124 CULTURE MEDIA AND THEIR STERILIZATION 



employed in the collection of an antitoxic or immune serum, it may be 

 here described. 



Method of Obtaining Sterile Blood Serum from a Horse. 1. Restrain 

 the horse so that it can be readily manipulated and cannot disturb 

 the operator. 



2. Shave a few square inches of skin a little above the middle of 

 the jugular vein. 



3. Sterilize the skin by scrubbing well with soap and water, then 

 with alcohol, next with solution (1 to 500 to 1000) of bichlorid of 

 mercury, and finally with sterile distilled water. (An alternate 

 method consists in shaving the skin on the previous day and applying 

 a 10 per cent, alcoholic solution of iodin to the entirely dry skin, half 

 an hour before the operation.) Cover the sterilized skin with sterile 

 cotton. 



4. Have ready a large, sterile hypodermic needle or small curved 

 trocar connected with a small rubber tube, leading into a sterile, 

 cotton-stoppered, cylindrical glass vessel. The entire apparatus must 

 be sterile. 



5. When the preparations are completed the operator must sterilize 

 his hands as carefully as for an important aseptic operation. An 

 assistant then compresses the jugular vein in the lower portion of 

 the neck. After removal of the sterile cotton from the previously 

 shaved and sterilized skin the trocar is pushed into the jugular 

 vein and the blood flows through it into the sterile receptacle. 



6. When a sufficient quantity has been collected it is placed on 

 ice for twenty-four to forty-eight hours and the serum is then de- 

 canted off from the coagulum. Too much stress cannot be laid upon 

 the importance of performing every step in an absolutely aseptic 

 manner. 



Sterilization of Bacterial Culture Media. This is generally accom- 

 plished by means of steam heat, generated in a suitable vessel called 

 a steam sterilizer. In the ordinary apparatus the steam passes out 

 without obstruction; in others it is retained under a pressure of 

 several atmospheres and the temperature rises above 100 C. Those 

 of the latter type are called autoclaves. Sterilization is completed in 

 them in a much shorter time (about one hour) than in the common ster- 

 ilizers with free streaming steam. When culture media are subjected 

 in the steam sterilizer to the temperature of boiling water (100 C.) 

 for a period of three to four hours or longer, all bacteria and their 

 spores are destroyed. This method is called continuous sterilization. 



Certain media, like blood serum or gelatin, however, are rendered 

 worthless by this method. The former becomes a grumous, broken-up 

 mass, the latter turns into a permanent fluid and refuses to coagulate 

 again. Continuous sterilization in the ordinary steam sterilizer or 

 the autoclave cannot, therefore, be employed in the case of these 

 substances or transudates like ascitic fluid, etc., but short periods of 

 discontinuous heating will answer the purpose. This method is 



