Salt Tolerance Test- Principle, Procedure, Results, Limitations

Objectives of Salt Tolerance Test

• To determine the ability of an organism to grow in high concentrations of salt.
• It is used for the differentiation of Enterococci from Non-Enterococci.

Principle of Salt Tolerance Test

Salt acts as a selective agent and interferes with membrane permeability and osmotic equilibrium. The salt tolerance medium is a selective and differential medium in which salt-tolerant organisms will produce heavy growth in the broth and on solid agar within 48 hours. Hajna devised the first formulation of a salt tolerance medium. The high salt concentration inhibits a range of bacteria but allows salt-tolerant organisms such as enterococci to grow in the medium. The Quadri formulation includes the fermentable carbohydrate, dextrose, and the color indicator, bromcresol purple. Organisms capable of growing in the high salinity medium, utilize the sugar and release acid as a by-product of their metabolism in the medium. As a result of the drop in pH, the indicator bromcresol purple change from purple to yellow. Enterococci are resistant to high salt concentration and show growth in mediums containing salt. E. faecalis, E. zymogenes, E. liquifaciens, and E. durans are among the Enterococcus species that are salt tolerant.

Media

Sodium chloride (NaCl) 6.5% media can be used or Brain-heart infusion broth (BHI) may be used in place of the individual components with the addition of NaCl and indicator dye.

Components: Heart digest (10 g), the enzymatic digest of animal tissue (10 g), NaCl (65 g), dextrose (1 g), bromocresol purple (0.016 g), per 1000 mL.

Procedure of Salt Tolerance Test

1. Inoculate one or two colonies from an 18 to 24-hour culture into 6.5% NaCl broth without an indicator or 6.5% NaCl broth containing indicator.
2. Incubate the tube at 35°-37°C in ambient air for 48 hours.
3. Examine media for turbidity/growth or the presence of colonies on media without an indicator or examine media with the indicator for a color change from purple to yellow.

Results of Salt Tolerance Test

Positive test: Visible turbidity in the broth, with or without a color change from purple to yellow

Negative test: No turbidity and no color change

Limitations of Salt Tolerance Test

• Infusion broth with 6.5% NaCl may produce slow reactions thereby making test interpretation difficult.
• Streptococcus agalactiae (group B) will grow in the media, however, it does not produce an acid reaction. Turbidity is observed in the medium but no color change will be present.
• A light inoculum must be used when inoculating broth. Inoculation of heavy inoculum may produce turbidity, thus resulting in a false-positive result.
• It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification.

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Quality Control

Positive: Enterococcus faecalis (ATCC29212): growth, color change to yellow

Negative: Streptococcus bovis (ATCC9809): inhibition, no color change

References

1. Tille P.M. 2014. Bailey and Scott’s diagnostic microbiology. Thirteen edition. Mosby, Inc., an affiliate of Elsevier Inc. 3251 Riverport Lane. St. Louis. Missouri 63043
2. Snyder JW, Atlas RM. Handbook of Media for Clinical Microbi­ology. CRC Press. Taylor & Francis Group. 6000 Broken Sound Parkway NW, Suite 300. Boca Raton, FL 33487-2742.
3. Hajna, A.A. and C.A. Perry. 1943. American Journal of Public Health; 33:550-58