326 PRODUCTION OF V^OLATILE FATTY ACIDS 



Methods. 



Media. — The medium for the growth of the organisms consisted of 

 1.0 per cent of "Difco" peptone, and 0.5 per cent of Na2HP04-2H20, 

 with or without 1.0 per cent of Merck's "highest purity" dextrose. 

 The reaction of this medium after sterilization ahvays lay between 

 pH 7.1 and 7.2. The hydrogen ion concentrations of the media and 

 cultures were determined colorimetrically. 



Cultures. — -The organisms were inoculated into sterile 10 cc. por- 

 tions of the media and incubated at 37°C. for 18 hours. These virile 

 cultures were then poured into 990 cc. of the sterile media contained 

 in 2 liter Florence flasks and incubated at 37°C. for the period of time 

 indicated. The pouring was conducted under conditions which 

 reduced the chance for contamination by this method. No control 

 plates were run to determine this factor, since it was held that the 

 bulky inoculation was sufficient to take care of any slight contami- 

 nation should such occur. The regularity in the content of volatile 

 fatty acids leads us to believe that there was no contamination. It 

 was found that the maximum production of total volatile acids in 

 sugar medium under these conditions occurred shortly before the 

 first 12 hour period and the total quantity remained quite stationary 

 during the following 60 hours (Fig. 1). Accordingly, the cultures 

 were allowed to become 48 hours old merely as an arbitrary period. 

 The cultures in non-sugar media were allowed a longer period before 

 examining for volatile acids, because volatile bases were investigated 

 in the same cultures. It is barely possible that the values for the 

 volatile acids obtained in the case of these cultures grown for a longer 

 period of time are somewhat too low because of the chance of volatile 

 acid utilization. 



Anaerobic Cultures. — -"Anaerobic" conditions were obtained by 

 evacuation of the inoculated flasks with a mercury-vapor pump (a 

 single Kraus modeled pump) in series with a motor-driven Geryke 

 vacuum pump. Gum stoppers, bearing a thick- walled tube drawn 

 out to a narrow neck to permit sealing with a fine tipped flame, were 

 cemented while hot with a heavy rubber-rosin cement into the necks 

 of the flasks. These stoppers had been previously sterilized, and the 

 glass tubes contained a plug of cotton to prevent contamination of the 



