10 c.c. pipette.

Gla.s.s 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 gla.s.s dish in the centre of the gla.s.s slab, and stand the cultivations inside this.

3. Place a sufficient number of pyrogallic acid tablets at one side of the gla.s.s 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 f.l.a.n.g.e of the bell jar with resin ointment and apply the jar firmly to the gla.s.s slab, covering the cultivations--so arranged that the long tube pa.s.ses with its lower end into the gla.s.s dish at a point directly opposite to the pyrogallic acid tablets. Lubricate the two stop-c.o.c.ks 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-c.o.c.ks.

6. Connect a long, straight piece of gla.s.s 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-c.o.c.k of the entry tube, then that of the exit tube b. Screw down the clamp and remove the gla.s.s 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-c.o.c.k of tube a and with two or three strokes of the air pump, aspirate a small quant.i.ty of gas, so creating a slight vacuum.

Then shut off the stop-c.o.c.k 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-c.o.c.k. (The water dissolves the soda and pyrogallic acid converting the latter into alkaline pyro. and so bringing its latent capacity for oxygen into action).

[Ill.u.s.tration: FIG. 137.--Bulloch's jar.]

10. Reverse the tubes from the tubulures so that they meet, out of harm's way, over the top of the bell gla.s.s; 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.

[Ill.u.s.tration: FIG. 138.--Haematocytometer cell, showing, a, section through the centre of the cell, and b, a magnified image of the cell rulings.]

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 circ.u.mstances, 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).