Salt Tolerance Test Introduction

Many of the biochemical test are performed in order to detect the ability of the organism to grow in a particular medium and to detect the utilization of the enzymes by them.

Once such biochemical test is salt tolerance test, which helps us to detect the ability of the bacteria to grow in the presence of the variable amount of sodium chloride and which is used to characterize the variety of bacteria.

Salt Tolerance Test also accounts the organism’s ability to tolerate the various osmotic concentrations. E. faecalis, E. zymogenes, E. liquifaciens, and E. durans, which are present among the species of Enterococcus, and are considered as salt tolerant.

What is Salt Tolerance Test?

Salt tolerance test is usually used to identify the group of enterococcal group D Streptococcus based on the ability to tolerate the salt contents.

Thus, the capability of the bacteria to grown in the variable amount of Sodium chloride containing medium, helps us to distinguish the variety of bacteria’s, including those of Viridians Streptococci.

This test is used particularly for identifying the Species of Enterococcal Group D organisms, as it has its specialized ability to grow in a medium containing of 6.5% of the Sodium chloride.

This test is often performed with Bile Esculin tests in most of the laboratories, to distinguish the species of Enterococcus from the Group D species of Streptococci and also from Streptococcus bovis and Streptococcus lactis.

Enterococci is considered as one of the Significant causes for endocarditis, which has a high degree of mortality rate.

Salt Tolerance Test and Broth

In Salt tolerance test, Brain Heart infusion broth is used most commonly, it is supplemented with 6.5% of Sodium chloride and the Bromocresol purple as the indicator to denote the pH of the medium.

The use of this indicator helps us to read the results easier.

The broth also consists of dextrose.

Where the fermentation of this dextrose results in acid production, which leads to the change in pH and the color of the medium changes from purple to yellow.

Salt Tolerance Test Objective

 The main aim of the test is to determine the ability of the organism to grow in high concentrations of the salt.

 It is also used to differentiate the variety of Enterococcus species from the Non- enterococcus species.

Salt Tolerance Test Principle

Generally, Salt has its own characteristic to act as a selective agent and it also has the capability to interfere with the membrane permeability and the osmotic equilibrium.

The salt tolerance in the medium is calculated as a selective and the differential one according to the capability of the organisms to produce heavy growth in the broth and on the solid agar medium within 48 hours.

The salt tolerance medium was first formulated by Hajna.

Where as the high salt concentration inhibits a range of the bacteria which allows the salt-tolerant organisms like enterococci to grow in the medium, the quadric formaulation includes the carbohydrates, that can be fermented, dextrose, color indicator, bromocresol purple.

Organisms which have the capability to grow in the slaine medium, utilize the sugar and it releases the acid as its by product during their metabolism in the medium.

Which results in the decrease in the pH, the indicator bromocresol changes its purple color into yellow color.

Enterococci are usually resistant to high concentration of the salts and it often shows growth in the medium which contains the species like Enterococcus faecalis, Enterococcus zymogenes, Enterococcus liquefactions and Enterococcus durans which are known as salt tolerant species of the Enterococcus.

Salt Tolerance Test Reagents

The medium is composed of Sodium chloride of about 6.5% which is often used in brain heart infusion broth, commonly called as BHI and it can be used instead of individual components along with the sodium chloride and the indicator.

Components Required:

 Herat digest of about 10 grams

 10 grams of Enzymatic digest of animal tissue

 Sodium chloride

 Bromocresol purple indicator (it is added Per 1000 ml)

Salt Tolerance Test Procedure

 Initially, one or two colonies are inoculated from 18 to 24-hour culture into the 6.5 of the sodium chloride broth without an indicator or 6.5 % of Nacl broth with indicator.

 Then the tube is further incubated at temperature of about 35 to 37ºC in an ambient air for about 48 hours.

 Then the incubated medium is examined for the presence of turbidity or growth or formation any colonies without an indicator. In some cases, there will also be a color change in the media with an indicator, here the color changes from purple to yellow.

Salt Tolerance Test Result

Positive Test: Here the positive result is indicated by visible turbidity or growth in the broth, and a color change from purple to yellow with the use of indicator.

Negative Test: Here the negative test is indicated by no turbidity or absence of color change, and the color remains the same.

Salt Tolerance Test Uses

 This test is often used to differentiate the species of enterococci from the non-enterococci species.

 It is also used to differentiate the species of non-beta hemolytic strains of the catalase from the negative gram-positive cocci based on their ability to grow in a medium containing 6.5% of sodium chloride broth.

 Where as the Aerococcus species such as the A. viridians and Aerococcus Urinae has also has the capability to grow in the medium containing 6.5% of sodium chloride, hence this salt tolerance broth has the capability to differentiate the different species of Aerococcus from the other similar organisms such as Stomatococcus and Helcoccus possessing the same characteristics.

Salt Tolerance Test Limitation

 It is often insisted that the biochemical immunological, molecular or mass spectrometry test is performed on the colonies from pure culture for complete identification of the test.

 Some strains of pseudococcus, Leuconostoc and Beta-hemolytic streptococcus species grows in the Salt tolerance Broth.

 Infusion broth having 6.5% Sodium chloride produces slow reactions in the medium which results in making the interpretations difficult.

 Usually, a light inoculum must be used while inoculating a broth. • If too heavy inoculum is used it produces turbidity and results in a false positive result.