XIII. 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 pure cultivations of various organisms. But before undertaking a systematic study of selected bacteria, it is 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 cultivation, in pus and other morbid exudations, infected tissues, or water or food-stuffs.

Fig. 111. Hæmatocytometer 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 hæmatocytometer, Fig. 111), diluting the fluid by the addition of sterile water or bouillon until a given volume (usually I c.c.) of the dilution should contain but one organism. By planting this volume of the fluid into several tubes or flasks of nutrient media, it occasion

ally happened that the resulting growth was the product of one individual microbe. A method so uncertain is now fortunately replaced by many others, both reliable and convenient, and in those methods selected for description here, the isolation of the required bacteria is effected

1. By plate cultivation:

(a) Gelatine.

(b) Agar.

(c) Serum agar.

(d) Blood agar.

2. By Esmarch's roll cultivation :

(a) Gelatine.

(b) Agar.

3. By serial cultivation.

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 they do an opportunity for the simultaneous isolation of more than one of the bacteria present in a mixture.

1. Plate Cultivations.

(a) Gelatine (vide page 145).

(b) Agar (vide page 149).

(c) Alkaline serum agar (vide page 157).

These plates are poured in a manner precisely

similar to that adopted for nutrient gelatine and nutrient agar plates (vide page 182).

(c') 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 1, 2, and 3, in a water-bath regulated at 45° C.; allow sufficient time to elapse for the temperature of the contents of each tube to reach that of the water-bath.

2. Make three dilutions of the inoculum in the three liquid serum tubes, treating them exactly, as if they were tubes of liquefied gelatine (vide page 183); replace them in the water-bath.

3. Take serum tube No. 1 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.

4. Mix thoroughly and pour plate No. 1 secundum

artem.

5. Treat the remaining dilutions in a similar fashion, and pour plates Nos. 2 and 3 in the usual way. 6. Label and incubate.

(d) Human Blood Agar.

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 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 (or a sterilised camel's hair brush) with a small quantity of the inoculum. Raise the cover of the plate, introduce the loop, mix

its contents with the drop of blood, and finally smear the mixture over the surface of the agar.

5. Withdraw the loop and close the plate.

6. Label and incubate.

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

2. Esmarch's Roll Cultivation.

(a) Gelatine.

1. Liquefy three tubes of gelatine by heat.

2. Prepare three dilutions of the inoculum (as described for plate cultivations).

3. Roll the tubes, held almost horizontally, in a groove made in a block of ice, until the gelatine has

Fig. 112.-Esmarch's roll culture on block of ice.

set in a thin film on the inner surface of tube (Fig. 112); or under the cold-water tap.

(b) Agar roll cultures are made in precisely the same way as gelatine roll cultures, but in order that the medium may adhere firmly to the glass, the agar used for the purpose 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 seldom prepared, and are in point of fact practically obsolete.

3. Serial Cultivations.-These are usually made

1

upon agar or blood-serum, although gelatine may also be used. The method is as follows:

1. Take at least six "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.

5. In like manner plant the remainder of the tubes in the series.

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. Towards 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 neighboring colonies. 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 torule and yeasts and must always 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 sufficient to inoculate wort from the mixture and incubate at 37° C. for twenty-four hours. Plant a fresh tube of wort from the resulting growth and incubate. Repeat the pro