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.

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.

In plate No. 1, probably, the colonies will be so numerous and crowded, and therefore so small, as to render it useless. In plate No. 2 they will be more widely separated, but usually No. 3 is the plate reserved for careful examination, as in this the colonies are usually widely separated, few in number, and large in size.

Agar plates are poured in a similar manner, but the agar must be melted in boiling water and then allowed to cool to 45 deg. C. or 42 deg. C. in a carefully regulated water-bath before being inoculated, and the entire process must be carried out very rapidly, otherwise the agar will have solidified before the operation is completed.

NOTE.—In pouring plates, since tube No. 1 (for the first dilution) rarely gives a plate that is of any practical value it is frequently replaced by a tube of bouillon or sterile salt solution, and in such case plate No. 1 is not poured.

Surface Plates.—

This method of pouring what may be termed "whole" plates (since colonies may appear both on the surface and in the depths of the medium) is essential to the accurate study of the formation of colonies under various conditions, but when the main object of the separation of the bacteria is to obtain subcultivations from a number of individual bacteria, "surface" plates must be prepared, since here colony formation is restricted to the surface of the medium. The method adopted varies slightly according to whether the medium employed is gelatine or agar, or one of the derivatives or variants of the latter.

(a) Gelatine Surface Plates.—

1. Liquefy three tubes of nutrient gelatine.

2. Pour each tube into a separate Petri dish and allow it to solidify. Then turn each plate and its cover upside down.



3. When quite cold raise the bottom of plate 1, revert it and deposit a drop of the inoculum (whether a fluid culture or an emulsion from solid culture) upon the surface of the gelatine with a platinum loop—close to one side of the plate; replace the bottom half of the Petri dish in its cover.

4. Take a piece of thin glass rod, stout platinum wire or best of all a piece of aluminium wire (say 2 mm. diameter) about 28 cm. long. Bend the terminal 4 cm. at right angles to the remainder, making an L-shaped rod (Fig. 126). Sterilise the short arm and adjacent portion of the long arm, in the Bunsen flame, and allow it to cool.

5. Now raise the bottom of the Petri dish in the left hand, leaving the cover on the laboratory bench, and holding it vertically, smear the drop of inoculum all over the surface of the gelatine with the short arm of the spreader by a rotatory motion, (Fig. 127). Replace the dish in its cover.

6. Raise the bottom of plate 2 and rub the infected spreader all over the surface of the gelatine—then go on in like manner to the third plate in the series.

7. Sterilise the spreader.

8. Label and incubate the plates.



After incubation, plate No. 1 will probably yield an enormous number of colonies; plate 2 will show fewer colonies, since only those bacteria adhering to the rod after rubbing over plate 1 would be deposited on its surface, and by the time the rod reached plate 3 but very few organisms should remain upon it. So that the third plate as a rule will only show a very few scattered colonies, eminently suitable for detailed study.

(b) Agar Surface Plates.—

1. Liquefy three tubes of nutrient agar—nutrose agar or the like.

2. Pour each tube into a separate Petri dish and allow it to solidify.

3. When quite solid invert each dish, raise the bottom half and rest it obliquely on its inverted cover (Fig. 128) and place it in this position in an incubator at 60 deg. C. for forty-five minutes (or in an incubator at 42 deg. C. for two hours). This evaporates the water of condensation and gives the medium a firm, dry surface.

4. On removing the plates from the incubator close each dish and place it—still upside down—on the laboratory bench.



5. Inoculate the plates in series of three, as described for gelatine surface plates 3-8.

Hanging-drop Cultivation.

Apparatus Required.—

Hanging-drop slides. Cover-slips. Section rack (Fig. 75). Blotting paper. Bell glass to cover slides. Original culture. Tubes of broth, or liquefied gelatine or agar. Forceps. Platinum loop. Bunsen burner. Grease pencil. Sterile vaseline. Lysol.

(a) Fluid Media.—

1. Prepare first and second dilutions of the inoculum as directed for plate cultivations (vide pages 228-229, sections 4 to 6), substituting tubes of nutrient broth for the liquefied gelatine.

2. Sterilise a hanging-drop slide by washing thoroughly in water and drying, then plunging it into a beaker of absolute alcohol, draining off the greater part of the spirit, grasping the slide in a pair of forceps, and burning off the remainder of the alcohol in the flame.

3. Place the hanging-drop slide on a piece of blotting paper moistened with 2 per cent. lysol solution and cover it with a small bell glass that has been rinsed out with the same solution and not dried.

4. Raise the bell glass slightly and smear sterile vaseline around the rim of the metal cell by means of a sterile spatula of stout platinum wire.

5. Remove a clean cover-slip from the alcohol pot with sterile forceps and burn off the alcohol; again raise the bell glass and place the sterile cover-slip on the blotting paper by the side of the hanging-drop slide.

6. Remove a drop of the broth from the second dilution tube with a large platinum loop; raise the bell glass and deposit the drop on the centre of the cover-slip. Sterilise the loop.

7. Raise the bell glass sufficiently to allow of the cover-slip being grasped with forceps, inverted, and adjusted over the cell of the hanging-drop slide. Remove the bell glass altogether and press the cover-slip firmly on to the cell.

8. Either incubate and examine at definite intervals, or observe continuously with the microscope, using a warm stage if necessary (Fig. 53).

(b) Solid Media.—Observing precisely similar technique, a few drops of liquefied gelatine or agar from the second dilution tube may be run over the surface of the sterile cover-slip and a hanging-drop plate cultivation thereby prepared.

This method is extremely useful in connection with the study of yeasts, when the circular cell on the hanging-drop slide should be replaced by a rectangular cell some 38 by 19 mm., and the gelatine spread over a cover-slip of similar size. After sealing down the preparation, the upper surface of the cover-slip may be ruled into squares by the aid of the grease pencil or a writing diamond and numbered to facilitate the subsequent identification of the colonies which are observed to develop from solitary germs.

Hanging-block Culture (Hill).—

Apparatus required: As for hanging-drop cultivation with the addition of a scalpel.

Carry out the method as far as possible under cover of a bell glass, to avoid aerial contamination.

1. Liquefy a tube of nutrient agar (or gelatine) and pour into a Petri dish to the depth of about 4 mm. and allow to set.

2. With a sharp scalpel cut out a block some 8 mm. square, from the entire thickness of the agar layer.

3. Raise the agar block on the blade of the scalpel and transfer it, under side down, to the centre of a sterile slide.

4. Spread a drop of fluid cultivation (or an emulsion of growth from a solid medium) over the upper surface of the agar block as if making a cover-slip film.

5. Place the slide and block covered by the bell glass in the incubator at 37 deg. C. for ten minutes to dry slightly.

6. Take a clean dry sterile cover-slip in a pair of forceps, and with the help of a second pair of forceps lower it carefully on the inoculated surface of the agar (avoiding air bubbles), so as to leave a clear margin of cover-slip overlapping the agar block.

7. Invert the preparation and with the blade of the scalpel remove the slide from the agar block.

8. With a platinum loop run a drop or two of melted agar around the edges of the block. This solidifies at once and seals the block to the cover-slip.

9. Prepare a sterile hanging-drop slide, and smear hard vaseline or melted white wax on the rim of the metal cell.

10. Invert the cover-slip with the block attached on to the hanging-drop slide, and seal the cover-slip firmly in place.

11. Observe as for hanging-drop cultivations.

ANAEROBIC CULTIVATIONS.

Numerous methods have been devised for the cultivation of anaerobic bacteria, the majority requiring the employment of special apparatus. The principle upon which any method is based and upon which it depends for its success falls under one or another of the following headings:

(a) Exclusion of air from the cultivation.

(b) Exhaustion of air from the vessel containing the cultivation by means of an air pump—i. e., cultivation in vacuo.

(c) Absorption of oxygen from the air in contact with the cultivation by means of pyrogallic acid rendered alkaline with caustic soda—i. e., cultivation in an atmosphere of nitrogen.

(d) Displacement of air by an indifferent gas, such as hydrogen or coal gas—i. e., cultivation in an atmosphere of hydrogen.

(e) A combination of two or more of the above methods.

A selection of the simplest and most generally useful methods is given here.

Whenever possible, the nutrient media that are employed in any of the processes should contain some easily oxidisable substance, such as sodium formate (0.4 per cent.) or sodium sulphindigotate (0.1 per cent.), which will absorb all the available oxygen held in solution by the medium. The further addition of glucose, 2 per cent., favors the growth of anaerobic bacteria (vide, pages 189-190).

Further, it is advisable to seal all joints between india-rubber stoppers and tubulures or the mouths of the tubes with melted paraffin; glass stoppers and taps should be lubricated with resin ointment or a mixture of beeswax 1 part, olive oil 4 parts.

(A) Method I (Hesse's Method).—

1. Make a stab culture in gelatine or agar, choosing for the purpose a straight tube containing a deep column of medium, and thrusting the inoculating needle to the bottom of the tube.

2. Pour a layer of sterilised oil (olive oil, vaseline, or petroleum), 1 or 2 cm. deep, upon the surface of the medium.

3. Incubate.

Method II.—This method is only available when dealing with pure cultivations.

1. Liquefy a tube of gelatine (or agar) by heat, pour it into a Petri dish, and allow it to solidify.

2. Inoculate the surface of the medium in one spot only.

3. Remove a cover-slip from the pot of absolute alcohol with sterile forceps; burn off the alcohol in the gas flame.

4. Lower the now sterile cover-slip carefully on to the inoculated surface of the medium, carefully excluding air bubbles, and press it down firmly with the points of the forceps. (A sterile disc of mica may be substituted for the cover-slip.)

5. Incubate.

Method III (Roux's Physical Method).—

1. Prepare tube cultures of fluid media (or solid media rendered fluid by heat) in the usual way.

2. Aspirate some of the inoculated media into capillary pipettes.

3. Seal both ends of each pipette in the blowpipe flame.

4. Incubate.

Method IV (Roux's Biological Method).—

1. Plant a deep stab, as in method I.

2. Pour a layer, 1 or 2 cm. deep, of broth cultivation of a vigourous aerobe—e. g., B. aquatilis sulcatus or B. prodigiosus—upon the surface of the medium; or an equal depth of liquefied gelatine, which is then inoculated with the aerobic organism.

3. Incubate.

The growth of the aerobe will use up all the oxygen that reaches it and will not allow any to pass through to the medium below, which will consequently remain in an anaerobic condition.

(B) Method V.—

1. Prepare tube or flask cultivations in the usual way.

2. Replace the cotton-wool plug by an india-rubber stopper perforated with one hole and fitted with a length of glass tubing which has a constriction about 3 cm. above the stopper and is then bent at right angles (Fig. 129). The stopper and glass tubing are sterilised by being boiled in a beaker of water for five minutes.



3. Connect the tube leading from the culture vessel with a water or air pump, interposing a Wulff's bottle fitted as a wash-bottle and containing sulphuric acid.

4. Exhaust the air from the culture vessel.

5. Before disconnecting the apparatus, seal the glass tube from the culture vessel at the constriction, using the blowpipe flame.

6. Incubate.

(C) Method VI (Buchner's Method).

Apparatus and Solutions Required.—

Buchner's tube (a stout glass test-tube 23 cm. long and 4 cm. in diameter, fitted with india-rubber stopper, Fig. 130).

Pyrogallic acid in compressed tablets each containing 1 gram.

Dekanormal solution of caustic soda.

METHOD.—

1. Prepare the tube cultivation in the usual way.

2. Moisten the india-rubber stopper of the Buchner's tube with water and see that it fits the mouth of the tube accurately.

3. Remove the stopper from the caustic soda bottle.

4. Drop one of the pyrogallic acid tablets[9] into the Buchner's tube (roughly, use 1 gramme pyrogallic acid for every 100 c.c. air capacity of the receiving vessel).

5. Add about 1 c.c. of the soda solution.

6. Place the inoculated tube inside the Buchner's tube. The pyrogallic tablet acts as a buffer and prevents damage to either the inoculated tube or the Buchner's tube even should it be slipped in hurriedly.

7. Fit the india-rubber stopper tightly into the mouth of the Buchner's tube.



The pyrogallic acid tablet dissolves slowly in the soda solution and its oxidation proceeds very slowly at first so that ample time is available when this method is adopted.

8. Restopper the caustic soda bottle.

9. Place Buchner's tube in a wire support, and incubate.

Method VII (Wright's Method).—

1. Prepare tube cultivation in the usual way.

2. Cut off that portion of the cotton-wool plug projecting above the mouth of the tube with scissors, then push the plug into the tube for a distance of 2 or 3 cm.

3. By means of a pipette drop about 1 c.c. of pyrogallic acid 10 per cent. aqueous solution on to the plug. It will immediately be absorbed by the cotton-wool.

4. With another pipette run in an equal quantity of the caustic soda solution.

5. Quickly close the mouth of the tube with a tightly fitting india-rubber stopper.

6. Incubate.



Method VIII (McLeod's Method).—

Apparatus and Solutions Required.—

McLeod's plate base (a hollow glazed earthenware disc 9 cm. in diameter and 2 cm. deep: the upper surface is pierced by a central hole, 2 cm. in diameter, giving access to the interior, the lower part of which is divided into two by a low partition. A shallow groove encircles the upper surface near to the edge).

Plasticine. Pyrogallic acid (1 gramme) compressed tablets. Sodic hydroxide (0.4 gramme) compressed tablets. Wash bottle of distilled water. Surface plates of one or other agar medium (in petri dishes of 8 cm. diameter). Surface plate spreader.

METHOD.—

1. Roll out a long cylinder of plasticine and fit it into the groove on the upper surface of the earthenware base.

2. Place a tablet of pyrogallic acid in one division of the interior of the plate base, and two tablets of sodic hydroxide in the other.

3. Prepare surface plate culture of the organism to be cultivated.

4. Run a few cubic centimetres of distilled water into that division of the plate base containing the sodic hydroxide.

5. Invert the bottom half of the surface plate over the plate base and press its edges firmly down into the plasticine filling the groove.

6. Label and incubate.

(D) Method IX.—

Apparatus Required.—

Small Ruffer's or Woodhead's flask (Fig. 33). Sterile india-rubber stopper. India-rubber tubing. Glass tubing. Metal screw clips. Cylinder of compressed hydrogen; or hydrogen gas apparatus

METHOD.—

1. Sterilise a glass vessel, shaped as in a Ruffer's or Woodhead's flask, in the hot-air oven. (The tubulure and the side tubes are plugged with cotton-wool.) After sterilisation, fix a short piece of rubber tubing occluded by a metal clip to each side tube.

2. Inoculate a large quantity (e. g., 200 c.c.) of the medium. Where solid media are employed they must first be liquefied by heat.

3. Remove the cotton-wool plug from the tubulure and pour the inoculated medium into the glass vessel.

4. Close the tubulure by means of an india-rubber stopper previously sterilised by boiling in a beaker of water.



5. Connect up the india-rubber tubing on one of the side tubes with a cylinder of compressed hydrogen (or the delivery tube of a Kipp's Fig. 132 or other hydrogen apparatus, Fig. 133), interposing a short piece of glass tubing; and in like manner connect a long piece of rubber tubing which should be led into a basin of water, to the opposite side tube.

6. Open both metal clips and pass hydrogen through the vessel until the atmospheric air is replaced by hydrogen. This is determined by collecting some of the gas which bubbles through the water in the basin in a test-tube and testing it by means of a lighted taper.

7. Close the metal clip on the tube through which the gas is entering; close the clip on the exit tube.

8. Disconnect the gas apparatus.

9. Incubate.

Method X (Botkin's Method).—

Apparatus Required.—

Large glass dish 20 cm. diameter and 8 cm. deep. Flat leaden cross slightly shorter than the internal diameter of the glass dish. Bell glass about 15 cm. diameter and 20 to 25 cm. high. Metal frame for plate cultivations. Or, glass battery jar for tube cultivations. Cylinder of compressed hydrogen. Rubber tubing. Two pieces of U-shaped glass tubing (each arm 8 cm. in length). Half a litre of glycerine (or metallic mercury).

METHOD.—

1. Place the leaden cross inside the glass dish, resting on the bottom.

2. Prepare the cultivations in the usual way.

3. Place the tube cultivations in a glass battery jar (or the plate cultivations on a metal frame), resting on the centre of the leaden cross.

4. Cover the cultivations with the bell jar.

5. Adjust the U-shaped pieces of glass tubing in a vertical position on opposite sides of the bell jar, one arm of each inside the jar, the other outside. These tubes are best held in position by embedding the U-shaped bends in two lumps of plasterine stuck on the bottom of the glass dish. Fix a short length of rubber tubing clamped with a metal clip to each of the outside arms (Fig. 134).

6. Fill the glass dish with glycerine or metallic mercury to a depth of about 5 cm.



7. Connect up one U-shaped tube with the hydrogen cylinder (or gas apparatus) by means of rubber tubing. Replace the atmospheric air by hydrogen, as in method IX.

8. Clamp the tubes and disconnect the gas apparatus.

9. Incubate.

Method XI (Novy's Method).—

Apparatus Required.—

Jar for plate cultivations (Fig. 135). Or, jar for tube cultivations (Fig. 136). Lubricant for stopper of jar. Rubber tubing. Cylinder of compressed hydrogen.

METHOD.—

1. Prepare cultivations in the usual way.

2. Place these inside the jar.

3. Lubricate the stopper and insert it in the mouth of the jar, with the handle in a line with the two side tubes.

4. Connect up the delivery tube a with the hydrogen gas supply by means of rubber tubing.



5. Attach a piece of rubber tubing to the exit tube b and collect samples of the issuing gas (over water) and test from time to time.

6. When the air is completely displaced by hydrogen, turn the handle of the stopper at right angles to the line of entry and exit tubes; this seals the orifice of both tubes.

7. Disconnect the gas apparatus and incubate.

(E) Method XII (Bulloch's Method).—

Apparatus Required.—

Bulloch's jar. Pot of resin ointment. Small glass dish 14 cm. diameter by 5 cm. deep. Vessel for tube cultures or metal rack for plate cultures. Pyrogallic acid tablets. Cylinder of compressed hydrogen. Geryk or other air pump. Rubber pressure tubing. 10 c.c. pipette. Glass tubing. Dry granulated caustic soda or compressed tablets each, containing 0.4 grammes sodic hydroxide. Small beaker of water.

METHOD.—

1. Prepare the cultivations in the usual way.

2. Place the glass dish in the centre of the glass slab, and stand the cultivations inside this.

3. Place a sufficient number of pyrogallic acid tablets at one side of the glass dish (i. e., 1 tablet for each 100 cubic centimeters air capacity of the bell jar). Place a small heap of dry granulated soda (or half a dozen tablets of sodic hydroxide) by the side of the pyro tablets.

4. Smear the flange of the bell jar with resin ointment and apply the jar firmly to the glass slab, covering the cultivations—so arranged that the long tube passes with its lower end into the glass dish at a point directly opposite to the pyrogallic acid tablets. Lubricate the two stop-cocks with resin ointment (Fig. 137).

5. Connect up the short tube a with the gas-supply by means of rubber pressure tubing and open both stop-cocks.

6. Connect a long, straight piece of glass tubing to the long tube b by means of a piece of rubber tubing interposing a screw clamp: and collect samples of the issuing gas from time to time and test.

7. When the air is displaced, shut off the stop-cock of the entry tube, then that of the exit tube b. Screw down the clamp and remove the glass tube from the rubber connection and connect up the short tube a to the air pump by means of pressure tubing.

8. Open the stop-cock of tube a and with two or three strokes of the air pump, aspirate a small quantity of gas, so creating a slight vacuum. Then shut off the stop-cock and disconnect the air pump.

9. Fill the 10 c.c. bulb pipette with water; insert its point into the rubber tubing on the long tube b as far as the screw clamp. Open the screw clamp and run in water until stopped by the internal pressure. Shut off stop-cock. (The water dissolves the soda and pyrogallic acid converting the latter into alkaline pyro. and so bringing its latent capacity for oxygen into action).



10. Reverse the tubes from the tubulures so that they meet, out of harm's way, over the top of the bell glass; again see that all joints are tight and transfer the apparatus to the incubator.

This last method is the most satisfactory for anaerobic cultivations, as by its means complete anaerobiosis can be obtained with the least expenditure of time and trouble.

FOOTNOTES:

[8] See also method of opening and closing culture tubes, pages 74-76.

[9] If compressed tablets of pyrogallic acid cannot be obtained make up a stock "acid pyro" solution

Pyrogallic acid, 10 grammes Hydrochloric acid, 1.5 c.c. Distilled water, 100 c.c.

and at step 4, run in 10 c.c. of the solution.



XV. METHODS OF ISOLATION.

The work in the preceding sections, arranged to demonstrate the chief biological characters of bacteria in general, is intended to be carried out by means of cultivations of various organisms previously isolated and identified and supplied to the student in a state of purity. A cultivation which comprises the progeny of a single cell is termed a "pure culture"; one which contains representatives of two or more species of bacteria is spoken of as an "impure," or "mixed" "cultivation," and it now becomes necessary to indicate the chief methods by which one or more organisms may be isolated in a state of purity from a mixture; whether that mixture exists as an impure laboratory cultivation, or is contained in pus and other morbid exudations, infected tissues, or water or food-stuffs.



Before the introduction of solid media the only method of obtaining pure cultivations was by "dilution"—by no means a reliable method. "Dilution" consisted in estimating approximately the number of bacteria present in a given volume of fluid (by means of a graduated-celled slide resembling a haematocytometer, Fig. 138), and diluting the fluid by the addition of sterile water or bouillon until a given volume (usually 1 c.c.) of the dilution contained but one organism. By planting this volume of the fluid into several tubes or flasks of nutrient media, it occasionally happened that the resulting growth was the product of one individual microbe. A method so uncertain is now fortunately replaced by many others, more reliable and convenient, and in those methods selected for description here, the segregation and isolation of the required bacteria may be effected—

A. By Mechanical Separation.

1. By surface plate cultivation:

(a) Gelatine. (b) Agar. (c) Serum agar. (d) Blood agar. (e) Hanging-drop or block.

[2. By Esmarch's roll cultivation:

This archaic method (see page 226) is no longer employed for the isolation of bacteria.]

3. By serial cultivation.

B. By Biological Differentiation.

4. By differential media.

(a) Selective. (b) Deterrent.

5. By differential incubation.

6. By differential sterilisation.

7. By differential atmosphere cultivation.

8. By animal inoculation.

The selection of the method to be employed in any specific instance will depend upon a variety of circumstances, and often a combination of two or more will ensure a quicker and more reliable result than a rigid adherence to any one method. Experience is the only reliable guide, but as a general rule the use of either the first or the third method will be found most convenient, affording as each of them does an opportunity for the simultaneous isolation of several or all of the varieties of bacteria present in a mixture.

1. Surface Plate Cultivations.—

(a) Gelatine (vide page 164).

(b) Agar (vide page 167).

(c) Alkaline serum agar (vide page 211).

These plates are prepared in a manner precisely similar to that adopted for nutrient gelatine and agar surface plates (vide pages 231-233).

(d) Serum Agar.—

1. Melt three tubes of nutrient agar, label them 1, 2, and 3, and place them, with three tubes of sterile fluid serum, also labelled 1a, 2a, and 3a, in a water-bath regulated at 45 deg. C.; allow sufficient time to elapse for the temperature of the contents of each tube to reach that of the water-bath.

2. Take serum tube No. 1a and agar tube No. 1. Flame the plugs and remove them from the tubes (retaining the plug of the agar tube in the hand); flame the mouths of the tubes, pour the serum into the tube of liquefied agar and replace the plug of the agar tube.

3. Mix thoroughly and pour plate No. 1 secundum artem.

4. Treat the remaining tube of agar and serum in a similar fashion, and pour plates Nos. 2 and 3.

5. Dry the serum agar plates in the incubator running at 60 deg. C. for one hour (see page 232).

6. Inoculate the plates in series as described for gelatine surface plates (page 231).

(e) Blood Agar, Human.—

1. Melt a tube of sterile agar and pour it into a sterile plate; let it set.

2. Collect a few drops of human blood, under all aseptic conditions, in a sterile capillary teat pipette.

3. Raise the cover of the Petri dish very slightly, insert the extremity of the capillary pipette, and deposit the blood on the centre of the agar surface. Close the dish.

4. Charge a platinum loop with a small quantity of the inoculum. Raise the cover of the plate, introduce the loop, mix its contents with the drop of blood, remove the loop, close the dish and sterilise the loop.

5. Finally smear the mixture over the surface of the agar with a sterilised L-shaped rod.

6. Label and incubate.

(If considered necessary, two, three, or more similar plates may be inoculated in series.)

(f) Blood Agar, Animal.—

When preparing citrated blood agar (page 171) it is always advisable to pour several blood agar tubes into plates, which can be stored in the ice chest ready for use at any moment for surface plate cultures.

(g) Hanging-drop or block culture, (vide page 233).

3. Serial Cultivations.—These are usually made upon agar or blood-serum, although gelatine may also be used.

The method is as follows:

1. Take at least four "slanted" tubes of media and number them consecutively.

2. Flame all the plugs and see that each can be readily removed.

3. Charge the platinum loop with a small quantity of the inoculum, observing the usual routine, and plant tube No. 1, smearing thoroughly all over the surface. If any water of condensation has collected at the bottom of the tube, use this as a diluent before smearing the contents of the loop over the surface of the medium.

4. Without sterilising or recharging the loop, inoculate tube No. 2, by making three parallel streaks from end to end of the slanted surface.

5. Plant the remainder of the tubes in the series as "smears" like tube No. 1.

6. Label with distinctive name or number, and date; incubate.

The growth that ensues in the first two or three tubes of the series will probably be so crowded as to be useless. Toward the end of the series, however, discrete colonies will be found, each of which can be transferred to a fresh tube of nutrient medium without risk of contamination from the neighbouring colonies.

"Working" up Plates.—

Having succeeded in obtaining a plate (or tube cultivation) in which the colonies are well grown and sufficiently separated from each other, the process of "working up," "pricking out," or "fishing" the colonies in order to obtain subcultures in a state of purity from each of the different bacteria present must now be proceeded with.

Occasionally it happens that this is quite a simple matter. For example, the original mixed cultivation when examined microscopically was found to contain a Gram positive micrococcus, a Gram positive straight bacillus and a Gram negative short bacillus. The third gelatine plate prepared from this mixture, on inspection after four day's incubation, showed twenty-five colonies—seven moist yellow colonies, each sinking into a shallow pit of liquefied gelatine, fourteen flat irridescent filmy colonies, and four raised white slimy colonies. A film preparation (stained Gram) from each variety examined microscopically showed that the yellow liquefying colony was composed of Gram positive micrococci; the flat colony of Gram positive bacilli and the white colony of gram negative bacilli. One of each of these varieties of colonies would be transferred by means of the sterilised loop to a fresh gelatine culture tube, and after incubation the growth in each subculture would correspond culturally and microscopically with that of the plate colony from which it was derived,—the object aimed at would therefore be achieved.

Usually, however, the colonies cannot be thus readily differentiated, and unless they are "worked up" in an orderly and systematic manner much labour will be vainly expended and valuable time wasted. The following method minimises the difficulties involved.

(A) Inspection.

a. Without opening the plate carefully study the various colonies with the naked eye, with the assistance of a watchmaker's lens or by inverting the plate on the stage of the microscope and viewing with the 1-inch objective through the bottom of the plate and the layer of medium.

b. If gross differences can be detected mark a small circle on the bottom of the plate around the site of each of the selected colonies, with the grease pencil.

c. If no obvious differences can be made out choose nine colonies haphazard and indicate their positions by pencil marks on the bottom of the plate.

(B) Fishing Colonies.—

a. Take a sterile Petri dish and invert it upon the laboratory bench. Rule two parallel lines on the bottom of the dish with a grease pencil, and two more parallel lines at right angles to the first pair—so dividing the area of the dish into nine portions. Number the top right-hand portion 1, and the central bottom portion 8 (Fig. 139). Revert the dish. The numbers 1 and 8 can be readily recognised through the glass and by their positions enable any of the other divisions to be localised by number. This is the stock dish.

b. Slightly raise the cover of the dish, and with a sterile teat-pipette deposit a small drop of sterile water in the centre of each of the nine divisions.

c. With the sterilised platinum spatula raise one of the marked colonies from the "plate 3" and transfer it to the first division in the ruled plate and emulsify it in the drop of water awaiting it. Repeat this process with the remaining colonies, emulsifying a separate colony in each drop of water.

(C) Preliminary Differentiation of Bacteria.—

a. Prepare a cover-slip film preparation from each drop of emulsion in the "stock dish" and number to correspond to the division from which it was taken. Stain by Gram's method.

b. Examine microscopically, using the oil immersion lens and note the numbers of those cover-slips which morphologically and by Gram results appear to be composed of different species of bacteria.



(D) Preparing Isolation Subcultures.—

a. Inoculate an agar slope and a broth tube from the emulsion in the stock dish corresponding to each of these specially selected numbers.

b. Ascertain whether the cover-slips from the nine emulsions in the stock dish include all the varieties represented in the cover-slip film preparation made from the original mixture before plating.

c. If some varieties are missing prepare a second stock dish from other colonies on plate 3, and repeat the process until each morphological form or tinctorial variety has been secured in subculture.

d. Place the stock dishes in the ice chest to await the results of incubation. (If any of the subcultures fail, further material can be obtained from the corresponding emulsion; or if it has dried, by moistening it with a further drop of sterile distilled water.)

e. Incubate all the subcultures and identify the organisms picked out.

4. Differential Media.—

(a) Selective.—Some varieties of media are specially suitable for certain species of bacteria and enable them to overgrow and finally choke out other varieties; e. g., wort is the most suitable medium-base for the growth of torulae and yeasts and should be employed when pouring plates for the isolation of these organisms. To obtain a pure cultivation of yeast from a mixture containing bacteria as well, it is often sufficient to inoculate wort from the mixture and incubate at 37 deg. C. for twenty-four hours. Plant a fresh tube of wort from the resulting growth and incubate. Repeat the process once more, and from the growth in this third tube plant a streak on wort gelatine, and incubate at 20 deg. C. The resulting growth will almost certainly be a pure culture of the yeast.

(b) Deterrent.—The converse of the above also obtains. Certain media possess the power of inhibiting the growth of a greater or less number of species. For instance, media containing carbolic acid to the amount of 1 per cent. will inhibit the growth of practically everything but the Bacillus coli communis.

5. Differential Incubation.—

In isolating certain bacteria, advantage is taken of the fact that different species vary in their optimum temperature. A mixture containing the Bacillus typhosus and the Bacillus aquatilis sulcatus, for example, may be planted on two slanted agar tubes, the one incubated at 40 deg. C., and the other at 12 deg. C. After twenty-four hours' incubation the first will show a pure cultivation of the Bacillus typhosus, whilst the second will be an almost pure culture of the Bacillus aquatilis.

6. Differential Sterilisation.—

(a) Non-sporing Bacteria.—Similarly, advantage may be taken of the varying thermal death-points of bacteria. From a mixture of two organisms whose thermal death-points differ by, say, 4 deg. C.—e. g., Bacillus pyocyaneus, thermal death-point 55 deg. C., and Bacillus mesentericus vulgatus, thermal death-point 60 deg. C.—a pure cultivation of the latter may be obtained by heating the mixture in a water-bath to 58 deg. C. and keeping it at that point for ten minutes. The mixture is then planted on to fresh media and incubated, when the resulting growth will be found to consist entirely of the B. mesentericus.

(b) Sporing Bacteria.—This method finds its chief practical application in the differentiation of a spore-bearing organism from one which does not form spores. In this case the mixture is heated in a water-bath at 80 deg. C. for fifteen to twenty minutes. At the end of this time the non-sporing bacteria are dead, and cultivations made from the mixture will yield a growth resulting from the germination of the spores only.

Differential sterilisation at 80 deg. C. is most conveniently carried out in a water-bath of special construction, designed by Balfour Stewart (Fig. 140). It consists of a double-walled copper vessel mounted on legs, and provided with a tubulure communicating with the space between the walls. This space is nearly filled with benzole (boiling-point 80 deg. C.; pure benzole, free from thiophene must be employed for the purpose, otherwise the boiling-point gradually and perceptibly rises in the course of time), and to the tubulure is fitted a long glass tube, some 2 metres long and about 0.75 cm. diameter, serving as a condensing tube (a tube half this length if provided with a condensing bulb at the centre will be equally efficient). The interior of the vessel is partly filled with water and covered with a lid which is perforated for a thermometer. This latter dips into the water and records its temperature. A very small Bunsen flame under the apparatus suffices to keep the benzole boiling and the water within at a constant temperature of 80 deg. C. The bath is thus always ready for use.

METHOD.—To use the apparatus.

1. Place some of the mixture itself, if fluid, containing the spores, or an emulsion of the same if derived from solid material, in a test-tube.

2. Immerse the test-tube in the water contained in the benzole bath, taking care that the upper level of the liquid in the tube is at least 2 cm. beneath the surface of the water in the copper vessel.

3. The temperature of the water, of course, falls a few degrees after opening the bath and introducing a tube of colder liquid, but after a few minutes the temperature will have again reached 80 deg. C.

4. When the thermometer again records 80 deg. C., note the time, and fifteen minutes later remove the tube containing the mixture from the bath.

5. Make cultures upon suitable media; incubate.



7. Differential Atmosphere Cultivation.—

(a) By adapting the atmospheric conditions to the particular organism it is desired to isolate, it is comparatively easy to separate a strict aerobe from a strict anaerobe, and vice versa. In the first case, however, it is important that the cultivations should be made upon solid media, for if carried out in fluid media the aerobes multiplying in the upper layers of fluid render the depths completely anaerobic, and under these conditions the growth of the anaerobes will continue unchecked.

(b) When it is desired to separate a facultative anaerobe from a strict anaerobe, it is generally sufficient to plant the mixture upon the sloped surface agar, incubate aerobically at 37 deg. C., and examine carefully at frequent intervals. At the first sign of growth, subcultivations must be prepared and treated in a similar manner. As a result of these rapid subcultures, the facultative anaerobe will be secured in pure culture at about the third or fourth generation.

(c) If, on the other hand, the strict anaerobe is the organism required from a mixture of facultative and strict anaerobes, pour plates of glucose formate agar (or gelatine) in the usual manner, place them in a Bulloch's or Novy's jar, and incubate at a suitable temperature. Pick off the colonies of the required organism when the growth appears, and transfer to tubes of the various media.

Incubate under suitable conditions as to temperature and atmosphere.

8. Animal Inoculation.—

Finally, when dealing with pathogenic organisms, it is often advisable to inoculate some of the impure culture (or even some of the original materies morbi) into an animal specially chosen on account of its susceptibility to the particular pathogenic organism it is desired to inoculate. Indeed, with some of the more sensitive and strictly parasitic bacteria this method of animal inoculation is practically the only method that will yield a satisfactory result.



XVI. METHODS OF IDENTIFICATION AND STUDY.

In order to identify an organism after isolation, tube, plate, and other cultivations must be prepared, incubated under suitable conditions as to temperature and environment, and examined from time to time (a) macroscopically, (b) by microscopical methods, (c) by chemical methods, (d) by physical methods, (e) by inoculation methods, and the results of these examinations duly recorded.

It must be stated definitely that no micro-organism can be identified by any one character or property, whether microscopical, biological or chemical, but that on the contrary its entire life history must be carefully studied and then its identity established from a consideration of the sum total of these observations.