260 LABORATORY EXERCISES IN BACTERIOLOGY. 



mospheres of hydrogen. After identification of the gross characteristics 

 as those of Bacillus tetani, study the minute appearances, sporulation, flag- 

 ellation, staining by Gram's method, coagulation of milk, acid production, 

 gas production, and indol production for comparison with the known prop- 

 erties of this organism. (Note. It is by no means necessary that Bacillus 

 tetani be the only anaerobe likely to be obtained from such source ; it is, 

 however, a common one, and others which may be obtained in mixture or 

 alone are to be carefully differentiated from it.) 



5. Cohn's Heating Method. This procedure is intended for the separation 

 of 'spore-forming bacteria from vegetative bacteria, and depends upon the well-known 

 resistance of spores to temperatures fatal to ordinary germs. An infusion known to 

 contain spores of some organism, as well as various vegetative varieties, is boiled for 

 one minute (or, when it is known that the spores are very resistive, for a longer time), 

 by which means all the adult types of bacteria are usually destroyed, most, if not all, 

 of the spores being uninjured by the heat. It is now quickly cooled and from it inocu- 

 lations are made in the usual manner in the usual media and the cultures placed in 

 proper surroundings for development of the spores (media best suited for germination 

 of the spores are those containing plenty of moisture, as bouillon). There may, of 

 course, be spores of several bacterial varieties present in the material, and in such 

 case the colonies resulting from development of the spores are to be separated from 

 each other by other suitable methods. 



Exercise 79. Make an infusion of hay by placing a bunch of hay in a jar 

 of water and allowing it to soak over night at incubator temperature. The 

 following morning the reddish infusion is strained from the hay through a 

 piece of cheese-cloth and boiled for one minute. Cool by placing the con- 

 tainer in a vessel of cool water. Plant from the boiled fluid to a number of 

 tubes of agar, gelatine, and bouillon, and allow to grow in the ordinary 

 atmosphere at room temperature. In a day or two the characteristic ap- 

 pearances of the hay bacillus will appear without contamination with the 

 other organisms which were probably present upon the hay. 



Or, having strained the infusion from the hay, dilute it with water until 

 it is about 1006 in specific gravity and neutralize by the addition of sodium 

 hydrate (litmus used as indicator). Distribute the fluid in a number of 

 sterile tubes, boil for one minute, and then place in the incubator or in 

 room temperature. In a day or two a dense white surface growth of Bacil- 

 lus subtilis will have been obtained. 



