1*4, 6 MANUAL OF METHODS FOR PURE CULTURE STUDY 



Adjusting reaction. The reaction of all these media is to be adjusted 

 to a hydrogen-ion concentration near neutrality (i.e. pH 7.0). The 

 beef -extract broth and agar normally require no adjustment to 

 bring them to this reaction; the others need the addition of alkali 

 ordinarily. In all cases the reaction should be tested, even though no 

 adjustment is thought to be necessary. For detailed instructions in 

 testing or adjusting the reaction one may consult Leaflet IX of this 

 Manual, entitled The Determination of pH and Titrable Acidity 

 or may follow the directions given by the American Public Health 

 Association (1936, p. 199). For ordinary purposes, however, good 

 results will be obtained by adjusting the media to the neutral point 

 of brom thymol blue;^ the medium is brought to such a reaction as to 

 turn this indicator a distinct grass-green (neither yellow green nor 

 blue green). This color corresponds closely to the desired reaction. 

 Another equally satisfactory method to bring the medium to this 

 reaction is to add suflBcient alkali to cause the first faint trace of 

 permanent pink to appear with phenol red.^ Reaction should always 

 be checked after final sterilization of each batch. 



Natural storage media. Recent years have shown quite a tendency 

 to employ natural media, particularly skim milk or soil, for the 

 storage of stock cultures. These materials are frequently used in their 

 natural state, without addition; but more often a small quantity of 

 calcium carbonate is added to neutralize acids formed. This addition 

 is absolutely necessary in the case of limestone-free soils or in the 

 case of milk when the organisms to be stored produce acid from 

 lactose. 



B. General Differential Media 



Plai7i gelatin for use in the determination of gelatin liquefaction. 

 This is made up like beef -extract gelatin but without the beef- 

 extract and peptone; it consists of 10% "Bacto-gelatin" (or some 

 other brand of the same jellying strength) dissolved in distilled water 

 and the reaction adjusted to pH 7.0. 



Sugar broths. Just before sterilization 0.5-1% of the required 

 carbohydrate is ordinarily added to beef-extract broth; the same 

 proportions are also usually to be employed in studying the 

 fermentation of any related carbon compound (e.g. alcohol or gluco- 



*Use 0.04% brom thymol blue or 0.02% phenol red. Alcoholic solutions may be 

 employed without neutralizing, or aqueous solutions of the sodium salts prepared as 

 directed by Clark (1928, p. 91-95) or as explained in Leaflet IX of this Manual (p. 



IX4X-11). 



