Wilson Blair Medium: Salmonella And Principle

Wilson Blair Medium helps for the isolation and differentiation of Salmonella typhi, Salmonella Arizonae, Salmonella Bongori, Salmonella Choleraesuis, Salmonella Enterica, Salmonella Enteritidis, Salmonella Paratyphi, Salmonella Typhi and Salmonella Typhimurium from clinical specimens, food, and water.

Wilson Blair Medium composition

CompositionGms / Litre
Peptone special10.000
Peptone B5.000
Dextrose (Glucose)10.000
Sodium chloride5.000
Agar30.000
Final pH ( at 25°C)7.3±0.2
Wilson Blair Medium composition

Wilson Blair Medium PRINCIPLE

Salmonella is a genus of gram-negative Enterobacteriaceae-commonly implicated in foodborne illness and the causative agent of typhoid and paratyphoid fever. Salmonella species have been isolated from humans and animals. More than 2000 serovars of Salmonella exist with each showing different host specificities. For example, humans are the only known natural reservoir for serotype Salmonella Typhi and serotypes Salmonella Paratyphi.

The organism can be transmitted by the fecal-oral route. It is excreted by humans in feces and may be transmitted by contaminated water, food, or by person-to-person contact (with inadequate attention to personal hygiene). Wilson and Blair Agar, formulated by Wilson Blair medium are recommended for isolating Salmonella species especially Salmonella Typhi from clinical specimens. The selective reagent formulation is a modification of the bismuth sulphite reagent described by Hajna and Perry.

This medium is particularly valuable for the isolation of S. Typhi. The medium is highly selective for Salmonella, being inhibitory to coliforms, Proteus, and Shigella; occasional strains of coliforms grow to form dull green or brown colonies but without a surrounding metallic sheen. The medium is also suitable for the isolation of lactose-fermenting strains of Salmonella (which can not be differentiated on lactose-containing differential media) since lactose is not the fermentable substrate used in this medium.

Wilson Blair Medium sample collection

For clinical samples follow appropriate techniques for handling specimens as per established guidelines. For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines. For water samples, follow appropriate techniques for sample collection, and processing as per guidelines and local standards. After use, contaminated materials must be sterilized by autoclaving before discarding. Clinical samples – feces; Food and dairy samples; and Water samples.

Wilson Blair Medium preparation

  • Suspend 60.0 grams in 1000 ml purified/distilled water.
  • Heat to boiling to dissolve the medium completely.
  • Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  • Cool to 45-50°C. To sterile molten base, add 4 ml of 1% Brilliant green solution and 70 ml of Selective reagent.
  • Solution 1: 40 gms Sodium sulphite in 100 ml distilled water.
  • Solution 2: 21 gms Dibasic sodium phosphate (Disodium hydrogen phosphate) in 100 ml distilled water.
  • Solution 3: 12.5 gms Bismuth ammonium citrate in 100 ml distilled water.
  • Solution 4: 0.96 gms Ferrous sulphate in 20 ml distilled water with 2 drops of Hydrochloric acid
  • Prepare each solution separately and boil the combined solution until a slate grey color develops.
  • Mix well and pour into sterile Petri plates.

Wilson Blair Medium uses

Peptone and Beef extract provide nitrogen, vitamins, minerals, and amino acids essential for growth. Dextrose is a fermentable carbohydrate providing carbon and energy, Bismuth sulfite indicator and Brilliant green are inhibitors of gram-positive bacteria and members of the coliform group. Disodium phosphate acts as a buffer system and bacteriological agar is the solidifying agent.

Ferrous sulfate is included for the detection of H2S production. When H2S is present, Salmonella spp reduces the iron salts to iron sulfate, which produces a black colony and turns the bismuth indicator to metallic bismuth, surrounding the area of the colonies with a bright seen.

Generally, Bismuth Sulfite Agar is inoculated by streaking the surface to obtain isolated colonies but the pour plate inoculation method can be also used, mixing the sample with the liquid medium and allowing the plate to solidify. All plates are incubated for 40- 48 hours at 35 ± 2°C. The solidified plates should have a uniform, opaque, cream-to-pale green appearance. If kept in refrigeration, the medium will slowly oxidize. It is recommended to keep the plates refrigerated for 4 days before use to reduce inhibition and thus to be able to isolate Salmonella in less heavily contaminated samples.

The colonies of S. typhi are black surrounded by a black or brownish zone, with a metallic sheen. In heavy-growth areas, these may appear as light green colonies. Other strains of Salmonella produce black to green colonies with little or no darkening of the surrounding medium. Shigella spp , other than Shigella flexneri and Shigella sonnei, do not grow. Those colonies that do grow are brown to green, raised with a crater-like appearance.

E.coli is partially inhibited, occasionally growing with brown or greenish glistening colonies. A few enterobacter strains may grow with raised, mucoid colonies, having a silvery sheen lighter than S. typhi. Colonies of coliforms that produce H 2 S form colonies similar in appearance to S. typhi. These may be readily differentiated as they produce gas with lactose media, e.g. TSI Agar (Cat.1046) or KliglerIron Agar (Cat. 1042). The hydrolysis of urea in Urea Broth (Cat. 1226) or Urea Agar Base (Cat. 1110) may be used to identify Proteus spp.

Wilson Blair Medium Result

The following results were obtained in the performance of the medium from type cultures after incubation at a temperature of 35 ± 2°C and observed after 40- 48 hours.

MicroorganismsGrowthColony 
Escherichia coliPartial InhibitionBrown-Green
Salmonella enteriditisGood Black with bright metallic
Salmonella typhiGood Black with bright metallic
Shigella flexneriPartial InhibitionBrown

Wilson Blair Medium Warning and Precautions :

In Vitro Diagnostic Use. For professional use only. Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred to in individual safety data sheets.

Wilson Blair Medium Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before the expiry date on the label. On opening, the product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in the dry ventilated area protected from extremes of temperature and sources of ignition. Seal the container tightly after use. Product performance is best if used within the stated expiry period.

Wilson Blair Medium Disposal

Users must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and materials that come into contact with clinical samples must be decontaminated and disposed of by current laboratory techniques.

Wilson Blair Medium Limitations

  • It is important to streak for well-isolated colonies.
  • With confluent growth typical colonial characteristics of Salmonella spp. will not develop.
  • Some Salmonella strains are markedly inhibited, for example, Salmonella Gallinarum, S. Sendai, S. Berta, and S. abortus-equi.

There for, when in doubt, almost any growth on the medium should be subject to further tests e.g. subculture onto a less selective medium in a manner to obtain well-isolated colonies. Use pure cultures for biochemical and serological confirmation.

Wilson Blair Medium Keynotes

Salmonella typhi is more frequently isolated from blood cultures than from fecal specimens. Blood cultures are positive for 80% of typhoid patients during the first week of fever but show decreasing positive results thereafter.

Gram-positive bacteria and coliforms are inhibited on Bismuth Sulfite Agar. It is a standard method medium for the clinical environment and industrial applications and it is accepted for routine detection of most Salmonella spp. The freshly prepared medium is strongly inhibitory and is suitable for heavily contaminated samples.

Peptone and beef extract provide nitrogen, vitamins, minerals, and amino acids essential for growth. Dextrose is a fermentable carbohydrate providing carbon and energy, Bismuth sulfite indicator and brilliant green are inhibitors of Gram-positive bacteria and members of the coliform group. Disodium phosphate acts as a buffer system and bacteriological agar is the solidifying agent.

Ferrous sulfate is included for the detection of H2S production. When H2S is present, Salmonella spp reduces the iron salts to iron sulfate, which produces a black colony and turns the bismuth indicator to metallic bismuth, surrounding the area of the colonies with a bright sheen. The colonies of S. typhi are black surrounded by a black or brownish zone, with a metallic sheen.

In heavy-growth areas, these may appear as light green colonies. Other strains of Salmonella produce black to green colonies with little or no darkening of the surrounding medium. Shigella spp, other than Shigella flexneri and Shigella sonnei, do not grow. Those colonies that do grow are brown to green, raised with a crater-like appearance.

E.coli is partially inhibited, occasionally growing with brown or greenish glistening colonies. A few Enterobacter strains may grow with raised, mucoid colonies, having a silvery sheen lighter than S. typhi. Colonies of coliforms that produce H2S form colonies similar in appearance to S. typhi.

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