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BISMUTH SULPHITE AGAR

Code: CM0201

A modification of the original Wilson and Blair Medium for the isolation of Salmonella typhi and other salmonellae. It is particularly useful for the isolation of lactose-fermenting salmonellae.

* Adjusted as required to meet performance standards

Directions
Suspend 20g in 500ml of distilled water in a 1 litre flask. Heat gently with frequent agitation until the medium just begins to boil and simmer for 30 seconds to dissolve the agar. Cool to 50-55°C, mix well to disperse suspension and pour thick plates (25 ml medium per plate). Allow the medium to solidify with the dish uncovered. Larger volumes may be prepared if great care is taken and adequate head space provided.
Dry the plates before use but take care to avoid overdrying. Correctly prepared plates should have a smooth, cream-like opacity with a pale straw colour. There should be no sedimentation of the indicator.
DO NOT OVERHEAT - DO NOT AUTOCLAVE

Description
Bismuth Sulphite Agar is a modification of the original Wilson and Blair¹ selective medium for the isolation and preliminary identification of Salmonella typhi and other salmonellae from pathological material, sewage, water supplies, food and other products suspected of containing these pathogens. In this medium freshly precipitated bismuth sulphite acts together with brilliant green as a selective agent by suppressing the growth of coliforms, whilst permitting the growth of salmonellae. Sulphur compounds provide a substrate for hydrogen sulphide production, whilst the metallic salts in the medium stain the colony and surrounding medium black or brown in the presence of hydrogen sulphide.
Atypical colonies may appear if the medium is heavily inoculated with organic matter. Such a situation may be prevented by suspending the sample in sterile saline and using the supernatant for inoculation.

The freshly prepared medium has a strong inhibitory action² and is suitable for heavily contaminated samples. Storing the poured plates at 4°C for 3 days causes the medium to change colour to green, making it less selective with small numbers of salmonellae being recovered³. However, for Salmonella typhi recovery the latter technique is not recommended ⁴.

Where the number of salmonellae is expected to be small, enrichment methods may be employed. The use of this medium is advocated by several authorities^5,6,7.

Technique
Bismuth Sulphite Agar may be used in conjunction with other selective enteric agars for the isolation of salmonellae by direct plating or from enrichment media⁸. Thus the following scheme may be adopted.

Inoculate directly on Bismuth Sulphite Agar and one or more of the following:
Desoxycholate Citrate Agar CM0227 or DCLS Agar CM0393
XLD Agar CM0469
Brilliant Green Agar CM0329
MacConkey Agar No.3 CM0115

At the same time inoculate an enrichment broth, such as Selenite Broth Base CM0395 + Sodium Biselenite LP0121, Mueller-Kauffmann Tetrathionate Broth CM0343 or Mueller-Kauffmann Tetrathionate-Novobiocin Broth (MKTT-n) CM1048. Subculture on to Bismuth Sulphite Agar and any other selective medium after 12-18 hours incubation. Examine the plates after 18 hours incubation and subculture suspect colonies to identification media, e.g. Kligler Iron Agar CM0033.

All negative plates should be incubated for 48 hours.

Salmonella typhi
Appearance
Black `rabbit-eye’ colonies with a black zone and metallic sheen surrounding the colony after 18 hours. Uniformly black after 48 hours incubation.

Other Salmonella species
Appearance
Variable colony appearance after 18 hours, they may be black, green or clear and mucoid. Uniformly black colonies are seen after 48 hours, often with widespread staining of the medium and a pronounced metallic sheen.

Other organisms, e.g. coliform bacteria, Serratia, Proteus species
Appearance
Usually inhibited but occasional strains give dull green or brown colonies with no metallic sheen or staining of the surrounding medium.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label. Note the following comments:
Due to its contents of reactive and hygroscopic substances, dehydrated Bismuth Sulphite Agar quickly deteriorates when exposed to the atmosphere. This is usually indicated by aggregation into a solid non-friable mass, and by the development of a brown coloration. Medium reconstituted from such material is brown, does not become green on storage, and is characterised by loss of differential and selective properties. For this reason the powder should be stored in a cool, dry place and after use the container should be properly closed.

Prepared medium
Prepared plates should not be stored for longer than two days at 2-8°C after which the brilliant green oxoidises to give a green medium which may be inhibitory to some salmonellae.

Appearance
Dehydrated medium: Light green coloured, free-flowing powder
Prepared medium: Pale green coloured gel

Quality control
Salmonella typhi should be used only in a Class 3 laboratory, not for routine testing or in food laboratories.

* This organism is available as a Culti-Loop®

Precautions
Prepared plates of medium should not be stored for longer than two days at 2-8°C; after which time the dye oxidises to give a green medium that can be inhibitory to some salmonellae.

Shigella species are usually completely inhibited.

Salmonella sendai, Salmonella cholera-suis, Salmonella berta, Salmonella gallinarum and Salmonella abortus-equi are markedly inhibited⁹.
It is important that the spreading technique yields well separated colonies. The typical colonial characteristics will not develop if the growth is too heavy or confluent; Salmonella typhi colonies will appear light green in these circumstances. Therefore, when in doubt, almost any growth on the medium should be subject to further tests.

References
1. Wilson W. J. and Blair E. M. McV (1927) J. Hyg. Camb. 26. 374.
2. Cook G. T. (1952) J. Path. Bact. 64. 559.
3. McCoy J. M. and Spain G. E. (1969) in Isolation Methods for Microbiologists, p. 20. Ed. by Shapton D. A. and Gould G. W. Academic Press London.
4. Hobbs B. C., King G. C. G. and Allison V. D. (1945) Monthly Bulletin of the Ministry of Health and Emergency Public Health Lab. Service 4. 40.
5. Anon (1981) Int. Standard ISO 6579-1981. Geneva. Internat. Organization for Standardization.
6. ICMSF (1978) Micro-organisms in Food 1. 2nd Edn. University of Toronto Press, Ontario.
7. Speck M. L. (1984) Compendium of methods for the micro-biological examination of foods. 2nd Edn. American Public Health Association.
8. Harvey R. W. S. and Price T. M. (1974) Public Health Laboratory Service Monograph Series No.8. Isolation of Salmonellas. HMSO London.
9. Hajna A. A. (1951) Pub. Hlth. Rep. 9. 48-51.