354 Tetanus 



twenty-four hours, during which time all of the contained micro- 

 organisms, including the tetanus bacillus, increased in number. 

 He then exposed it for an hour to a temperature of 8o°C., by which 

 all fully developed bacteria, tetanus as well as the others, and the 

 great majority of the spores, were destroyed. As scarcely anything 

 but the tetanus spores remained alive, their subsequent growth 

 gave a fairly pure culture. 



A method more certain in its results has been suggested by Theobald 

 Smith* and depends upon increasing the number of tetanus spores 

 in the inoculation material before the actual isolation of the organ- 

 ism is attempted. Fermentation tubes are employed for the pur- 

 pose, and are filled with dextrose-free culture bouillon. Into each 

 tube a small bit of sterile rabbit- or guinea-pig liver is introduced so 

 as to occupy the constriction between the bulb and upright arm. 

 After sterilization— or after incubation long enough to show that 

 the introduced tissue has caused no contamination— the suspected 

 material is introduced. The tube is kept at 37°C. for forty-eight 

 hours, when it will be found that a great increase of tetanus spores 

 has obtained about the implanted tissue. When examination of 

 stained drops, removed with a sterile pipet, shows plenty of spores, 

 the culture is heated to 8o°C. for thirty minutes to kill any non- 

 sporulating contaminating organisms in the originally implanted 

 media, and" transfers made with a pipet from the neighborhood of 

 the bit of tissue, to fresh appropriate media for tubing or plating. 



Cultivatiori. — The tetanus bacillus is difficult to cultivate because 

 it will not grow where the smallest amount of free oxygen is present. 

 It is hence a typical obHgatory anaerobe. Farranf and Grixoni 

 believe it to have originally been an optional anaerobe, and it is said 

 by these writers that the organism can gradually be accustomed to 

 oxygen so as to grow in its presence. When this is achieved, it Ibses 

 its virulence. These observations have not been confirmed. 



The general methods for the cultivation of anaerobic organisms, 

 are given under the appropriate heading (Anaerobic Cultures), and 

 need not be repeated here. 



The cultures grow best at 37°C., and at this temperature the spore- 

 formation is at a maximum. Growth takes place between the 

 extremes of 15° and 4S°C. 



The cultures usually give off a disagreeable odor resulting from 

 H2S and mercaptan. 



The colonies of the tetanus bacillus, when grown upon gelatin 

 plates in an atmosphere of hydrogen, resemble those of the well- 

 known hay bacillus. There is a rather dense, opaque central mass 

 surrounded by a more transparent zone, the margins of which con- 

 sist of a fringe of radially projecting baciUi. Liquefaction occurs 

 slowly. 



* Tour. Med. Research, 1905, xiv, 193. 



t Centrabl. f. Bakt. u. Parasitenk.," July 15, 1898, p. 28. 



