172 DIFCO MANUAL 



of Salmonella and Shigella cultures for serologic classification. Typical colonies 

 suspected of being pathogens were picked from primary plating media into Triple 

 Sugar Iron Agar. AH tubes showing acid and gas in the slant and butt were dis- 

 carded. Transfers were made from tubes showing an alkaline slant and acid or 

 acid and gas butt onto Urea Agar. A preliminary reading was made at the end 

 of 2-4 hours at 37°C. and all tubes showing alkaline reactions were discarded. 

 The tubes were reincubated and reactions were generally complete in 24 hours. 

 All cultures producing an alkaline reaction were Proteus or members of the para- 

 colon aerobacter group. Salmonella and Shigella cultures failed to produce any 

 increase in alkalinity in the medium. Hydrogen sulfide positive cultures, as 

 determined on the previously inoculated Triple Sugar Iron Agar, were investi- 

 gated with Salmonella polyvalent antisera, while hydrogen sulfide negative 

 cultures were investigated with Shigella polyvalent or Salmonella polyvalent 

 antisera. Urea Agar is used to determine the decomposition of urea by organ- 

 isms in the examination of specimens for Salmonella and Shigella according to 

 the method given in "Diagnostic Procedures and Reagents"^ of the American 

 Public Health Association. 



Bacto-Urea Agar Base, when prepared for use, gives reactions similar to those 

 described by Christensen^ and Ewing and Bruner.* Proteus attacks the urea 

 rapidly and after 2-4 hours incubation the color change, due to ammonia pro- 

 duction, has penetrated deep into the medium. After 24 hours incubation the 

 entire butt of the tube is alkaline in reaction. Urease positive paracolons, in con- 

 trast, hydrolyze urea much more slowly, showing only slight penetration of the 

 alkaline reaction into the butt of the medium in 6 hours and requiring 3 to 5 

 days to change the reaction of the entire butt. According to Christensen^ most 

 paracolon aerobacter and paracolon intermediate cultures are urease positive and 

 paracolon Escherichia cultures are urease negative. Salmonella and Shigella 

 species fail to produce any trace of alkalinity on this medium. Urea Agar may 

 also be used to show urease production by other organisms. Some Gram positive 

 cocci and diphtheroids, as well as certain pigment producing members of the 

 pyocyaneous group and others give a positive reaction on this medium. 



To prepare the medium, dissolve 29 grams of Bacto-Urea Agar Base, Dehy- 

 drated, in 100 ml. of distilled water. Filter sterilize this concentrated base. Dis- 

 solve 15 grams Bacto-Agar in 900 ml. distilled water by boiling and sterilize in 

 the autoclave for 15 minutes at 15 pounds pressure (121°C.). Cool to 50-55°C. 

 and add 100 ml. filter sterilized concentrated Bacto-Urea Agar Base, under 

 aseptic conditions. Mix thoroughly and distribute in sterile tubes. Slant the tubes 

 so as to have a butt of about 1 inch in depth and a slant of about 1.5 inches in 

 length. After solidification the slants are heavily inoculated by spreading growth 

 from an agar culture over the entire surface. Do not inoculate into the butt. Final 

 reaction of the medium will be pH 6.8. 



Bacto-Urea Agar Base cannot be sterilized by heat because of the presence of 

 urea. The complete Urea Agar Medium must be slanted before the medium 

 solidifies to avoid the necessity of remelting the agar and causing hydrolysis of 

 the urea in the medium. 



One pound of Bacto-Urea Agar Base, Dehydrated, will make 15.6 liters of 

 medium. 



^ J. Bact., 52:461:1946. * Am. J. Clin. Path., 17:1:1947. 



3 Proc. Soc. Exp. Biol. Med., 47:108:1941. ^Diagnostic Procedures and Reagents, 3rd Edi- 



*J. Bact., 51 =433: 1946. tion: 227: 1950. 



