Selenite broth: Composition, Preparation and Uses

Selenite broth is used as an enrichment medium for the isolation of Salmonella and Shigella from stool, urine, water and food products. It is the most widely used enrichment medium to isolate Salmonella Typhi from fecal specimen of suspected patients and/or from carriers during Salmonella outbreaks.  Selenite is inhibitory to coliforms and certain other microbial species found in faecal specimens. After the inoculation of the sample, selenite broth is incubated for 12-18 hours at 35°C -37°C and then subculture is done in selective agar (e.g., bismuth sulfite or desoxycholate citrate agar).

Rappaport-Vassiliadis (RV) medium is now being recommended as an alternative of Selenite Broth/Tetrathionate broth for the selective enrichment of Salmonella spp. from food and sewage polluted water.

Composition*:

Ingredients Gms/litre
Casein enzymic hydrolysate 5.00
Lactose 4.00
Sodium phosphate 10.00
Sodium hydrogen selenite 4.00

Casein enzymic hydrolysate provides nitrogenous substances and carbon compounds required for bacterial growth.

Lactose (fermentable carbohydrate) serves to maintain the pH of medium.  Any increase in pH will reduce the selective activity of selenite. Proteus and Pseudomonas species which are non-lactose fermenter appear to be resistant to effects of selenite.

Sodium phosphate maintains a stable pH and also lessens the toxicity of selenite.
Selenite exerts selective inhibitory effects. It is suggested that it reacts with sulphur and sulphydral groups of in critical cell components of microorganisms. Coliforms, fecal streptococci, and Gram-positive organisms are inhibited by sodium selenite.

Caution: Sodium hydrogen selenite (Sodium biselenite) is very toxic, corrosive agent and causes teratogenicity. Handle with greatcare. If there is contact with skin, wash immediately with lot of water.

Final pH (at 25°C): 7.0 ± 0.2

*The formula may vary slightly from manufacturer to manufacturer.

NOTE: L-cystine supplement may be available (depending upon the manufacturer) to use as an enrichment agent to increase the recovery of Salmonella.

Preparation of Selenite Broth:

  1. Suspend 23 g of the dehydrated powder in 1 litre of purified water (if twin pack is available for use; dissolve 4g of sodium biselenite in 1 litre of distilled water and then add 19g of Selenite Broth Base- check manufacturer’s instruction labeled/available in the pack).
  2. Sterilise in a boiling water bath, or in free flowing steam, for 10 minutes. Avoid overheating (as it is detrimental). Do not autoclave.
  3.  Distribute in sterile test tubes. (Note: Discard the prepared medium if large amount of red precipitate is seen at the bottom of the tube/bottle)
  4. Label the side of each tube with date of preparation and batch number.
  5. Perform sterility testing.
  6. Store at 2-8°C
  7. Test samples of the finished product for performance using stable, typical control cultures.

Appearance of Selenite Broth

  • Dehydrated medium: Straw coloured, free-flowing powder
  • Prepared medium: Light straw coloured solution

Storage conditions and Shelf life

  • Dehydrated medium: Store at 10-30°C and use before the expiry date on the label.
  • Prepared medium:  Store at 2-8°C away from light. It can be used up to eighteen months provided there is no change in the appearance of the medium to suggest contamination or deterioration.

Inoculation of Selenite Broth

  • Inoculate the tube of selenite with 1-2 gm of stool sample or other sample (approximately 10-15% by volume) and emulsify the broth.
  • Incubate in ambient air at 35-37°C for up to 24 hours (Coliforms may overgrow the pathogens if incubated for longer than 24 hours).
  • After 12-18 hours of incubation, subculture the broth into selective and differential enteric culture medium such as XLD, HE Agar. Streak for isolation.
  • Incubate the culture plates in ambient air at 35°C-37°C for 18-24 hours.
  • Search for typical colony morphology (which differs according to media used)

NOTE:  Selenite broth should not be used as the sole isolation medium for the isolation of Salmonella. It should be used in conjunction with selective and nonselective plating media to increase the probability of isolating pathogens, especially when they may be present in small numbers.

Limitations 

  • The inhibitory effect of selenite diminishes after the first 6-12 hours of incubation.
  • Discard media if selenite oxidizes and forms large amounts of a red precipitate. A small amount of colored precipitate is not detrimental.

Further reading and references 

About Nisha Rijal 19 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.