7. Open the pericardium with another set of sterile instruments.

8. Sear the surface of the left ventricle with a red-hot iron and remove fluid blood from the heart by means of sterile pipettes (e. g., those shown in Fig. 13, c).

9. Deliver a small quant.i.ty of the blood on the slanted surface of the agar in each of the tubes, and allow it to run over the entire surface of the medium.

10. Place the tubes in the slanting position and allow the blood to coagulate.

11. Return the "blood agar" to the hot incubator for forty-eight hours and eliminate any contaminated tubes. Store the remainder for future use.

_Media for the Study of Mouth Bacteria Generally._

~Potato Gelatine (Goadby).~--

1. Prepare glycerine potato broth (see page 203, sections 1 to 5).

2. Add 10 per cent. gelatine to the potato decoction and bubble live steam through the mixture for ten minutes.

3. Estimate the reaction; adjust the reaction of the medium to +5.

4. Cool the medium to below 60C., clarify with egg as for nutrient gelatine.

5. Filter through papier Chardin.

6. Tube, and sterilise as for nutrient gelatine.

_Media for the Study of Protozoa._

~Tissue Medium (Noguchi).~--_For spirochaetes (cultivations must be grown anaerobically)._

1. Plug and sterilise test-tubes 20 2 cm.

2. Kill a small rabbit with chloroform vapour. Open the abdomen with all aseptic precautions, remove kidneys and t.e.s.t.i.c.l.es and transfer to a sterile gla.s.s dish. Cut up the organs with sterile scissors into small pieces--say 4 millimetre cubes. The four organs should yield from 25 to 30 pieces of tissue.

3. Drop a small piece of sterile tissue into the bottom of each sterilised tube.

4. Take a flask containing about 400 c.c. nutrient agar (+10 reaction), liquefy the medium by heat and cool in a water bath to 50C.

5. Add 200 c.c. ascitic or hydrocele fluid (horse or sheep serum may be employed, but is not so good) to the liquid agar and mix carefully to avoid formation of air bubbles.

6. Fill about 20 c.c. of the ascitic agar into each of the sterilised tubes which already contains a piece of sterile rabbit's tissue, stand all the tubes upright in racks or a jar, and allow agar to set.

7. After solidification pour sterile paraffin oil on the surface of the medium in each tube to the depth of 3 centimetres.

8. Incubate tubes at 37 C. for several days and discard any which prove to be contaminated.

9. Store such tubes as are sterile for future use.

XIII. INCUBATORS.

[Ill.u.s.tration: FIG. 113.--Incubator.]

An incubator (Fig. 113) consists essentially of a chamber for the reception of cultivations, etc., surrounded by a water jacket, the walls of which are of metal, usually copper, and outside all an asbestos or felt jacket, or wooden casing. The water in the jacket is heated by gas or electricity and maintained at some constant temperature by a thermo-regulator. The cellular incubator (Fig. 114) which was made for me[7] some years ago is of the greatest practical utility. Here the central cavity is subdivided by five double-walled part.i.tions (in which water circulates in connection with the water tanks at the top and base of the incubator) and again by iron shelves to form twenty-four pigeon holes. Into each of these slides an iron drawer 35 cm. long 12 cm.

wide 22 cm. high forming a self-contained incubator. The drawer is fitted with a wooden form to which is fixed a handle and a numbered label. The thermo-regulating apparatus is the well-known Hearson capsule.

[Ill.u.s.tration: FIG. 114.--Cellular incubator.]

Two incubators at least are required in the laboratory, for the cultivation of bacteria the one regulated to maintain a temperature of 37C., and known as the "hot" incubator; the other, 20 C. to 22C., and known as the "cool" or "cold" incubator.

Two other incubators, regulated to 42 C. and 60C. respectively, whilst not absolutely, necessary very soon justify their purchase.

~Thermo-regulators.~--The thermo-regulator is the most essential portion of the incubator, as upon its efficient working depends the maintenance of a constant temperature in the cultivation chamber. It is also used in the fitting up of water and paraffin baths, and for many other purposes.

[Ill.u.s.tration: FIG. 115.--Reichert's thermo-regulator.]

Of the many forms and varieties of thermo-regulator (other than electrical), two only are of sufficiently general use to need mention.

In one of these the flow of gas to the gas-jet is controlled by the expansion or contraction of mercury within a gla.s.s bulb; in the other, by alterations in the position of the walls of a metallic capsule containing a fluid, the boiling-point of which corresponds to the temperature at which the incubator is intended to act. They are:

(a) _Reichert's_ (Fig. 115), consists of a bulb containing mercury which is to be suspended in the medium, whether air or water, the temperature of which it is desired to regulate. Gas enters at A, and pa.s.ses out to the jet by B. As the temperature rises the mercury expands and cuts off the main gas supply. As the temperature falls the mercury contracts and reopens the narrow tube C. By means of a thumbscrew D (which mechanically raises or lowers the column of mercury irrespective of the temperature) and the aid of a thermometer the apparatus can be set to keep the incubator at any desired temperature. With this form a special gas burner is required, with separate supply of gas to a pilot jet at the side.

(b) _Hearson's capsule regulator_ consists of a metal capsule hermetically sealed and filled with a liquid which boils at the required temperature, this is adjusted in the interior of the incubator. Soldered to the upper side of the capsule is a thick piece of metal having a central cup to receive the lower end of a rigid rod, through which the movements of the walls of the capsule are transmitted to the gas valve fixed outside the incubator.

The gas valve or governor is shown in figure 116. A is the inlet for gas, C the outlet to burner heating the water jacket, B D a lever pivoted to standards at G, and acted upon by the capsule, through the rigid rod which enters the socket below the screw P.

[Ill.u.s.tration: FIG. 116.--Capsule thermo-regulator.]

The construction of the valve is such that, whenever the short arm of the lever B D presses on the disc below the end B, the main supply of gas is entirely cut off. At such times, however, a very small portion of gas pa.s.ses from A to C, through an aperture inside the valve, the size of which aperture can be adjusted by the screw needle S, hence the gas flame below the incubator is never extinguished.

The expansion of the metal walls of the capsule, which takes place upon the boiling of its contents, provides the motive force, transmitted through the rigid rod to raise the long arm of the lever B D, and as this expansion only takes place at a predetermined temperature, the lever will only be acted upon when the critical temperature is reached, no sensible effect being produced at even 1 C. below that at which the capsule is destined to act.

W is a weight sliding on the lever rod D; by increasing the distance between the weight and the fulcrum of the lower increased pressure is brought to bear upon the walls of the capsule with the result that the boiling-point of the liquid in the capsule is slightly raised, and a range of about two degrees can thus be obtained with any particular capsule.

FOOTNOTES:

[7] Made by the firm of Chas. Hearson & Co., 235 Regent St., London, W.

XIV. METHODS OF CULTIVATION.

Cultivations of micro-organisms are usually prepared in the laboratory in one of three ways: