156 



with water containing carbon dioxide are always less acid than desired, 

 in fact some media are alkaline, note columns headed (4) and (5). 



Distilled water is believed by so many to be carbon dioxide free, no 

 matter whether the water from which it is made is hard or soft, that, 

 as a rule bacteriologic culture media has been adjusted to a less degree 

 of acidity than planned for. Litmus is not sensitive to carbonic acid, 

 thus it seems fair to assume that acidities of culture media, observed 

 with phenolphthalein, but which do not prove out with litmus may be 

 partly due to the carbon dioxide present in the dilution water added to 

 the aliquot titrated. Anthony and Ekroth (6) make statements con- 

 cerning the work of MacNeal, Muir and Ritchie, Stilt, and others con- 

 cerning comparisons between litmus and phenolphthalein titrations. Ti- 

 trations with phenolphthalein carried out near the boiling point of the 

 media are unreliable, due to the inci'eased hydrolysis of the media and 

 to the fact that phenolphthalein is more sensitive in cold solutions (15). 



Hot and Cool Titrations With Especially Prepared Media. 



An experiment was conducted to find out the effect of temperature 

 on acidity titrations when agar agar plus gelatin were present with 

 salts that undergo changes in hydrolysis with increasing temperature. 

 The agar agar and gelatin used were selected because of their small 

 changes in acidity when autoclaved or heated. They were selected by a 

 procedure described by the author (16) in another article. Unfiltered 

 water solutions of the agar and gelatin used were free from pi-e- 

 cipitates and thus by themselves did not even need filtering. 



Two basic media were made up according to the following procedure: 



Agar agar Media. — Thirty grams of agar agar were dissolved in 

 the inner part of a double boiler in 2,000 cc. of carbon dioxide free dis- 

 tilled water. When solution was complete distilled water (carbon diox- 

 ide free) was added to make the weight of agar and water up to 2,000 

 gms. 



Agar plus Gelatin Media. — This was made up exactly as the agar 

 media except that 7.5 grams of gelatin were added per 1,000 grams of 

 media. 



Fifty gram aliquots of each media were weighed out into clean 

 250 cc. erlenmeyer flasks. Thirty-four aliquots of each media were 

 taken. The chemicals were previously prepared by making water solu- 



