Salt Tolerance Test: Principle, Procedure, Results, Uses

Salt Tolerance Test is the biochemical test used to determine the ability of bacteria to tolerate a high salt concentration (of 6.5%).

Different genera of bacteria have different requirements for their ambient growth. Besides temperature, pH, water activity, and nutritional requirements, salinity is an important factor affecting bacterial growth. Every organism needs sodium ions (Na⁺). Hence a certain amount of sodium chloride (NaCl) is essential for the organism’s survival.  However, the amount needed and the maximum amount of salt tolerating capacity varies among different species.

Such bacteria that can tolerate a high amount of salt (salt concentration equal to or more than in seawater) are called halotolerant bacteria or halophiles. Bacteria that grow ambiently in a salt concentration of <1.7% (<0.2 to 0.3 M) are considered non-halophiles. Bacteria that require NaCl concentration of 1.7 to 4.8% (0.3 to 0.7 M), 4.8% to 20% (0.7 to 3.4 M), and 20% to 30% (3.4 to 5.9 M) salt for their ambient growth are called slight halophiles, moderate halophiles, and extreme halophiles respectively. If the non-halophiles tolerate salt concentrations of up to 2.5M, they are called halo-tolerant bacteria.

Characterizing bacteria based on their salt-tolerating capacity is an important biochemical testing scheme used in clinical and research labs for bacterial identification. Generally, a 6.5% salt tolerance test is performed in clinical laboratories to differentiate Enterococcus spp. from other Gamma-hemolytic (non-hemolytic) Streptococcus spp.

Salt tolerance test with a wide range of NaCl concentration is used for research purposes and bacterial identification purposes such as differentiating Mycobacterium spp., characterizing Actinobacteria, studying extremophiles (like halotolerant and halophiles), characterizing Vibrio spp., etc. In this article, we will discuss the 6.5% salt tolerance test as it is an important and common salt tolerance test used in diagnostic laboratories.

Objectives

• To determine the ability of bacteria to tolerate a high salt concentration (of 6.5%)  

Principle of Salt Tolerance Test

Adding salt to a culture medium increases the ionic concentration of the medium and affects the osmotic pressure and membrane permeability across the bacterial cell membrane. Thus, salt acts as a selective agent and allows only the growth of salt-tolerant or halophilic organisms.

Salt-tolerant (and halophilic) bacteria have developed several mechanisms in order to adapt and thrive in high salt concentration environments. The major four mechanisms include increasing cytoplasmic ion concentration equal to the exterior environment, increasing concentration of compatible solutes (solute similar to Na⁺ ions like K⁺) within the cytoplasm, physiological modification of intracellular water movement process, and modification of nucleotides and proteins to function in high salt concentration. These mechanisms make salt-tolerant bacteria able to survive in salt concentration of 6.5%; but, unfortunately, non-halophile bacteria have none of these mechanisms, and hence, they become a victim of osmotic shock, plasmolysis and specific ion effect and get inhibited and/or killed in the environment of high salt concentration.

Requirements for Salt Tolerance Test

Culture Media

The modified salt broth is available, containing 6.5% NaCl for performing a 6.5% salt concentration test. Apart from this, Brain Heart Infusion (BHI) Broth supplemented with 6.5% NaCl and 0.1% bromo cresol purple indicator (modified BHI Broth) is widely used for this test. (Alternatively, Tryptic Soy Broth supplemented with 6.5% NaCl and 0.1% bromo cresol purple indicator can be used for this purpose).

Composition of Modified Salt Broth per 1000 mL

HMH Peptone (Heart Digest)- 10.00 grams

Peptone- 10.00 grams

Glucose (Dextrose)- 1.00 grams

Sodium Chloride- 65.00 grams

Bromo cresol purple- 0.016 grams

Final pH 7.2 ±0.2 at 25°C

(References: Modified Salt Broth (himedialabs.com))

Preparation of Modified Salt Broth

• Measure the appropriate amount of modified salt broth powder (or the media components) and mix in the water of the required volume in a conical flask (or glass bottle) according to the instruction of the manufacturing company (8.6 grams for 100 mL water for the above media). 
• Stir well using a magnetic stirrer or manually and heat to boiling so that all the components dissolve completely in water. 
• Dispense 5 mL (or desired volume) of the broth in test tubes and loosely put it the cap or cotton plug.
• Autoclave the test tubes at 121°C and 15 lbs pressure for 15 minutes and let them cool to around 40-45°C before inoculation.

Composition of HI Broth per 1000 mL

Calf Brain Infusion from (HM infusion powder)- 12.50 grams

BHI Powder- 5.00 grams

Protease Peptone- 10.00 grams

Glucose (Dextrose)- 2.00 grams

Disodium Hydrogen Phosphate- 2.50 grams

Sodium Chloride- 5.00 grams

Final pH 7.4 ±0.2 at 25°C

(Reference: BHI Broth (Brain Heart Infusion Broth) (himedialabs.com))

Preparation of Modified BHI Broth

• Measure the appropriate amount of BHI broth powder (or the media components) according to the manufacturer’s instruction (37.0 grams per 1000 mL for above) and mix 60.00 grams of sodium chloride and 0.016 grams of Bromocresol Purple per 1000 mL media.
• Add the mixture in a conical flask (or glass bottle) with 1000 mL water.
• Stir well using a magnetic stirrer or manually and heat to boiling so that all the components dissolve completely in water. 
• Dispense 5 mL (or desired volume) of the broth in test tubes and loosely put it on the cap or cotton plug.
• Autoclave the test tubes at 121°C and 15 lbs pressure for 15 minutes and let them cool to around 40 – 45°C before inoculation.

Reagents

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

Equipment

PPE and other general laboratory materials

Test Organisms (Sample Bacteria)

Positive Control: Enterococcus faecalis ATCC 29212

Negative Control: Streptococcus bovis ATCC 33317

Procedure of Salt Tolerance Test

1. Using a sterile inoculating loop, pick up well-isolated colonies of sample bacteria from fresh (18 to 24 hours old) culture and inoculate the broth. (Transfer 1 to 2 loops full of bacterial suspension/ fresh broth culture.)
2. Incubate the tubes aerobically for 24 hours at 35±2°C and observe for visible growth (turbidity) or color change. 
3. If no growth is seen, re-incubate the tubes and observe for growth for up to 72 hours and check for turbidity or color change at 48 and 72 hours.  

Result and Interpretation of Salt Tolerance Test

• A positive test is indicated by bacteria growth (increase in turbidity) and/or change in the color of broth from purple to yellow. (usually within 24 hours, but may take up to 48 to 72 hours for some Enterococci strains, and up to 14 days for Mycobacterium spp. and Actinobacteria) 
• A negative test is indicated by no bacterial growth (turbidity) and no color change of the medium.

Quality Control

Enterococcus faecalis ATCC 29212 causes an increase in turbidity and the development of yellow color in the medium after 24 hours of incubation. 

Streptococcus bovis ATCC 33317 shows no growth and no color change even after 72 hours of incubation.

Common Salt Tolerant Pathogenic Cocci Bacteria

Enterococcus spp., Staphylococcus aureus, Streptococcus agalactiae  

Precautions

• While supplementing (adding salt) readymade culture medium, look at the amount of NaCl already present in the medium and add accordingly to make a final NaCl concentration of 6.5%.
• Don’t report negative results after 24 hours; re-incubate for 72 hours before reporting negative.
• Use light inoculum because heavy inoculum may cause turbidity and give a false positive result.

Applications of Salt Tolerance Test

• Differentiation of Enterococcus spp. from other non-hemolytic Streptococci/Group D Streptococci (non-enterococci). 
• Differentiation of Aerococcus spp. (6.5% salt-tolerant species) from other similar characters showing cocci like Stomatococcus and Helococcus (both non-salt tolerant). 

Limitations of Salt Tolerance Test

• It is not a confirmatory test; hence requires other biochemical test results to confirm bacterial genera and species.
• Some β-hemolytic Streptococci (like Streptococcus agalactiae) also grow in 6.5% NaCl concentration and may create confusion with Enterococci identification. 
• Little growth without acid production may create confusion while reading turbidity. 
• If bacteria grow without producing acid, the color of the medium doesn’t change, making it difficult to read.
• Require at least 72 hours for reporting the negative result.

References

1. Leber, Amy L., editor in chief. (2016). Clinical microbiology procedures handbook (Fourth edition). Washington, DC : ASM Press 1752 N St., N.W., [2016]
2. Tille, P. M., & Forbes, B. A. (2014). Bailey & Scott’s diagnostic microbiology (Thirteenth edition.). St. Louis, Missouri: Elsevier.
3. Vreeland RH. Mechanisms of halotolerance in microorganisms. Crit Rev Microbiol. 1987;14(4):311-56. doi: 10.3109/10408418709104443. PMID: 3308318.
4. Greenway, H & Munns, Rana. (1980). Mechanisms of Salt Tolerance in Nonhalophytes. Annual Review of Plant Physiology. 31. 149-190. 10.1146/annurev.pp.31.060180.001053.
5. Edbeib, Mohammed & Wahab, Roswanira & Huyop, Fahrul. (2016). Halophiles: Biology, adaptation, and their role in decontamination of hypersaline environments. World Journal of Microbiology and Biotechnology. 32. 135. 10.1007/s11274-016-2081-9.
6. Peters AC, Thomas L, Wimpenny JW. Effects of salt concentration on bacterial growth on plates with gradients of pH and temperature. FEMS Microbiol Lett. 1991 Jan 15;61(2-3):309-14. doi: 10.1016/0378-1097(91)90571-q. PMID: 1903754.
7. Conville, P. S., & Witebsky, F. G. (1998). Variables Affecting Results of 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
8. Salt Tolerance Test: Principle, Procedure, and Result (researchtweet.com)
9. Salt Tolerance Test – Principle, Procedure, Uses and Interpretation (microbiologyinfo.com)
10. What Are Halophiles – Water Content – Bedford Astronomy Club
11. How does salt concentration affect bacterial growth? – WisdomAnswer
12. Salt Tolerance Test Principle, Purpose, Procedure, Result (microbiologynote.com)
13. Salt Tolerance Test: Introduction, Principle, Procedure and Result (universe84a.com)
14. Streptococci, groups A, B, and D. Enterococcus faecalis (microbiologybook.org)
15. temperature-salt 2019 (ysu.edu)