DEHYDRATED CULTURE MEDIA 137 



which should be evenly distributed over the entire surface of the medium. The 

 plates are incubated at 35-3 7 °G. for a full 24 hours, at which time at least three 

 or four of each type of suspected colonies are picked from each plate for 

 identification. 



Bacto-S S Agar inhibits the formation of colonies of contaminating types but 

 does not destroy them. In the identification procedures it is necessary to have a 

 pure culture, and, for this reason, only the center of the colony should be picked 

 for transfer. If the suspected colonies are not well isolated, they should be puri- 

 fied by subculturing on some non-selective medium such as Bacto-MacConkey 

 Agar. Suspected colonies are transferred to appropriate differential tube media, 

 such as Bacto-Kligler Iron Agar, Bacto-Triple Sugar Iron Agar or Bacto- 

 Krumwiede Triple Sugar Agar, for ascertaining the group to which they belong. 

 The use of Bacto-Triple Sugar Iron Agar is particularly recommended as a single 

 medium for separation of the Salmonella and Shigella groups. Salmonella may 

 be further differentiated by their inability to produce indole from Bacto-Tryptone, 

 as the experience of recent investigators indicates that indole is not formed by 

 pathogenic Salmonella. The hydrolysis of urea as demonstrated on Urea Broth 

 or Urea Agar, as discussed on pages 170 and 171, may be used to identify Proteus 

 or Paracolobactrum (paracolon) organisms. Agglutination tests for further 

 identification of isolated strains may be run on organisms from any of these 

 differential tube media. 



Bacto-S S Agar is particularly adapted for the isolation of Shigella and 

 Salmonella but as this is a selective medium, a non-inhibitive medium such as 

 Bacto-MacConkey Agar, which permits the growth of even the most fastidious 

 Gram negative intestinal pathogens, should be run in conjunction with it. If 

 typhoid is suspected, it is recommended that a poured plate of Bismuth Sulfite 

 Agar be run on each specimen. Enrichment of the specimen in Tetrathionate 

 Broth or Selenite Broth followed by streaking on Bismuth Sulfite Agar or S S 

 Agar is also recommended. This latter procedure is especially desirable in the 

 isolation of Salmonella. 



A procedure designed to show the largest number of pathogens from a speci- 

 men would be: 



A. Streak or smear a large inoculum on one plate of S S Agar and one plate of 

 Bismuth Sulfite Agar. 



B. Streak or smear a light inoculum on one plate of MacConkey Agar. 



C. Prepare Bismuth Sulfite Agar poured plates with a 5 ml. and a one drop 

 inoculum. 



D. Enrich for 12-18 hours in Tetrathionate Broth or Selenite Broth followed 

 by streaking on one plate of Bismuth Sulfite Agar or Brilliant Green Agar 

 and one plate of S S Agar. 



Saccharose in 1 per cent concentration may be added to isolation media, such 

 as Bacto-S S Agar to permit the detection of certain members of the coliform 

 group which ferment saccharose more readily than lactose. This principle was de- 

 scribed by Holt-Harris and Teague^^ and has been employed by many other 

 bacteriologists. In some laboratories pathogenic significance is assigned to these 

 organisms, and under such conditions, saccharose should not be added to the 

 medium. 



To rehydrate the medium, suspend 60 grams of Bacto-S S Agar in 1000 ml. of 

 cold distilled water and heat to boiling to dissolve the medium completely. Do 

 not sterilize in the autoclave. About 20 ml. of the medium should be poured into 

 standard petri dishes of 90-100 mm. in diameter. It is important that the surfaces 

 of the medium be quite dry when inoculated and this may be accomplished by 

 allowing the medium to solidify and to stand for about 2 hours with the covers 



