture for ten minutes.

3. Estimate the reaction; adjust the reaction of the medium to +5.

4. Cool the medium to below 60 deg. C., clarify with egg as for nutrient gelatine.

5. Filter through papier Chardin.

6. Tube, and sterilise as for nutrient gelatine.

_Media for the Study of Protozoa._

~Tissue Medium (Noguchi).~--_For spirochaetes (cultivations must be grown anaerobically)._

1. Plug and sterilise test-tubes 20 x 2 cm.

2. Kill a small rabbit with chloroform vapour. Open the abdomen with all aseptic precautions, remove kidneys and testicles and transfer to a sterile glass dish. Cut up the organs with sterile scissors into small pieces--say 4 millimetre cubes. The four organs should yield from 25 to 30 pieces of tissue.

3. Drop a small piece of sterile tissue into the bottom of each sterilised tube.

4. Take a flask containing about 400 c.c. nutrient agar (+10 reaction), liquefy the medium by heat and cool in a water bath to 50 deg. C.

5. Add 200 c.c. ascitic or hydrocele fluid (horse or sheep serum may be employed, but is not so good) to the liquid agar and mix carefully to avoid formation of air bubbles.

6. Fill about 20 c.c. of the ascitic agar into each of the sterilised tubes which already contains a piece of sterile rabbit's tissue, stand all the tubes upright in racks or a jar, and allow agar to set.

7. After solidification pour sterile paraffin oil on the surface of the medium in each tube to the depth of 3 centimetres.

8. Incubate tubes at 37 deg. C. for several days and discard any which prove to be contaminated.

9. Store such tubes as are sterile for future use.

XIII. INCUBATORS.

An incubator (Fig. 113) consists essentially of a chamber for the reception of cultivations, etc., surrounded by a water jacket, the walls of which are of metal, usually copper, and outside all an asbestos or felt jacket, or wooden casing. The water in the jacket is heated by gas or electricity and maintained at some constant temperature by a thermo-regulator. The cellular incubator (Fig. 114) which was made for me[7] some years ago is of the greatest practical utility. Here the central cavity is subdivided by five double-walled partitions (in which water circulates in connection with the water tanks at the top and base of the incubator) and again by iron shelves to form twenty-four pigeon holes. Into each of these slides an iron drawer 35 cm. long x 12 cm. wide x 22 cm. high forming a self-contained incubator. The drawer is fitted with a wooden form to which is fixed a handle and a numbered label. The thermo-regulating apparatus is the well-known Hearson capsule.

Two incubators at least are required in the laboratory, for the cultivation of bacteria the one regulated to maintain a temperature of 37 deg. C., and known as the "hot" incubator; the other, 20 deg. C. to 22 deg. C., and known as the "cool" or "cold" incubator.

Two other incubators, regulated to 42 deg. C. and 60 deg. C. respectively, whilst not absolutely, necessary very soon justify their purchase.

~Thermo-regulators.~--The thermo-regulator is the most essential portion of the incubator, as upon its efficient working depends the maintenance of a constant temperature in the cultivation chamber. It is also used in the fitting up of water and paraffin baths, and for many other purposes.

Of the many forms and varieties of thermo-regulator (other than electrical), two only are of sufficiently general use to need mention. In one of these the flow of gas to the gas-jet is controlled by the expansion or contraction of mercury within a glass bulb; in the other, by alterations in the position of the walls of a metallic capsule containing a fluid, the boiling-point of which corresponds to the temperature at which the incubator is intended to act. They are:

(a) _Reichert's_ (Fig. 115), consists of a bulb containing mercury which is to be suspended in the medium, whether air or water, the temperature of which it is desired to regulate. Gas enters at A, and passes out to the jet by B. As the temperature rises the mercury expands and cuts off the main gas supply. As the temperature falls the mercury contracts and reopens the narrow tube C. By means of a thumbscrew D (which mechanically raises or lowers the column of mercury irrespective of the temperature) and the aid of a thermometer the apparatus can be set to keep the incubator at any desired temperature. With this form a special gas burner is required, with separate supply of gas to a pilot jet at the side.

(b) _Hearson's capsule regulator_ consists of a metal capsule hermetically sealed and filled with a liquid which boils at the required temperature, this is adjusted in the interior of the incubator. Soldered to the upper side of the capsule is a thick piece of metal having a central cup to receive the lower end of a rigid rod, through which the movements of the walls of the capsule are transmitted to the gas valve fixed outside the incubator.

The gas valve or governor is shown in figure 116. A is the inlet for gas, C the outlet to burner heating the water jacket, B D a lever pivoted to standards at G, and acted upon by the capsule, through the rigid rod which enters the socket below the screw P.

The construction of the valve is such that, whenever the short arm of the lever B D presses on the disc below the end B, the main supply of gas is entirely cut off. At such times, however, a very small portion of gas passes from A to C, through an aperture inside the valve, the size of which aperture can be adjusted by the screw needle S, hence the gas flame below the incubator is never extinguished.

The expansion of the metal walls of the capsule, which takes place upon the boiling of its contents, provides the motive force, transmitted through the rigid rod to raise the long arm of the lever B D, and as this expansion only takes place at a predetermined temperature, the lever will only be acted upon when the critical temperature is reached, no sensible effect being produced at even 1 deg. C. below that at which the capsule is destined to act.

W is a weight sliding on the lever rod D; by increasing the distance between the weight and the fulcrum of the lower increased pressure is brought to bear upon the walls of the capsule with the result that the boiling-point of the liquid in the capsule is slightly raised, and a range of about two degrees can thus be obtained with any particular capsule.

FOOTNOTES:

[7] Made by the firm of Chas. Hearson & Co., 235 Regent St., London, W.

XIV. METHODS OF CULTIVATION.

Cultivations of micro-organisms are usually prepared in the laboratory in one of three ways:

~Tube cultures.~
~Plate cultures.~
~Hanging-drop cultures.~

These may be incubated either ~aerobically~ (i. e., in the presence of oxygen) or ~anaerobically~ (i. e., in the absence of oxygen, or in the presence of an indifferent gas, such as hydrogen, nitrogen, or carbon dioxide).

With regard to the temperature at which the cultivations are grown, it may be stated as a general rule that all media rendered solid by the addition of gelatine are incubated at 20 deg. C., or at any rate at a temperature not exceeding 22 deg. C. (that is, in the "cold" incubator); whilst fluid media and all other solid media are incubated at 37 deg. C. (that is, in the "hot" incubator). Exceptions to this rule are numerous. For instance, in studying the growth of the psychrophylic bacteria, the yeasts and the moulds, the cold incubator is employed for all media.

Tube cultivations are usually packed in the incubator in small tin cylinders, such as those in which American cigarettes are sold, or in square tin boxes. Beakers or tumblers may be used for the same purpose, but being fragile are not so convenient. Metal test-tube racks, long enough to just fit into the interior of the incubator and each accommodating two rows of tubes, are also exceedingly useful.

~AEROBIC.~

~The Preparation of Tube Cultivations.~

The preparation of a tube cultivation consists in:

(a) Inoculating a tube of sterile nutrient medium with a portion of the material to be examined.

(b) Incubating the inoculated tube at a suitable temperature.

The details of the first of these processes must be varied somewhat according to whether the tubes of nutrient media are inoculated or "planted" from--

1. Pre-existing cultivations.

2. Morbid material previously collected (_vide_ page 373).

3. Fluids, tissues, etc., or from the animal body direct.

The method of preparing tube cultivations from pre-existing cultivations is as follows:

~1. Fluid Media~ (e. g., Nutrient Bouillon).--

1. Flame the cotton-wool plug of the tube containing the cultivation and also that of the tube of sterile bouillon.

2. Hold the two tubes, side by side, between the left thumb and the first and third fingers, allowing the sealed ends to rest on the dorsum of the hand, and separating the mouths of the tubes (which are pointed to the right) by the tip of the second finger. Keep the tubes as nearly horizontal as is possible without allowing the fluid in the bouillon tube to reach the cotton-wool plug (Fig. 117).

3. Sterilise the platinum loop and allow it to cool.[8]

4. Grasp the plug of the tube containing the cultivation between the little finger and palm of the hand and remove it from the tube.

5. Grasp the plug of the bouillon tube between the fourth finger and the ball of the thumb and remove it from the tube.

6. Pass the platinum loop into the tube containing the culture--do not allow the loop to touch the sides of the tube, or the handle to touch the medium--and remove a small portion of the growth; withdraw the loop from the tube, keeping the infected side of the loop downward.

7. Pass the loop into the bouillon tube almost down to the level of the fluid, reverse the loop so that the infected side faces upward, emulsify the portion of the growth in the moisture adhering to the side of the tube which is uppermost. Withdraw the loop.

8. Replug both tubes.

9. Sterilise the platinum loop.

10. Label the bouillon tube with (a) the name of the organism and (b) the date of inoculation.

11. Incubate.

~2. Solid Media.~--Solid media are stored in tubes in one of two ways:

1. Oblique tube or slanted tube (Fig. 118), in which the medium has been allowed to solidify whilst the tube was retained in an inclined position, so forming an extensive surface of medium extending from the bottom of the tube almost to its mouth.

This is employed for "streak" or "smear" cultivations (_Strichcultur_).

2. Straight tube (Fig. 119), in which the medium forms a cylindrical mass in the lower portion of the tube and presents an upper surface which is at right angles to the long axis of the tube.

This is employed for "stab" or "stick" cultivations (_Stichcultur_), or by inoculating the medium whilst fluid, and allowing to solidify in this position, for "shake" cultivations.

_Streak Culture._--

1. Flame the plugs, sterilise the platinum loop (or spatula). Open the tubes and charge the loop as in previous inoculation.

2. Pass the infected loop to the bottom of the tube to be inoculated and draw it, as lightly as possible, along the centre of the surface of the medium, terminating the "streak" over the thin layer of medium near the mouth of the tube.

3. Replug the tubes, sterilise the platinum loop.

4. Label the newly inoculated tube and incubate.

_Smear Culture._--Proceed generally as in streak culture, but rub the infected loop all over the surface of the medium, instead of restricting the inoculation to a narrow line.

NOTE.--Gelatine and agar oblique tubes should be freshly
"slanted" before use.

_Stab Culture._--

1. Flame the plugs, open the tubes, sterilise the platinum needle and charge it with the inoculum as in the previous cultivations.

2. Pass the platinum needle into the tube to be inoculated until it touches the centre of the surface of the medium. Now thrust it deeply into the substance of the medium, keeping the needle as nearly as possible in the axis of the cylinder of medium. Then withdraw the needle.

3. Replug the tubes. Sterilise the platinum needle.

4. Label the newly planted tube and incubate.

NOTE.--When gelatine is stored for some time the upper
surface of the cylinder becomes concave owing to
evaporation. Tubes showing this appearance should be
liquefied and again allowed to set before use for stab
culture, otherwise when the needle enters the medium, the
surface tension will cause the gelatine cylinder to split.

_Shake Culture._--

1. Liquefy a tube of nutrient gelatine (or agar, or other similar medium), by heating in a water-bath (Fig. 121).

2. Inoculate the liquefied medium and label it, etc., precisely as if dealing with a tube of bouillon.

3. Place the newly planted tube in the upright position (e. g., in a test-tube rack) and allow it to solidify.

4. Label the tube; when solid, incubate.

_Esmarch's Roll Cultivation._--

1. Liquefy three tubes of gelatine by heat.

2. Prepare three dilutions of the inoculum (as described for
plate cultivations, page 228, steps 4 to 7).

3. Roll the tubes, held almost horizontally, in a groove
made in a block of ice, until the gelatine has set in a thin
film on the inner surface of tube (Fig. 120); or under the
cold-water tap.

In order that the medium may adhere firmly to the glass, the
agar used for roll cultivation should have 1 per cent.
gelatine or 1 per cent. gum arabic added to it before
sterilisation.

Roll cultivations, which served a most important purpose in
the days before the introduction of Petri dishes for plate
cultivations, are now obsolete in modern laboratories and
are merely mentioned for the benefit of students, since
examiners who are interested in the academic and historical
aspects of bacteriology sometimes expect candidates to be
acquainted with the method of preparing them.

The Preparation of Plate Cultures.

If a small number of bacteria are suspended in liquefied gelatine, agar, or other similar medium, and the infected medium spread out in an even layer over a flat surface and allowed to solidify, each individual micro-organism becomes fixed to a certain spot and its further development is restricted to the vicinity of this spot. After a variable interval the growth of this organism becomes visible to the naked eye as a "colony." This is the principle upon which the method of plate cultivation is based and its practice enables the bacteriologist to study the particular manner of development affected by each species of microbe when growing (a) unrestricted upon the surface of the medium, (b) in the depths of the medium. The method itself is as follows:

~Apparatus Required.~--

1. Tripod levelling stand.

2. Large shallow glass dish, with a square sheet of plate
glass to cover it.

3. Spirit level.

4. Case of sterile Petri dishes.

5. Tubes of sterile nutrient media, gelatine (or agar)
previously liquefied by heating in the water-bath and cooled
to 42 deg. C., otherwise the heat of the medium would destroy
many, if not all, of the bacteria introduced.

6. Tube of cultivation to be planted from.

7. Platinum loop.

8. Bunsen burner.

9. Grease pencil.

Method of "Pouring" Plates.--

1. Place the glass dish on the levelling tripod (Figs. 122, 123); if gelatine plates are to be poured fill the dish with ice water--gelatine solidifies so slowly that it is necessary to hasten the process; if agar is to be used fill with water at 50 deg. C.--agar sets almost immediately at the room temperature and by slightly retarding the process lumpiness is avoided; cover the dish with the square sheet of glass.

2. Place the spirit level on the sheet of glass and by means of the levelling screws adjust the surface of the glass to the horizontal.

This leveling is an important matter since the development of a colony is to some extent proportionate to the supply of medium available for its nutrition. Thus in a "smear" on sloped tube culture, the colonies at the upper part of the medium are stunted and small but increase in size and luxuriance of growth the nearer they approach to the bottom of the tube, where there is the greatest depth of medium.

3. Place three sterile Petri dishes in a row on the surface of the glass plate and number them 1, 2, and 3, from left to right.

4. Number the previously liquefied tubes of nutrient media 1, 2, and 3. Flame the plugs and see that each plug can be readily removed from the mouth of its tube.

5. Add one loopful of the inoculum to tube No. 1, treating the liquefied medium as bouillon. After replugging, grasp the tube near its mouth by the thumb and first finger of the right hand, and with an even circular movement of the whole arm, diffuse the inoculum throughout the medium; avoid jerky movements, as these cause bubbles of air to form in the medium.

The knack of mixing evenly without producing air bubbles, is not always easily acquired, by this method. An alternative plan is to hold the inoculated tube vertically upright between the opposed palms and to rotate it between them by rapid backward and forward movements of the two hands (Fig. 124).

6. Sterilise the platinum loop, and add two loopfuls of diluted inoculum to tube No. 2, and mix as before.

7. In a similar manner transfer three loopfuls of liquefied medium from tube No. 2 to tube No. 3, and mix thoroughly.

8. Flame the plug of tube No. 1, remove it, then flame the lips of the tube; slightly raise the cover of Petri dish No. 1, introduce the mouth of the tube; then, elevating the bottom of the tube, pour the liquefied medium into the Petri dish, to form a thin layer. Remove the mouth of the tube and close the "plate." If the medium has failed to flow evenly over the bottom of the plate, raise the plate from the levelling platform and by tilting in different directions rectify the fault.

9. Pour plates No. 2 and No. 3, in a similar manner, from tubes Nos. 2 and 3.

10. Label the plates with the distinctive name or number of the inoculum, also the date; the number of the dilution having been previously indicated (step 3).

11. Place in the cool incubator for three or more days, as may be necessary.

In this way colonies may be obtained quite pure and separate from each other.