152 BACTERIOLOGY. 



4. Nutrient gelatin. To one liter of bouillon add 100 grams of 

 finest gelatin. Heat twenty minutes in steam sterilizer. Alkalize 

 with solution of c. p. sodium carbonate and filter with filter paper 

 in steam sterilizer. Fill tubes. Sterilize for three successive days, 

 fifteen minutes each day. 



5. Blood serum. Collect blood in a sterilized container. When 

 coagulated, loosen clot and let stand twenty-four hours in a cool 

 place. Remove clear serum and fill sterile test-tubes with the same. 

 Coagulate at 65 degrees to 75 degrees Centigrade. Sterilize at 58 

 degrees Centigrade, one hour each day for six days. 



6. Potato medium. After thoroughly washing potatoes in soap 

 and water and removing eyes and spots, immerse in mercuric chlo- 

 ride solution, 1 to 1,000, for ten minutes. Make cylinders of the 

 potatoes the size of tubes; bisect obliquely, placing each half in a 

 tube. Sterilize for three successive days, a half hour each day. 



As already suggested, each medium, when prepared, is to be 

 placed in cotton-plugged tubes, and then sterilized. The cotton 

 proves a perfect filter for the bacteria. Agar-agar may be kept for 

 months without any indication of growth of any kind, the cotton 

 plug preventing all access of germs from the outside. 



INOCULATION. 



The inoculation of the media is accomplished by means of a ster- 

 ilized platinum wire. A small portion of the pure culture of the 

 species desired is caught upon the loop at the end of the wire and 

 then drawn over the surface of the medium. Care should be taken 

 to sterilize the wire both before and after using. 



PLATING. 



A pure culture of a species is obtained by plating. This is ac- 

 complished by gently heating, until melted, the nutrient gelatin, or 

 agar-agar, in three tubes. With a sterilized platinum wire, a small 

 portion of the substance containing the bacteria is transferred to 

 the liquefied gelatin, or agar-agar, in tube number one. Then, after 

 sterilizing the wire again, a small quantity of the contents of tube 

 one is transferred to tube two. In like manner, tube three is inocu- 

 lated from tube two. Then the contents of the tubes are trans- 



