BISMUTH SULPHITE AGAR (U.S.P.)

 

Selective medium for the isola­tion of Salmonella, especially S.typhi and lactose positive Salmonellae.

 

Code: CB1/1009

 

Typical Formula g/l

 

Beef extract	5.00
Tryptone	5.00
Peptone	5.00
D-Glucose	5.00
Disodium phosphate	4.00
Bi-sulphite indicator	8.00
Ferrous sulphate	0.30
Brilliant green	0.025
Agar	20.00

 

Final pH 7.7 +/- 0.2

 

Directions

Suspend 52.3 g in 1000 ml of cold distilled water; heat to boiling, cool to 50^_°C, disperse the cloudy precipitate by stirring and pour into sterile petri dishes. Do not overheat or autoclave. Store the plates in the refrigerator, in the dark, for no more than 2 days.

 

Description

Bismuth Sulphite Agar is a selective medium used for the isola­tion of Salmonella, especially Salmonella typhi, and lactose positive Salmonella spp. from faeces, urine, water and other specimens contaminated with these pathogens. The medium is a modification of the original Wilson and Blair formula recommended, by APHA and USP. The medium acts as a selective agent for Salmonella typhi, which reduces the sodium sulphite to sulphide in the presence of glucose. Escherichia coli is inhibited by the action of the brilliant green, the ammonium citrate and the bismuth, in the presence of a high sodium concentration. The Salmonella colonies are black surroun­ded by a shiny metallic zone which is clearly visible when the pla­te is put in front of a light source. The colonies appear after 24 hours, and are complete after 48 hours. The medium has the advantage of allowing the direct plating of faeces or other specimens contaminated with enteric bacteria. The freshly prepared medium has strong inhibitory properties and it is suitable for heavily contaminated specimens. The storage of prepared plates for 3 days causes the medium to change colour to green, making it less selective.

 

In Use

 

Bismuth Sulphite Agar may be used in conjunction with other selective enteric agars (XLD Agar, Brilliant Green Agar, SS Agar, Hektoen Enteric Agar). For Salmonella isolation, Edward and Ewing recommend the ino­culation of two plates of Bismuth Sulphite Agar, one by streaking the specimen (or specimen enriched in Selenite Broth or Tetra­thionate Broth), on the medium, and the second by using the pou­red plate technique. Inoculate directly one plate of Bismuth Sulphite Agar and one plate of a second enteric agar.

Inoculate with the specimen on an enrichment broth and subculture onto Bismuth Sulphite Agar and also onto the second enteric agar after 12-18 hours of incubation.

Examine the plates after 18 hours of incubation and subculture the suspect colonies to identification media.  All negative plates should be incubated for 48 hours.

S.typhi grows with black colonies, with black zone and metallic sheen after 18 hours, uniformly black after 48 hours. Other Salmonella sp. grow with variable colony appearance after 18 hours: black, clear or mucoid; uniformly black after 48 hours. Other organisms are usually inhibited or grow with green or brown colonies, with no metallic sheen.

 

User quality assurance   (37°C-24 h)

Productivity control

S.enteritidis  ATCC 13076: good growth, black colonies with metallic sheen

S.typhimurium  ATCC 14028: good growth, black colonies with metallic sheen

Selectivity control

E.coli ATCC 25922: partially or completely inhibited

E.faecalis ATCC 29212: partially or completely inhibited

 

Storage

Dehydrated media: 15-30°C

User prepared plates: up to 4 days at 2-8°C

 

References  

APHA (1985) - Standard Methods for the Examination of Water and Wastewater, 16 ed. pp. 919-926.

U.S. Pharmacopoeia 24, NF 19 (2000)

Wilson J.W. (1938) - J. Hyg, 38, 507-519.

Wilson J.W. & Blair E.M. (1931) -  J. Hyg. 31, 138.