H. a. Thornton , 269 



however, there is a remarkable fall in colony development on media 

 having an acidity higher than pH 6-8. 



Thus, in preparing a standard medium, some latitude for changes in 

 reaction during sterilisation is permissible, if its reaction be kept within 

 a range of from ^H 7 to ^H 7-8. On the other hand, if the medium be on 

 the acid side of neutrality, a slight increase in its acidity may cause its 

 H-ion concentration to reach the critical point involving a marked fall 

 in colony development. 



For this reason, the use of an ammonium salt in a standard medium 

 is a disadvantage, for if such a medium be brought to the alkaline side 

 of neutrality with NaOH, some of the ammonia is liberated, and, during 

 sterilisation, is driven off, bringing the reaction back to neutral point. 

 A slight hydrolysis of the carbohydrate constituent of the medium during 

 sterilisation is now sufficient to bring the H-ion concentration up to a 

 dangerous point. Thus if Conn's sodium asparaginate agar(3) (containing 

 ammonium phosphate) be adjusted to a slightly alkaline reaction before 

 sterilisation, the H-ion concentration of the medium after autoclaving 

 is found to be approximately ^H 6-8, so that a slight further increase 

 in acidity, arising from any cause, would harmfully affect the medium. 



From these considerations, it was decided to use nitrate as the in- 

 organic nitrogen source in the medium. Trials showed that the colony 

 development was as good on a neutral medium containing nitrate as on 

 one containing an ammonium salt, while, in the former case, the risk of 

 a detrimental increase in acidity, during autoclaving, need not be in- 

 curred. The following comparison shows the advantage of nitrate in this 

 connection. The medium CV (see p. 259) was made up with 0-05 per cent, 

 asparagine, and divided into two portions to one of which 0-1 per cent. 

 (NH4)2S04 was added, and to the other equivalent nitrogen in the form 

 of KNO3. The reaction of both media was standardised to ^H 7-4 and 

 the media were autoclaved at 15 lbs. pressure for 15 minutes. After 

 sterilisation the reactioii of the ammonium sulphate medium was found 

 to be pH 6-7, while that of the nitrate medium was ^H 7-2. 



The percentage of KNO3 used in the count medium was finally fixed 

 at 0-05 per cent, as a result of work above described on the control of 

 spreading colonies. 



The chief cause of the development of acidity in media during sterili- 

 sation is believed to be the hydrolysis of the carbohydrate constituent. 

 I therefore made tests of media containing various sugars and related 

 compounds to discover which source of energy material was most 

 suitable. 



