160 MANUAL OF MICROBIOLOGICAL METHODS 



Conn and Hucker (1920). Agar slants may be inoculated either on the 

 surface alone or partly on the surface and partly in a stab at the base. 

 It has been found in practice that if much gas is produced, it may occur 

 at the very base of the column of agar even when all the growth seems to 

 occur on the surface, but if there is reason to suspect that gas production 

 is being overlooked, shake cultures may be used in addition to the agar 

 slant. 



Demonstration of cleavage. Utilization of the sugar (or other fer- 

 mentable substance) may be indicated by a chemical determination show- 

 ing its partial or complete disappearance or by the demonstration of the 

 end products of fermentation. These end products are generally organic 

 acids, sometimes accompanied with the evolution of gases, e.g., free 

 hydrogen, carbon dioxide, or occasionally methane. Determinations of 

 the amount of sugar remaining or of the nature of the organic acids pro- 

 duced are very valuable in discriminating investigations but require 

 time-consuming chemical work that is difficult to employ in the routine 

 examination of large numbers of cultures. These chemical methods are 

 referred to in more detail elsewhere (Chap. VIII). In many instances, 

 however, a sufficient amount of information is obtained merely by demon- 

 strating an increase in acid or the presence of gas. 



For routine work in the case of organisms concerning which little 

 advance information is at hand, the use of indicators is especially valuable 

 in determining whether or not production of acid has occurred. It must 

 be remembered, however, that in many instances more useful and sig- 

 nificant information can be obtained by means of titration. For rapid 

 tests, useful with some organisms, see Hannan and Weaver (1948). 

 Cowan (1953a and h) has developed a rapid method to replace an earlier 

 micromethod which proved unreliable. 



When the indicator method is employed, the indicators may be incorpo- 

 rated with the media in the first place or may be added subsequently when 

 the final reaction is being determined. If they are added when determin- 

 ing final reaction, the color obtained should be compared with color 

 standards (see Chap. IV) in order to secure accuracy. The use of indi- 

 cator media is less accurate but is a much more rapid procedure; when 

 the cultures are growing on agar, moreover, it is the only satisfactory 

 procedure. 



When using indicator media, make them up according to the directions given on 

 page 53 of Chap. III. The indicator most commonly added is bromcresol purple, 

 but with organisms producing considerable acid, bromcresol green or even brom- 

 phenol blue may be employed. When studying a series of unknown organisms it is 

 often advisable to inoculate all onto the prescribed sugar medium with bromcresol 

 purple; later those that show acid may be reinoculated onto the same medium with 

 bromcresol green; and subsequently those positive to this indicator upon the same 



