Biochemical Test

Salt Tolerance Test Principle, Purpose, Procedure, Result

The ability to develop in the presence of varying concentrations of sodium chloride (NaCl) has been used to distinguish between various bacteria.

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This article writter by MN Editors on September 04, 2022

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Salt Tolerance Test Principle, Purpose, Procedure, Result
Salt Tolerance Test Principle, Purpose, Procedure, Result
  • The ability to develop in the presence of varying concentrations of sodium chloride (NaCl) has been used to distinguish between various bacteria.
  • It considers the organism’s tolerance for a range of osmotic concentrations. Enterococcus faecalis, Enterococcus zymogenes, Enterococcus liquifaciens, and Enterococcus durans are all salt-tolerant Enterococcus species.
  • A salt tolerance test can be used to identify enterococcal group D Streptococcus.
  • Multiple bacteria, notably viridans streptococci, have been defined by their tolerance for varying concentrations of sodium chloride (NaCl) in their growth medium.
  • The enterococcal group D germs can only develop when the broth or agar medium contains 6.5% NaCl.
  • Many labs employ this test in conjunction with the bile-esculin test to tell group D streptococci, Streptococcus bovis, and Streptococcus lactis apart from Enterococcus species. Endocarditis caused by enterococci is a leading cause of hospitalisation and death.
  • For this reason, a broth called brain-heart infusion (BHI) is employed, and it is augmented with 6.5% sodium chloride and bromocresol purple, which acts as a pH indicator.
  • A visual aid is provided to facilitate interpretation of the test outcomes.
  • Dextrose is also present in the soup base.
  • The sugar dextrose is fermented into an acid by microorganisms.
  • The media’s pH changes, transforming the colour from purple to yellow.

Objective of Salt Tolerance Test

  • This test is used to find out if an organism can grow where there is a lot of salt.
  • It is used to differentiate enterococci (positive) from nonenterococci (negative).

Salt Tolerance Test Principle

  • Salt is a selective agent that disrupts osmotic balance and membrane permeability.
  • In about 48 hours, salt-tolerant microorganisms will produce abundant growth in the broth and on solid agar in the salt tolerance medium because of the medium’s selective and differential properties.
  • The first successful salt-tolerant medium was created by Hajna. A wide variety of bacteria are killed off by the high salt concentration, although salt-tolerant species like enterococci can thrive there.
  • Dextrose, a fermentable carbohydrate, and bromcresol purple, a colour indicator, are both components of the Quadri formula.
  • Sugar is metabolised by organisms that can thrive in the high salinity medium, and acid is produced as a byproduct of the metabolism.
  • Reduced pH causes a colour change in bromcresol purple indicator from purple to yellow.
  • To the contrary, enterococci thrive in salty environments. Enterococcus faecalis, Enterococcus zymogenes, Enterococcus liquifaciens, and Enterococcus durans are all salt-tolerant Enterococcus species.

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

Media required for Salt Tolerance Test 

Media: Brain-heart infusion (BHI) broth can be used instead of the individual parts by adding NaCl and indicator dye.

Components

IngredientsGms / Litre
Peptic digest of animal tissue 10.000
Heart infusion10.000
Glucose1.000
Sodium chloride65.000
Bromocresol purple0.016
Final pH ( at 25°C)7.2±0.2

Salt Broth Preparation

  1. Suspend 86.01 grammes in 1000 ml distilled water.
  2. If you need to, heat the mixture to dissolve it completely.
  3. Dispense as desired.
  4. Use an autoclave for 15 minutes at 15 pounds of pressure (121°C) and 121°F.

Key Fact

  • Enterococcal group D streptococci can be distinguished from non-enterococcal group D streptococci using Salt Broth, Modified. 
  • Testing the salt tolerance of bile esculin-positive and catalase-negative Enterococci in a medium containing 6.5% sodium chloride is a reliable way to distinguish between them. 
  • The high concentration of salt in this medium serves as a differential and selective agent by disrupting membrane permeability and osmotic balance. 
  • The 6.5% sodium chloride tolerance test is useful for distinguishing enterococcal group D Streptococcus species (Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, and Enterococcus avium) from non-enterococcal species like Streptococcus bovis and Streptococcus equines. 
  • Carbohydrates come from glucose, while nitrogenous nutrients come from heart infusion and peptic digest of animal tissue. 
  • The pH indicator bromocresol purple changes colour from purple to yellow when exposed to an acidic environment. 
  • Salt water is a good example of sodium chloride’s use as a selective and differential agent. 
  • The indicator’s turbidity and, at times, its shifts in colour, both point to growth. 
  • Glucose usage and acid generation may also cause a shift in colour, from purple to yellow. 
  • Enterococcus species can be readily identified as a serological group D streptococci or biliary esculin positive isolate.

Recommended Media for Salt Tolerance Test

  • Commercial HI broth with 6.5% salt from BD Diagnostic Systems’ Remel SF broth with 5% salt and sodium azide
  • The Hardy Diagnostics NaCl 6.5% is found in BHI broth with a high salt content.
  • The Hardy Diagnostics Tryptic Soy Broth (TSB) with 6.5% NaCl is a recommended growth medium for separating group D streptococci from enterococci.

Salt Tolerance Test Procedure

Preparation of 100 ml  6.5% NaCl Peptone Water

  1. Weigh 1.5 g of peptone water that has been dehydrated and 6.25 g of sodium chloride.
  2. Mix them with 100 ml of distilled water.
  3. Mix well and divide between the test tubes.
  4. Sterilize by putting it in an autoclave for 15 minutes at 15 pounds of pressure and 121°C.

 Test Procedure

  1. Put one or two colonies from a culture that has been going for 18 to 24 hours into 6.5% NaCl broth.
  2. Keep the tube at 35°C to 37°C for 48 hours in normal air.
  3. Check for growth every day.
Salt Tolerance Test Principle, Purpose, Procedure, Result
Salt Tolerance Test Principle, Purpose, Procedure, Result

Results of Salt Tolerance Test

Results of Salt Tolerance Test
Salt tolerance (6.5% NaCl) test. A, Positive. B, Negative.
  • Salt Tolerance Positive test: You can see that the broth is cloudy, even if the colour doesn’t change from purple to yellow. Note: Turbidity is a sign of a positive test on its own.
  •  Salt Tolerance Negative test: After 72 hours, there is no cloudiness and no change in colour. 

Quality Control

  • Positive: Enterococcus faecalis (ATCC29212) is growing; the colour is changing from red to yellow.
  • Negative: Streptococcus bovis (ATCC9809) is inhibited, as shown by little to no growth and no change in colour. 

Limitations of Salt Tolerance Test

  • Colonies grown in sterile conditions should be tested using biochemical, immunological, molecular, or mass spectrometry techniques for definitive identification.
  • It may be difficult to interpret test results while using 6.5% NaCl infusion broth because of the potential for sluggish reactions.
  • Strains of Streptococcus agalactiae (group B) can multiply in medium without triggering an acid response. The medium will be turbid, but it won’t change colour.
  • When inoculating broth, it’s best to use a small amount of bacteria. A turbidity-induced false-positive result could be the result of an inoculation with a large inoculum.

Salt Tolerance Test Uses

  • In addition to being able to distinguish Aerococcus species from Stomatococcus and Helcococcus, salt tolerance broth can be used to distinguish Aerococcus species like A. viridans and A. urinae.
  • Used to distinguish enterococci (positive) from other bacteria (negative).
  • The capacity to develop in a 6.5% sodium chloride broth is used to distinguish non-beta-hemolytic strains of catalase-negative, gram-positive cocci (such as Enterococcus and Aerococcus).

References

  • 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
  • https://www.austincc.edu/microbugz/salt_tolerance.php
  • https://www.bioscience.com.pk/topics/microbiology/item/239-salt-tolerance-test
  • https://himedialabs.com/TD/M1290.pdf
  • https://universe84a.com/collection/salt-tolerance-test/
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