184 IVAN V. SHUNK 



as otherwise grease from the fingers may get on the cover- 

 glasses which should never be touched with the fingers except 

 around the edges. When wiped, the cover-glasses are placed in 

 a well cleaned Petri dish and heated in an oven at a tempera- 

 ture of about 275 to 300°C. for one hour. In addition to these 

 precautions I frequently flame the cover-glasses a little just 

 before using. 



In obtaining cultures to stain, it is usually necessary to have 

 a culture eighteen to twenty hours old. However, in the case of 

 certain organisms which multiply very rapidly, and which begin 

 to form spores in a few hours, cultures not more than twelve 

 hours old are sometimes desirable, as for example in the case of 

 Bacillus subtilis. Certain other organisms which develop only 

 at relatively low temperatures, and grow slowly, may give good 

 results from much older cultures, even a week or more, as for 

 example Pseudomonas janthina. For all of my work which has 

 given favorable results, cultures on slanted agar-agar have been 

 used. 



The tube of slanted agar-agar is inoculated, and incubated at 

 37° if it will grow under those conditions; otherwise it is allowed 

 to remain at room temperatures. When the culture is ten to 

 twenty hours old or older, depending upon the species, some of 

 the bacteria are carefully removed from the surface of the agar- 

 agar, and placed in a test tube containing 2 or 3 cc. of sterilized 

 tap water. Care is taken to remove none of the culture media 

 while transferring the bacteria. It is best to have the tap water 

 at the same temperature as the culture, and to accomplish this 

 I usually place the tubes of sterile water in the incubator while 

 the cultures are developing. After the transfer to the water, 

 the tube is allowed to stand fifteen to twenty minutes, or longer, 

 at room temperatures, or preferably in the incubator, in order 

 to allow the bacteria to diffuse through the water, and to permit 

 of some slight development. 



Several small drops of this water are then placed on a clean 

 cover-glass with a very small platinum loop, and without any 

 attempt at spreading them, the drops are allowed to dry in the 

 air. If the drops are small they will dry more rapidly. Good 



