The Elements of Bacteriological Technique Part 61

_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.

[Ill.u.s.tration: FIG. 120. Esmarch's roll culture on block of ice.]

In order that the medium may adhere firmly to the gla.s.s, 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 gla.s.s dish, with a square sheet of plate gla.s.s 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 42C., 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.

[Ill.u.s.tration: FIG. 121.--Handy form of water-bath for melting tubes of agar and gelatine previous to slanting them; or to making shake cultures or pouring plates.]

Method of "Pouring" Plates.--

1. Place the gla.s.s 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 50C.--agar sets almost immediately at the room temperature and by slightly r.e.t.a.r.ding the process lumpiness is avoided; cover the dish with the square sheet of gla.s.s.

2. Place the spirit level on the sheet of gla.s.s and by means of the levelling screws adjust the surface of the gla.s.s 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.

[Ill.u.s.tration: FIG. 122.--Plate-levelling stand.]

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

[Ill.u.s.tration: FIG. 123.--Plate-levelling stand, side view.]

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.

[Ill.u.s.tration: FIG. 124.--Mixing emulsion for plates.]

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).

[Ill.u.s.tration: FIG. 125.--Pouring plates.]

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 C. or 42 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.~--