and Laboratory Methods. 1^13 



shows that the addition of peptone greatly favors anaerobic growth. Bouillon 

 without peptone proved to be a very poor medium, the addition of one per cent, 

 peptone very materially improved it, and the maximum effect was reached with 

 bouillon containing two per cent, peptone. Any addition above two per cent, 

 did not have any noticeable effect on, or lowered the power of nutrition of such 

 a medium. The same author also highly recommends the addition of small 

 amounts of litmus to the nutrient medium. 



Smith (1899) adds small sterile pieces of internal organs of rabbits, guinea- 

 pigs, etc., to ordinary sterile bouillon in fermentation tubes, and successfully uses 

 these tubes for anaerobic cultures without re-sterilizing the bouillon. In this 

 way the medium contains some albumin in solution, and therefore offers favora- 

 ble conditions for bacterial growth. (For method see V. f. Smith, 1899.) 



As regards the action of sugars and other reducing agents, the reader is 

 referred to IV. 



2. Reaction of Medium. 



Most of the scientists, who have studied anaerobic bacteria, agree that this 

 group of organisms generally requires a medium with a slightly alkaline reaction. 

 Kitasato observed that the bacillus of Rauschbrand grows best in a slightly acid 

 medium. According to Novy, 1 893, anaerobic bacteria thrive well in strongly alka- 

 line bouillon, but in such cultures their vitality decreases rapidly and the organisms 

 die in a comparatively short time. Thus, Bacillus oedamatis maligni No. 2 died 

 within two days after inoculation, although it showed very vigorous growth at the 

 start. 



3. Age of Medium. 



Since it is of great importance, if good results are to be obtained, that the 

 medium should be as nearly free from oxygen as possible, it is obvious that old 

 media are not suited for anaerobic cultures. The older the medium, the longer 

 has been the time during which the latter has been absorbing oxygen from the 

 air. It is, therefore, necessary that, for anaerobic cultures, fresh media be used. 

 Novy (1893) Hammerl (1901) and other investigators found that in a given series 

 of bouillon cultures the anaerobes developed much more vigorously in freshly 

 made than in stale medium. If stale bouillon is used it should be reboiled for 

 the purpose of driving out the oxygen present, before the medium is inoculated. 

 The same holds true in the case of agar, while gelatin tubes evidently do not 

 require this precaution. Thus Novy was able to cultivate anaerobic bacteria in 

 gelatin that was six months old just as readily as in freshly made gelatin. 



4. Age and Quantity of the Inoculating Material. 



a. Age. Novy (1893), in a series of experiments with bouillon cultures of 

 ages ranging from one day to four months, observed that where old cultures are 

 used as inoculating material generally negative results are obtained, while, where 

 he made the inoculations with young cultures, no difficulty was encountered in 

 obtaining vigorous growth. He therefore concludes that the failure to secure 

 the desired growth is often due to the age of the culture used. 



b. Quantity. For the successful cultivation of anaerobic bacteria it is gener- 

 ally advisable and necessary to use large quantities of inoculating material in 

 order to start growth. Hence, where inoculations with small amounts of culture 

 material have proved unsuccessful, positive results may be obtained by using 

 larger amounts of it. After the bacteria have begun to grow there is little diffi- 

 culty in continuing their development. Otto F. Hunziker. 

 New York State Veterinary College, Cornell University. 



