162 APPENDIX 



in the steam sterilizer. Distribute into tubes and sterilize once 

 in the autoclave for thirty minutes at 115 C. 



Glycerine Agar. Made like ordinary nutrient agar except 

 that 6 per cent, of glycerine is added to the medium after 

 filtration. 



Litmus milk. When machine-separated milk can be obtained 

 fresh it may be used. Frequently however it cannot be obtained 

 fresh; it is then so heavily bacterially contaminated as to be 

 unsatisfactory. In this case ordinary milk must be used and the 

 cream separated in the laboratory centrifuge. It should always 

 be tested to be certain that it is free from preservatives. Steam 

 for one hour in the steamer. Remove any coagulum or scum. 

 Estimate the reaction and bring to a + 1 per cent, reaction. 

 Add pure litmus to a suitable standard tint. Distribute into 

 tubes and sterilize in current steam for one hour on three suc- 

 cessive days. 



Peptone water. Boil together 10 grammes peptone (Witte's), 

 5 grammes sodium chloride and one litre of rain or distilled 

 water. Filter tube, and sterilize in the autoclave for thirty 

 minutes at 1 15 C. 



Blood Serum. Collect the blood from a slaughter house in 

 a clean glass or enamelled metal cylinder. Cleanliness must be 

 exercised, but it is not necessary that special precautions should 

 be taken to collect the blood under sterile conditions. Remove 

 to the laboratory with as little agitation of the blood as possible, 

 and place in the ice chest for 24 hours. Pipette off the separated 

 serum into a clean flask. Transfer to previously sterilized and 

 plugged test tubes, adding about 5 to 6 c.c. to each tube. Place 

 the tubes in the steam sterilizer in a slanting position. With 

 the square form of sterilizer, which is the most useful shape, this 

 can be readily done by making a little frame to support them in 

 this position. Generate a moderate amount of steam, and leave 

 the cover only loosely fitting, until the serum has quite solidified. 

 This takes from one to three hours and must not be hurried. 

 Then fit on the cover tightly and subject to current steam at 

 ioo c C. for one hour. Sterilize again for the two following days 

 at 1 00 C. for thirty minutes each day. 



