168 DIFCO MANUAt 



37°C. a preliminary examination was made and all tubes showing an alkaline 

 reaction were discarded. The remaining tubes were reincubated and their reac- 

 tions were generally complete in 24 hours. All cultures showing an alkaline 

 reaction were Proteus or members of the paracolon-aerobacter group. The Sal- 

 monella and Shigella cultures failed to produce increased alkalinity in the Urea 

 Medium. Hydrogen sulfide positive cultures, as determined on the previously 

 inoculated Triple Sugar Iron Agar, were investigated with Salmonella poly- 

 valent antiserums, while hydrogen sulfide negative cultures were investigated with 

 Shigella polyvalent or Salmonella polyvalent antiserums. In a survey of methods 

 used for the collection and preservation of stool specimens for the isolation and 

 identification of Salmonella, Shigella and intestinal protozoa Felsenfeld^^ ^t- 

 ported that the use of Triple Sugar Iron Agar was increasing while the use of 

 double sugar agar was decreasing. The use of a single differential medium indi- 

 cates a trend towards standardization of laboratory techniques. 



Bacto-Triple Sugar Iron Agar is essentially the formula originally described 

 by Sulkin and Willett.^ The Bacto-Tryptone has been replaced by a combination 

 of Bacto-Peptone and Proteose Peptone, Bacto-Yeast Extract has been added, the 

 agar concentration increased to 1.5 per cent and phenol red used as an indicator 

 instead of brom thymol blue. On this medium, cultural reactions are clear-cut, 

 and readings may be made at 18-24 hours incubation. For typical cultural reac- 

 tions in 18 hours, it is recommended that the medium be inoculated heavily with 

 growth from a solid culture medium. If inoculated from a suspension of organ- 

 isms, or from broth culture, typical reactions of hydrogen sulfide production, and 

 reversion, may not be obtained until 36-40 hours at 37 °C. To obtain true difTer- 

 ential cultural reactions on this medium it is necessary to have a pure culture. 

 In inoculating directly from isolation media such as MacConkey Agar, S S Agar 

 or Bismuth Sulfite Agar plates, select well isolated colonies and pick only the 

 very center of the colony. If there is any question as to the ability to obtain a 

 pure culture from a certain colony, it is recommended that the suspicious colony 

 be purified by streaking on MacConkey Agar before inoculating into Triple Sugar 

 Iron Agar. This procedure is always recommended to insure culture purity when 

 picking from poured plates of Bismuth Sulfite Agar. It is often possible to detect 

 contaminated cultures on Triple Sugar Iron Agar slants, and when this is the case 

 it is necessary to isolate the organism in pure culture before its typical cultural 

 reactions can be determined. 



The cultural reactions on Bacto-Triple Sugar Iron Agar are similar to those 

 obtained on Kligler Iron Agar, but to it is added the information of the ability 

 of an organism to ferment saccharose. A table of typical reactions on this and 

 other differential media is given on page 161. Results obtained on Triple Sugar 

 Iron Agar, as on Kligler Iron Agar, constitute presumptive evidence only, and 

 must be confirmed biochemically and serologically. 



To rehydrate the medium, suspend 65 grams of Bacto-Triple Sugar Iron Agar 

 in 1000 ml. cold distilled water and heat to boiling to dissolve the medium com- 

 pletely. The solution is distributed in tubes which are stoppered with cotton plugs 

 or loosely fitting caps. Tube and sterilize in the autoclave for 15 minutes at 15 

 pounds pressure (121°C.). Slant in such a manner as to allow a generous butt. 

 The final reaction of the medium will be pH 7.4. 



One pound of Bacto-Triple Sugar Iron Agar will make 6.6 liters of medium. 



^ J. Med. Research, 25:217:1911. ' Bact. Rev., 3:1:1939- 



3 Am. J. Pub. Health, 7:1042:1917. * J. Bact., 45:101:1943. 



J. Exp. Med., 28:319:1918. * J. Bact., 50:609:1945. 



'J; 



Med. Research, 37:225:1917. 10 Am. J. Clin. Path., 17:1:1947. 



"25:649:1c 



s J. Lab. Clin. Med., 25:649:1940. ^1 J. Bact., 52:461:1946. 



« J. Bact., 49:516:1945. "Public Health Reports, 65:1075:1950. 



