Salt Tolerance Test in Microbiology: Principle, Procedure, Results, and Applications

Table of Contents

• Introduction to Salt Tolerance
• Objectives
• Principle of Salt Tolerance Test
• Requirements for Salt Tolerance Test
• Procedure of Salt Tolerance Test
• Result and Interpretation of Salt Tolerance Test
• Quality Control
• Precautions
• Applications of Salt Tolerance Test
• Limitations of Salt Tolerance Test
• References

Introduction to Salt Tolerance

• Salt Tolerance Test is a biochemical test used to evaluate the ability of bacteria to tolerate high salt concentrations (6.5%).
• Growth requirements vary among bacterial genera, including temperature, pH, water activity, nutritional needs, and salinity.
• Sodium ions (Na⁺) are essential for all organisms, but the required amount and salt tolerance levels differ by species.
• Halotolerant bacteria or halophiles can tolerate high salt levels, equivalent to or exceeding seawater concentration.
• Non-halophiles thrive in salt concentrations below 1.7% (<0.2 to 0.3 M).
• Slight halophiles require NaCl concentrations between 1.7% and 4.8% (0.3 to 0.7 M).
• Moderate halophiles require NaCl concentrations between 4.8% and 20% (0.7 to 3.4 M).
• Extreme halophiles thrive at NaCl concentrations between 20% and 30% (3.4 to 5.9 M).
• Halo-tolerant bacteria are non-halophiles that can tolerate up to 2.5M salt concentration.
• Salt tolerance tests are used in clinical and research laboratories for bacterial identification.
• These tests differentiate bacterial species based on their salt tolerance levels.
• Salt tolerance tests help identify halotolerant bacteria, halophiles, and extremophiles.
• The 6.5% Salt Tolerance Test differentiates Enterococcus spp. from other gamma-hemolytic (non-hemolytic) Streptococcus spp. in clinical labs.
• It aids in bacterial identification and characterization, including differentiation of Mycobacterium spp., study of Actinobacteria, research on extremophiles, and characterization of Vibrio spp.
• The 6.5% Salt Tolerance Test is widely used in diagnostic laboratories and plays a critical role in understanding bacterial adaptations to high-salinity environments.

Objectives

• To assess the capability of bacteria to withstand a high salt concentration (6.5%).

Principle of Salt Tolerance Test

Adding salt to a culture medium increases its ionic concentration, which impacts osmotic pressure and membrane permeability across bacterial cell membranes. This makes salt a selective agent, allowing only salt-tolerant or halophilic organisms to grow.

Salt-tolerant (halophilic) bacteria have evolved specific mechanisms to adapt and thrive in high-salt environments, including:

• Increasing cytoplasmic ion concentration to match the external environment.
• Accumulating compatible solutes (e.g., solutes like K⁺ ions that mimic Na⁺).
• Modifying intracellular water movement processes.
• Altering nucleotides and proteins to maintain functionality in high-salt conditions.

These adaptations enable salt-tolerant bacteria to survive in 6.5% salt concentration. In contrast, non-halophilic bacteria lack these mechanisms and are susceptible to osmotic shock, plasmolysis, and specific ion effects, leading to their inhibition or death in high-salt environments.

Requirements for Salt Tolerance Test

Culture Media

• Modified salt broth containing 6.5% NaCl is commonly used for the 6.5% salt concentration test.
• Brain Heart Infusion (BHI) Broth supplemented with 6.5% NaCl and 0.1% bromo cresol purple indicator (modified BHI Broth) is also widely utilized.
• Alternatively, Tryptic Soy Broth with 6.5% NaCl and 0.1% bromo cresol purple indicator can serve the same purpose.

Composition of Modified Salt Broth per 1000 mL

• HMH Peptone (Heart Digest): 10.00 g
• Peptone: 10.00 g
• Glucose (Dextrose): 1.00 g
• Sodium Chloride: 65.00 g
• Bromo cresol purple: 0.016 g
• Final pH: 7.2 ± 0.2 at 25°C

(Reference: himedialabs.com)

Preparation of Modified Salt Broth

• Weigh the required amount of modified salt broth powder (8.6 g for 100 mL water) or individual components.
• Mix with the required volume of water in a conical flask or glass bottle.
• Stir well manually or using a magnetic stirrer and heat until all components dissolve.
• Dispense 5 mL (or desired volume) of the broth into test tubes. Cap loosely or use a cotton plug.
• Autoclave at 121°C and 15 lbs pressure for 15 minutes.
• Allow the broth to cool to 40-45°C before inoculation.

Composition of HI Broth per 1000 mL

• Calf Brain Infusion (HM infusion powder): 12.50 g
• BHI Powder: 5.00 g
• Protease Peptone: 10.00 g
• Glucose (Dextrose): 2.00 g
• Disodium Hydrogen Phosphate: 2.50 g
• Sodium Chloride: 5.00 g
• Final pH: 7.4 ± 0.2 at 25°C

(Reference: himedialabs.com)

Preparation of Modified BHI Broth

• Measure BHI broth powder (37.0 g per 1000 mL) along with 60.0 g sodium chloride and 0.016 g bromo cresol purple.
• Add the mixture to 1000 mL of water in a conical flask or glass bottle.
• Stir and heat until all components dissolve completely.
• Dispense 5 mL (or desired volume) into test tubes, leaving caps loose or using cotton plugs.
• Autoclave at 121°C and 15 lbs pressure for 15 minutes.
• Cool to 40-45°C before inoculation.

Reagents

• Sodium Chloride (extra pure or laboratory grade).
• Bromocresol purple indicator.

Equipment

• Test tubes.
• Incubator.
• Weighing machine.
• Autoclave.
• Bunsen burner.
• Inoculating loop.
• Personal protective equipment (PPE) and general laboratory materials.

Test Organisms (Sample Bacteria)

• Positive Control: Enterococcus faecalis ATCC 29212.
• Negative Control: Streptococcus bovis ATCC 33317.

Procedure of Salt Tolerance Test

• Using a sterile inoculating loop, collect well-isolated colonies of the sample bacteria from a fresh culture (18 to 24 hours old).
• Transfer 1 to 2 loops of bacterial suspension or fresh broth culture into the test broth.
• Incubate the inoculated tubes aerobically at 35 ± 2°C for 24 hours.
• Observe the tubes for visible growth (turbidity) or a change in color.
• If no growth is observed, continue incubating the tubes.
• Check for turbidity or color change at 48 and 72 hours.

Result and Interpretation of Salt Tolerance Test

• Positive Result: Bacterial growth, indicated by increased turbidity and/or a color change in the broth from purple to yellow.
• Typically observed within 24 hours but may take up to 48–72 hours for certain Enterococci strains, and up to 14 days for Mycobacterium spp. and Actinobacteria.
• Negative Result: No bacterial growth (no turbidity) and no color change in the medium.

Quality Control

• Enterococcus faecalis ATCC 29212 exhibits increased turbidity and a yellow color change in the medium after 24 hours of incubation.
• Streptococcus bovis ATCC 33317 shows neither growth nor any color change, even after 72 hours of incubation.

Common Salt Tolerant Pathogenic Cocci Bacteria

Enterococcus species, Staphylococcus aureus, Streptococcus agalactiae

Precautions

• When adding salt to the ready-made culture medium, consider the amount of NaCl already in the medium and adjust accordingly to achieve a final NaCl concentration of 6.5%.
• Do not report negative results at 24 hours; re-incubate for up to 72 hours before concluding negative results.
• Use a light inoculum, as a heavy inoculum can cause turbidity, leading to a false positive result.

Applications of Salt Tolerance Test

• Distinguishing Enterococcus spp. from other non-hemolytic Streptococci/Group D Streptococci (non-enterococci).
• Differentiating Aerococcus spp. (salt-tolerant at 6.5%) from other similar cocci, such as Stomatococcus and Helococcus (both non-salt tolerant).

Limitations of Salt Tolerance Test

• It is not a definitive test and should be complemented with other biochemical tests for accurate bacterial genus and species identification.
• Certain β-hemolytic Streptococci (e.g., Streptococcus agalactiae) may also grow in 6.5% NaCl, potentially leading to confusion in identifying Enterococci.
• Minimal growth without acid production can complicate the interpretation of turbidity.
• If bacteria grow without producing acid, the medium's color remains unchanged, making it challenging to interpret results.
• A minimum of 72 hours is required before reporting a negative result.

References

• Leber, Amy L., editor in chief. (2016). Clinical Microbiology Procedures Handbook (Fourth edition). Washington, DC: ASM Press.
• Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s Diagnostic Microbiology (13th edition). St. Louis, Missouri: Elsevier.
• Vreeland, R. H. (1987). Mechanisms of halotolerance in microorganisms. Critical Reviews in Microbiology, 14(4), 311-356. doi: 10.3109/10408418709104443. PMID: 3308318.
• Greenway, H., & Munns, R. (1980). Mechanisms of salt tolerance in nonhalophytes. Annual Review of Plant Physiology, 31, 149-190. doi: 10.1146/annurev.pp.31.060180.001053.
• Edbeib, M., Wahab, R., & Huyop, F. (2016). Halophiles: Biology, adaptation, and their role in the decontamination of hypersaline environments. World Journal of Microbiology and Biotechnology, 32, 135. doi: 10.1007/s11274-016-2081-9.
• Peters, A. C., Thomas, L., & Wimpenny, J. W. (1991). Effects of salt concentration on bacterial growth on plates with gradients of pH and temperature. FEMS Microbiology Letters, 61(2-3), 309-314. doi: 10.1016/0378-1097(91)90571-Q. PMID: 1903754.
• Conville, P. S., & Witebsky, F. G. (1998). Variables affecting results of the sodium chloride tolerance test for identification of rapidly growing mycobacteria. Journal of Clinical Microbiology, 36(6), 1555-1559. https://doi.org/10.1128/JCM.36.6.1555-1559.1998
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