8. Place the tubes in the incubator at 37 C. for forty-eight hours in order to eliminate those that have been contaminated. Store the remainder in a cool place for future use.

_Alternative Method._

_Steps 1-5 as above._

6. Sterilise the serum by the fractional method--that is, by exposure in a water-bath to a temperature of 56 C. for half an hour on each of six consecutive days; store in the fluid condition.

7. Coagulate in the insp.i.s.sator when needed.

[Ill.u.s.tration: FIG. 110.--Serum insp.i.s.sator.]

~Serum Water.~--

This forms the basis of many useful media, and is prepared as follows:

1. Collect blood in the slaughterhouse (see page 168) and when firmly clotted collect all the expressed serum and measure in a graduated cylinder.

2. For every 100 c.c. of serum add 300 c.c. distilled water and mix in a flask.

3. Heat the mixture in the steamer at 100 C. for thirty minutes. (This destroys any diastatic ferment present in the serum and partially sterilises the fluid.)

4. Filter if turbid.

5. If not needed at once complete the sterilisation of the serum water by two subsequent steamings at 100 C. for twenty minutes at twenty-four hour intervals.

~Citrated Blood Agar. Guy's.~--

1. Kill a small rabbit with chloroform vapour, and nail it out on a board (as for a necropsy); moisten the hair thoroughly with 2 per cent.

solution of lysol.

2. Sterilise several pairs of forceps, scissors, etc. by boiling.

3. Reflect the skin over the thorax with sterile instruments.

4. Open the thoracic cavity by the aid of a fresh set of sterile instruments.

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

6. Sear the surface of the left ventricle with a red-hot iron.

7. Take a sterile capillary pipette (Fig. 13, c); break off the sealed extremity with a pair of sterile forceps.

8. Steady the heart in a pair of forceps and thrust the point of the pipette through the wall of the ventricle and through the seared area, apply suction to the plugged end of the pipette and fill it with blood.

9. Transfer the entire quant.i.ty of blood collected from the rabbit's heart to a small Erlenmeyer flask containing a number of sterile gla.s.s beads and 5 c.c. concentrated sod. citrate solution. (See page 378.)

10. Agitate thoroughly and set aside for a couple of hours.

11. Melt up several tubes of nutrient agar (see page 167) and cool to 42 C.

12. With a sterile 10 c.c. graduated pipette transfer 1 c.c. citrated blood from the Erlenmeyer flask to each tube of liquefied agar. Rotate the tube between the hands in order to diffuse the citrated blood evenly throughout the agar.

13. Place the tubes in a sloping position and allow the medium to set.

14. Place tubes of blood agar for forty-eight hours in the incubator at 37 C. and at the end of that time eliminate any contaminated tubes.

15. Store such tubes as remain sterile for future use.

~Milk.~--

1. Pour 1 litre of fresh cow's or goat's milk into a large separating funnel, and heat in the steamer at 100 C. for one hour.

2. Remove from the steamer and estimate the reaction of the milk (normal cows' milk averages +17). If of higher acidity than +20, or lower than +10, reject this sample of milk and proceed with another supply of milk from a different source.

Reject milk to which antiseptics have been added as preservatives.

3. Allow the milk to cool, when the fat or cream will rise to the surface and form a thick layer.

4. Draw off the subnatant fat-free milk into sterile tubes (10 c.c. in each).

5. Sterilise in the steamer at 100 C. for twenty minutes on each of five successive days.

6. Incubate at 37 C. for forty-eight hours and eliminate any contaminated tubes. Store the remainder for future use.

~Litmus Milk.~--

1. Prepare milk as described above, sections 1 to 3.

2. Draw off the subnatant fat-free milk into a flask.

3. Add sterile litmus solution, sufficient to colour the milk a deep lavender.

4. Tube, sterilise, etc., as for milk.

~Nutrose Agar (Eyre).~--

(This is a modification of the well known Drigalski-Conradi medium originally introduced for the isolation of B. typhosus).

1. Collect 250 c.c. perfectly fresh ox serum (_vide_ Blood Serum, page 168, steps 1 to 5) and add to it 450 c.c. sterile distilled water.

2. Weigh out agar powder, 20 grammes, and emulsify it with 250 c.c. of the cold serum water.

3. Weigh out