54 MANUAL OF MICROBIOLOGICAL METHODS 



extracts contain fermentable carbohydrate. In all instances, control 

 tubes to which carbohydrate has not been added must be inoculated to 

 check changes of pH due to the breakdown of carbohydrates or other 

 substances. 



The synthetic medium of Ayers, Rupp, and Johnson (1919) may be 

 used if a peptone-free medium is desired and if the organism being 

 studied can utilize ammonium salts as a source of nitrogen. It is pre- 

 pared as follows: NH4H2PO4, 1.0 g; KCl, 0.2 g; MgS04-7H20, 0.2 g; 

 water, 1,000 ml; carbohydrate, 10.0 g; adjust the pH by addition of 

 1 N NaOH. 



The soluble carbohydrates or polyalcohols are added to the basal 

 medium at a level of 0.5-1 .0 per cent. (For precautions taken in sterihza- 

 tion see page 39.) The indicator (commonly 1 ml of a 1.6 per cent alco- 

 holic solution per 1,000 ml of medium) is added before sterilization. 

 Although litmus and Andrade's indicator (acid fuchsin decolorized with 

 alkali) have been used widely, they do not give accurate results in terms 

 of H-ion concentration; thus, except for special purposes, (see Chap. 

 VII, page 165) it is recommended that sulfon-phthalein indicators be 

 employed. Select the appropriate indicator from the list given in 

 Table 2, governing choice by following considerations: phenol red, with 

 pH range 6.9-8.5, is useful for indication of changes on the alkahne side 

 of neutrality and slight changes to acid ; bromthymol blue has a sensitive 

 range extending slightly in either direction from neutrality; bromcresol 

 purple, with a pH range of 5.4-7.0, is useful in synthetic media and for 

 pronounced pH changes in more highly buffered media. Bromthymol 

 blue is frequently the most useful of these but must be used with caution 

 in synthetic media, since it indicates even the minor pH changes due to 

 CO2 absorption. 



Double and triple sugar agars are of particular value in the rapid 

 identification of the gram-negative enteric bacteria. These media are 

 tubed in columns sufficiently deep to allow a 1.5-in. butt in addition to a 

 slant. They should be inoculated by smearing the slant and stabbing 

 the butt with a straight inoculating needle. RusselVs double sugar agar is 

 prepared as follows: to a liter of peptone broth or beef-extract peptone 

 broth add glucose, 1.0 g; lactose, 10.0 g; NaCl, 5.0 g; phenol red, 0.025 g; 

 and agar, 15 g. After incubation, those organisms {Salmonella typhosa) 

 which attack glucose but not lactose will show an acid reaction (yellow) 

 in the butt but not in the slant, whereas those types (Escherichia coli) 

 which ferment lactose will give an acid reaction throughout the entire 

 medium. Gas production is indicated by formation of gas bubbles or 

 splitting of the medium. Krumwiede^s triple sugar agar is prepared by 

 adding 10.0 g of sucrose to the above formula. Fermentation of either 

 sucrose or lactose, or both, will give rise to an acid reaction throughout 



