Bismuth Sulfite Agar: Composition, Preparation, Uses and Colony Morphology

Bismuth Sulfite Agar (BS) is a selective as well as differential medium for isolation and presumptive identification of Salmonella spp especially Salmonella Typhi. Salmonella spp are the causative agent of various diseases like gastroenteritis, sepsis, enteric fever. Salmonella can be isolated from wide range of clinical, food, sewage and other environmental samples. Bismuth Sulfite Agar is a modification of original Wilson and Blair Medium

Composition and purpose of ingredients used:

Bismuth Sulfite Agar is selective due to the presence of inhibitors, and is differential on the basis of hydrogen sulfide ( H2S)  production. The presence of Bismuth sulfite indicator and brilliant green inhibits the growth of Gram-positive bacteria and members of the coliform group, while allowing Salmonella to grow luxuriantly.

Beef extract and peptones provide nitrogen, vitamins and minerals needed for growth of microorganisms.

Dextrose is the carbohydrate present which acts as a source of carbon.

Disodium phosphate is the buffering agent which maintains the osmotic equilibrium.

Bismuth Sulfite Agar
Bismuth Sulfite Agar

Ferrous sulfate is an indicator for hydrogen sulfide production, which occurs when the H2S produced by Salmonella reacts with the iron salt. This reaction causes a black or green metallic colony and brown or black precipitate whilst the reduction of bismuth ions to metallic bismuth produces a metallic luster around of the colonies. Agar is the solidifying agent.

Composition:

Ingredients Gm / Litre
Peptone 10
Beef extract 5
Dextrose (Glucose) 5
Disodium phosphate 4
Ferrous sulphate 0.3
Bismuth sulphite indicator 8
Brilliant green 0.025
Agar 20
Final pH ( at 25°C) 7.7±0.2

Note: Composition may vary according to manufacturer. Some use enzymatic digest of casein and enzymatic digest of animal tissue instead of peptone. Glucose may be used as an alternate to dextrose. However, the inclusion purpose of each ingredient and quantity are same.

Procedure for preparation:

  1. Suspend required amount of dry powder (as per manufacturer’s instruction) in 1000 ml of distilled water.
  2. Heat gently with frequent agitation until the medium just begins to boil and simmer for 30 seconds to dissolve the agar.
  3. DO NOT OVERHEAT – DO NOT AUTOCLAVE
  4. Cool to 50-55°C, mix well to disperse suspension and pour plates (25 ml medium per plate).

NOTE: The sensitivity of the medium depends largely upon uniform dispersion of precipitated bismuth sulphite in the final medium; therefore the medium should be mixed well in a way that there should be no sedimentation of the indicator.

  • Allow the medium to solidify, store at 2-8°C until use and dry the prepared plates before use.

Sample processing: Clinical specimens may be directly inoculated in the medium, or if the sample is considered to have low organism load, it can be sub-cultured from an enrichment broth. For environmental samples appropriate dilution or enrichment may be necessary before plating.

Result and interpretation:

The colony morphology of certain bacteria are illustrated as follows

Organism Appearance
Salmonella Typhi Black `rabbit-eye’ colonies with a black zone and metallic sheen surrounding the colony after 18 hours. Uniformly black after 48 hours incubation Note: When a heavy inoculum is used, S. Typhi appears light green and could be misinterpreted as negative growth.
Salmonella Paratyphi A and Other Salmonella spp 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 like Coliform bacteria, Serratia, Proteus Usually inhibited but occasional strains give dull green or brown colonies with no metallic sheen or staining of the surrounding medium.

Note: S. typhi and S. arizonae are the only enteric organisms to exhibit typical brown zones in the medium; although, Sarizonae is usually inhibited

Limitations

1. Autoclaving/over heating the medium should be avoided as it destroys the selectivity of the medium.

2. Due to its selectivity, Shigella species (except S. flexneri and S. sonnei) and certain Salmonella like S. Sendai, S. Berta, S. Gallinarum, S. Abortus are also inhibited.

3. Incubation of Bismuth Sulfite Agar plates at higher temperatures (i.e., 43ºC.) results in small, atypical Salmonella colonies and also reduces sensitivity showing significantly lower recovery.

4. Although, Typical S. Typhi colonies usually develop within 24 hours, a further incubation up to 48 hours is required for growth of all typhoid strains.

5. Prepared Bismuth Sulfite Agar plates should be used within 2 days. After 3 days of storage there is a reduction in selectivity of the media, decreasing the number of Salmonella recovered.

6. Colonies on Bismuth Sulfite Agar may be contaminated with other viable organisms; therefore, isolated colonies should be sub cultured onto a less selective medium (i.e., MacConkey Agar)

About Nisha Rijal 17 Articles
I am working as Microbiologist in National Public Health Laboratory (NPHL), government national reference laboratory under the Department of health services (DoHS), Nepal. Key areas of my work lies in Bacteriology, especially in Antimicrobial resistance.

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