Salt Tolerance Test for Enterococcus species: Principle, Procedure and results

Salt Tolerance Test is used in the identification of enterococcal group D Streptococcus on the basis of their salt tolerance. The ability of the bacteria to grow in the presence of variable amount of Sodium Chloride (NaCl) has been used to characterize several bacteria, including viridians streptococci.

This test is particularly useful for presumptive identification of the enterococcal group D organisms, which have the specific ability to grow in the presence of 6.5% NaCl incorporated into either a broth or an agar medium. This test along with Bile-Esculin test, is used in many laboratories to distinguish Enterococcus species from the group D streptococci, Streptococcus bovis and Streptococcus lactis. Enterococci  is a significant cause of endocarditis with a high degree of mortality.

Brain Heart infusion (BHI) broth supplemented with 6.5% sodium chloride and bromcresol purple as a pH indicator is used for this purpose.   The indicator is included to make reading the test results easier. The broth also includes dextrose. The fermentation of dextrose (glucose) results in the production of acid. This changes the pH of the media causing the media to turn from purple to yellow.

Procedure of the Salt Tolerance Test

  1. Select no more than 2-3 colonies (preferably from an overnight culture) to inoculate a tube of salt tolerance broth

Note: It is important to lightly inoculate the tube otherwise you may get a false positive.

  1. Loosen the cap and incubate aerobically for 24 hours at 37°C.
  2. Continue incubation up to 72 hours if you get a negative result at 24 hours.

Results and Interpretation

  1. A positive test is the presence of obvious bacterial growth (turbidity in liquid medium) in the medium, with or without a color change in the indicator. If the organism is bile-esculin-positive and grows in the 6.5% NaCl broth, the organisms are an Enterococcus species. If the organism is bile-esculin-positive and fails to grow in the 6.5% NaCl broth, the organism is a group D streptococcus.
  2. A negative result is indicated by no growth after 72 hours. Enterococcus spp. typically changes the media color within 24 hours.

Organisms and their results:

  1. Positive growth: Enterococcus faecalis
  2. Negative growth: Streptococcus bovis

Limitation of the Procedure

  1. Salt tolerance media was intended to differentiate catalase negative gram-positive cocci. Be sure to perform a catalase test before you proceed with the salt tolerance broth test. As many staphylococci can grow in media containing even 10% salt.
  2. If the medium is inoculated too heavily, inoculums may be interpreted as growth, resulting in a false positive reaction.
  3. To prevent interpretation of the test falsely as negative, agitate the tube gently before reading, as the growth may settle out during incubation.
  4. Other species of catalase negative gram-positive organisms can grow in this media.
  5. Up to 80% of group B streptococci may be salt tolerant, as will occasional isolates of group A streptococci. Aerococci may also be bile-esculin-positive and may grow in 6.5% NaCl

2 thoughts on “Salt Tolerance Test for Enterococcus species: Principle, Procedure and results

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