~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 20C., or at any rate at a temperature not exceeding 22 C. (that is, in the "cold" incubator); whilst fluid media and all other solid media are incubated at 37 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:

[Ill.u.s.tration: FIG. 117.--Inoculating tubes, seen from the front.]

~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. Pa.s.s 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. Pa.s.s 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 ma.s.s 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. Pa.s.s 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. Pa.s.s 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.

[Ill.u.s.tration: FIG. 118.--Sloped or slanted medium for streak or smear culture.]

[Ill.u.s.tration: FIG. 119.--Straight tube.]