Salt Tolerance Test: Introduction, Principle, Procedure and Result Interpretation

Growth of Enterococcus in 6.5% sodium chloride

Introduction of Salt Tolerance Test

The salt tolerance test is useful to differentiate Enterococcus from non-enterococcal group D streptococci. Growth of Enterococcus faecalis in 6.5% sodium chloride peptone water as shown above image.

Principle of Salt Tolerance Test

The medium is helpful to aid in the differentiation of Enterococcus spp. from streptococci by determining the ability of enterococci to grow in the presence of 6.5% NaCl. Peptone provides nitrogenous and carbonaceous compounds, long-chain amino acids, and vitamins which provide essential nutrients.  A high concentration of salt acts as a selective agent and interferes with membrane permeability and osmotic equilibrium. Salt tolerant organisms will produce heavy growth in the broth and on solid agar within 48 hours. For example, salt-tolerant species of Enterococcus are  E. faecalis, E. zymogenes, E. liquifaciens, and E. durans.

Test Requirements for Salt Tolerance Test:

  • 6.5% sodium chloride peptone water
  • Overnight culture of the test organism
  • Inoculating loop or wire
  • Solid culture media
  • Incubator
  • Bunsen burner
  • Test tube rack
  • Quality control strains ( Enterococcus faecalis ATCC  29212 for Positive control Streptococcus bovis ATCC 9809 negative control)

Composition of Test Medium

Ingredients   Gms / L

Peptone : 10.0

Sodium chloride :  5.0

Final pH (at 25°C) : 7.2±0.2

Add from outside,

Sodium Chloride : 62.5

Preparation of 100 ml  6.5% NaCl Peptone Water

  1.  Weigh 1.5.0 grams of dehydrated peptone water and 6.25 gm sodium chloride.
  2. Suspend those in 100 ml distilled water.
  3. Mix thoroughly and distribute into the test tubes.
  4. Sterilize by autoclaving at 15 lbs pressure and 121°C temperature for 15 minutes.

The Test Procedure for Salt Tolerance Test

  1. Warm the medium to room temperature prior to inoculation.
  2. Take a light inoculum from an overnight culture and inoculate the medium.
  3. Incubate aerobically at 35ºC  for 24-48 hours.
  4. A similar procedure for negative and positive control while omitting steps 1 and 2 for the uninoculated tube.
  5. Observe for turbidity after incubation.

Result Interpretation of Salt Tolerance Test:

As shown above picture-

Salt tolerance test: Positive ( Presence of turbidity or growth )

Salt tolerance test: Negative (absence of turbidity or lacking growth)

Uninoculated tube:  Negative ( tested for checking the quality of media)

Negative control tube: Test negative

Positive control tube: Test positive

Test tube: Positive

Note: This single test is not a confirmatory assay to say the organism is Enterococcus. Therefore bile aesculin and other accessories tests should be performed for confirmation. If there is confusion for turbidity, we should inoculate such broth onto a solid/ agar culture medium. Incubate this medium and observe after 48 hours for final result interpretation.

Recommended Salt Tolerance Test Media are:

  • Commercial HI broth with 6.5% salt  from  Remel
  • SF broth which contains 5% salt and sodium azide from BD Diagnostic Systems
  • Hardy Diagnostics NaCl 6.5% Media that contains high salt BHI broth.
  • Hardy Diagnostics Tryptic Soy Broth (TSB) with 6.5% NaCl is a growth medium recommended for the differentiation of enterococci from the group D streptococci.

Limitations of Salt Tolerance Test

  • It is recommended that biochemical, immunological, molecular, or mass spectrometry testing be performed on colonies from pure culture for complete identification but not from this medium.
  • This broth with 6.5% NaCl may produce slow reactions thereby making test interpretation difficult.
  • A light inoculum must be used when inoculating broth. Too heavy an inoculum may produce turbidity and therefore resulting in a false-positive result.
  • The growth of Staphylococcus aureus can not be omitted in this broth.

Further Readings

  1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr and Sommers H.M.
  5. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  6. https://catalog.hardydiagnostics.com/cp_prod/Content/hugo/TSBwith6_5N Cl.htmlh
  7. ttps://catalog.hardydiagnostics.com/cp_prod/Content/hugo/NaCl65Media.hm
  8. https://himedialabs.com/TD/M028.pdf
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