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 hÆmatocytometer, 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:

[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.

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° 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° 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 torulÆ 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° 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°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°C., and the other at 12° 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°C.—e. g., Bacillus pyocyaneus, thermal death-point 55°C., and Bacillus mesentericus vulgatus, thermal death-point 60°C.—a pure cultivation of the latter may be obtained by heating the mixture in a water-bath to 58° 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° 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° 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°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° 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°C.

4. When the thermometer again records 80°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°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.