11^4-8 MANUAL OF METHODS FOR PURE CULTURE STUDY 



carefully adjusted to pH 7.0 with brom thymol blue before adding 

 any indicator; then 1 ml. of a saturated aqueous solution of each 

 indicator should be added. This mixture of indicators changes very 

 slowly from purple to yellow through a long range (from about 

 pH = 8.0 to about pH = 5,2) extending for a considerable distance on 

 each side of neutrality. By comparing with a blank tube of the 

 neutral medium it is easy to detect an increase either in acidity or in 

 alkalinity. 



Brom cresol green (introduced by Cohen, 1922) indicates moder- 

 ately great changes to the acid side of neutrality as its range is pH = 

 3.8-5.4. It is best kept for this purpose in a 2% alcoholic solution, 

 adding 2.0 ml. to each litre of medium. Used in agar media it shows 

 appreciable change from green to yellow if the reaction is as high as 

 pH = 5.2; and from that point to the acid end of its range it is very 

 satisfactory. 



Brom phenol blue is now suggested by Cohen (1927) to replace 

 brom-chlor-phenol blue which he described earlier. As its range is 

 from pH = 3.0 to pH = 4.6 it is of value in indicator media only with 

 organisms showing a very high final hydrogen-ion concentration. 

 For this reason it is very seldom called for; but it is valuable in dis- 

 tinguishing the most vigorous acid formers. 



In spite of all the arguments in favor of the sulphonphthaleins as 

 H-ion indicators, litmus still remains popular among bacteriologists, 

 aad no perfect substitute for it has been obtained. Its advantages 

 are that it is a long-range (even if not highly accurate) indicator, 

 showing changes on both sides of the neutral point, and at the same 

 time indicates changes in oxidation-reduction potential. This makes 

 it useful for diagnostic purposes when employed in certain media, 

 notably in milk; and no combination of indicators showing all the 

 characteristics of litmus has yet been proposed. Unfortunately, at 

 the time when this (9th) edition of this leaflet goes to press, the source 

 of the lichens from which litmus is manufactured has been cut off 

 from the United States, and this indicator is becoming harder and 

 harder to obtain. 



Nitrate broth. For routine work 0.1% ICNO3 is added to the regular 

 formula for beef-extract broth and reaction adjusted as usual. 

 Similarly routine nitrate agar should contain 0.1% KNO3 added to 

 the ordinary formula for beef-extract agar, with the reaction properly 

 adjusted. Modification of these formulae is often necessary as ex- 

 plained on p. V42-IO Leaflet V of this Manual. A synthetic nitrate 

 medium often found useful is given below (p. 1144-I4). 



