ii.] PREPARATION OF CULTURE MATERIAL. 13 



simultaneously with the turning off of the burner. This broth 

 so prepared is placed in the incubator at 32 38 C. and kept 

 there from one to three weeks. If, as is generally the case, it 

 remains limpid, it is considered completely sterile. 



2. Peptone and Sugar Solution. Beef peptone (Savory and 

 Moore's) is dissolved in distilled water, over a burner, to the 

 amount of about 2 per cent. ; to the solution is added cane 

 sugar to the amount of about 1 per cent. ; so that every 100 

 ccm. of the fluid contains two grammes of peptone and one 

 gramme of sugar. When dissolved it is well neutralised and 

 then filtered (the vessels being of course also in this, as in all 

 other cases, sterilised by overheating) into flasks, and treated 

 in the same manner as the broth. 



The same fluid can be used without the addition of the 

 cane-sugar; or peptone dissolved to the amount of 1 to 2 

 per cent, in broth. 



3. Bucfaier's Fluid. 10 parts of Liebig's extract, and 8 parts 

 of peptone, in 1,000 parts of water. 



4. Hydrocele Fluid (Koch). A new or well sterilised (by 

 over-heating) trocar and cannula are used for the tapping ; to 

 the cannula is fixed an india-rubber tube that has been soaking 

 in strong carbolic acid solution for forty-eight hours. The distal 

 end of the tube is introduced carefully and rapidly into the 

 neck of a sterilised flask plugged with sterile cotton-wool, and 

 the fluid thus allowed to flow into the flask to about two-thirds 

 of its volume. This is then exposed in a water- or sand-bath 

 to a temperature of from 58 to 62 C. for three to five hours 

 on five or six consecutive days. Placed then into the incubator 

 at 32 38 C. for from one to three weeks, the fluid remains 

 limpid. 



5. Blood Serum (Koch). A glass cannula and india-rubber 

 tubing are soaked for forty-eight hours in strong carbolic acid ; 

 the cannula is tied into the carotid artery of a healthy sheep, 

 and the arterial blood, after opening the clip at the proximal 

 end of the artery, is allowed to flow into a sterile flask, the 

 distal end having been introduced into the neck of the flask as 

 above. After letting it stand for twelve to twenty-four hours the 

 clear serum is taken off by means of a large sterilised glass 

 pipette or glass syphon ; this is carefully introduced between 

 the cotton-wool plug and glass neck, and then discharged into a 

 sterile plugged flask or large test-tube, the pipette or syphon 

 being also here carefully introduced between cotton-wool plug 

 and glass. 



This stock serum is then heated for successive days in the 

 same manner as the hydrocele fluid, 



