Salt Tolerance Test- Principle, Procedure, Results, Uses

A salt tolerance test is a microbiological test that determines the ability of bacteria to grow in a high salt concentration environment. It is based on the principle that salt affects the osmotic pressure and membrane permeability of bacterial cells, and only some bacteria have developed mechanisms to adapt and survive in such conditions. The test is useful for differentiating some bacterial groups, such as enterococci, aerococci, and staphylococci, from other similar-looking bacteria that are not salt tolerant .

The test is performed by inoculating a broth medium that contains 6.5% sodium chloride (NaCl) and a pH indicator, such as bromocresol purple, with the bacterial culture. The broth also contains a fermentable carbohydrate, such as glucose or dextrose, that serves as a substrate for bacterial metabolism. After incubation, the growth and color of the broth are observed. A positive result is indicated by turbidity (cloudiness) and/or a color change from purple to yellow, which means that the bacteria can grow and produce acid in the high salt medium. A negative result is indicated by no growth and no color change, which means that the bacteria are inhibited or killed by the high salt concentration .

Some advantages of the salt tolerance test are:

• It is simple, inexpensive, and easy to perform.
• It can provide a rapid presumptive identification of some bacterial groups.
• It can help in the diagnosis of some infections caused by salt-tolerant pathogens.

Some limitations of the salt tolerance test are:

• It is not a confirmatory test and requires other biochemical or molecular tests for complete identification of bacteria.
• It may give false positive results with some bacteria that can grow in high salt but do not belong to the target groups, such as some beta-hemolytic streptococci or pediococci.
• It may give false negative results with some bacteria that are slow growing or have low inoculum in the high salt medium.
• It may be difficult to interpret the results if there is no color change but slight turbidity in the broth .

The salt tolerance test is an important tool for microbiologists and clinicians to differentiate and identify some bacteria that have clinical significance. For example, enterococci are common causes of urinary tract infections, endocarditis, wound infections, and bacteremia, and they are often resistant to many antibiotics. Aerococci are emerging pathogens that can cause urinary tract infections, endocarditis, septicemia, and meningitis. Staphylococci are well-known causes of skin and soft tissue infections, abscesses, pneumonia, osteomyelitis, septic arthritis, endocarditis, toxic shock syndrome, and food poisoning .

In this article, we will discuss the objectives, principle, requirements, procedure, results, quality control, precautions, applications, and limitations of the salt tolerance test in detail. We will also provide some examples of common salt-tolerant pathogenic cocci bacteria that can be identified by this test.

The salt tolerance test is a simple and useful method to determine the ability of bacteria to tolerate a high salt concentration (of 6.5%) and to differentiate some bacterial groups based on this characteristic. The main objectives of the test are:

• To identify enterococci (positive) from non-enterococci (negative) among the catalase-negative, gram-positive cocci . Enterococci are important pathogens that can cause infections of the urinary tract, bloodstream, heart valves, and surgical sites.
• To identify aerococci (positive) from other similar cocci such as stomatococci and helcococci (negative). Aerococci are opportunistic pathogens that can cause urinary tract infections, endocarditis, and bacteremia.
• To identify some salt-tolerant species of streptococci, pediococci, leuconostoc, mycobacteria, and actinobacteria . These bacteria may have clinical or environmental significance depending on the context .

The principle of salt tolerance test is based on the ability of bacteria to grow in a medium with a high concentration of sodium chloride (NaCl). NaCl acts as a selective agent that affects the osmotic pressure and membrane permeability of bacterial cells. Only salt-tolerant or halophilic bacteria can survive and grow in such a medium, while non-halophilic bacteria are inhibited or killed by the high salt concentration.

Salt-tolerant bacteria have developed various mechanisms to adapt to high salt environments, such as:

• Increasing the cytoplasmic ion concentration to match the external environment
• Increasing the concentration of compatible solutes (such as potassium ions) within the cytoplasm
• Modifying the intracellular water movement process
• Modifying the nucleotides and proteins to function in high salt conditions

These mechanisms allow salt-tolerant bacteria to maintain their osmotic balance and cellular integrity in the presence of 6.5% NaCl or higher. On the other hand, non-halophilic bacteria lack these mechanisms and suffer from osmotic shock, plasmolysis and specific ion effects that damage their cell membranes and enzymes.

The salt tolerance test uses a broth medium that contains 6.5% NaCl as well as a fermentable carbohydrate (such as glucose) and a pH indicator (such as bromocresol purple). The broth medium can be modified from brain-heart infusion (BHI) broth or tryptic soy broth by adding NaCl and bromocresol purple. The broth medium is inoculated with a pure culture of the test organism and incubated at 35°C for 24 to 72 hours. The growth and fermentation of the test organism are indicated by turbidity and color change of the broth medium.

A positive result is observed when the test organism grows (increases turbidity) and ferments the carbohydrate (produces acid) in the broth medium. The acid production lowers the pH of the medium and changes the color of bromocresol purple from purple to yellow. A positive result indicates that the test organism is salt-tolerant and belongs to Enterococcus spp. or Aerococcus spp.

A negative result is observed when the test organism does not grow (no turbidity) or does not ferment the carbohydrate (no acid production) in the broth medium. The pH and color of the medium remain unchanged. A negative result indicates that the test organism is not salt-tolerant and belongs to other non-enterococcal group D streptococci or other gram-positive cocci.

The salt tolerance test is a simple and useful method to differentiate enterococci from other non-hemolytic streptococci based on their ability to grow in a high salt concentration. However, it is not a confirmatory test and requires other biochemical tests for complete identification of bacterial species. Some limitations of this test are:

• Some beta-hemolytic streptococci (such as Streptococcus agalactiae) can also grow in 6.5% NaCl and may be confused with enterococci
• Some enterococci may grow slowly or weakly in 6.5% NaCl and may require longer incubation time or heavier inoculum
• Some bacteria may grow without producing acid in 6.5% NaCl and may not change the color of the medium
• The broth medium should be prepared carefully by adjusting the amount of NaCl according to the original composition of the base medium

To perform the salt tolerance test, you will need the following materials and equipment:

• Culture media: You can use either modified salt broth or modified BHI broth as the culture medium for this test. Both media contain 6.5% NaCl and bromocresol purple indicator. Modified salt broth also contains peptone and glucose, while modified BHI broth contains brain-heart infusion, peptone and glucose. You can prepare these media from scratch or use ready-made powder according to the manufacturer`s instructions .
• Test organisms: You will need pure cultures of the bacteria that you want to test for salt tolerance. You can use Enterococcus faecalis ATCC 29212 as a positive control and Streptococcus bovis ATCC 33317 as a negative control .
• Inoculating loop or wire: You will need a sterile inoculating loop or wire to transfer the bacterial colonies from the solid culture media to the broth tubes.
• Solid culture media: You will need solid culture media such as blood agar or nutrient agar to grow the bacterial colonies for 18 to 24 hours before inoculating them into the broth tubes.
• Incubator: You will need an incubator to incubate the broth tubes at 35±2°C for up to 72 hours .
• Bunsen burner: You will need a Bunsen burner to sterilize the inoculating loop or wire before and after each inoculation.
• Test tube rack: You will need a test tube rack to hold the broth tubes during incubation and observation.

The culture media used in salt tolerance test are modified salt broth and modified BHI broth. Both media contain 6.5% sodium chloride (NaCl) to create a high salt concentration environment for the bacteria. In addition, both media also contain bromocresol purple as a pH indicator to detect acid production by the bacteria.

Modified salt broth is a liquid medium that consists of peptone, glucose, NaCl and bromocresol purple. Peptone provides nitrogenous compounds and amino acids for bacterial growth. Glucose serves as a fermentable carbohydrate source that can be utilized by some bacteria to produce acid. NaCl creates osmotic stress for the bacteria and selects for salt-tolerant or halophilic organisms. Bromocresol purple changes color from purple to yellow when the pH of the medium drops below 6.8 due to acid production.

Modified BHI broth is another liquid medium that consists of brain heart infusion (BHI) powder, protease peptone, glucose, NaCl and bromocresol purple. BHI powder is a rich source of nutrients derived from calf brain and beef heart that supports the growth of a wide range of bacteria. Protease peptone provides additional nitrogenous compounds and amino acids for bacterial growth. Glucose serves as a fermentable carbohydrate source that can be utilized by some bacteria to produce acid. NaCl creates osmotic stress for the bacteria and selects for salt-tolerant or halophilic organisms. Bromocresol purple changes color from purple to yellow when the pH of the medium drops below 6.8 due to acid production.

Both media are prepared by dissolving the appropriate amount of powder (or the media components) in water and autoclaving at 121°C and 15 lbs pressure for 15 minutes. The media are then dispensed in test tubes and inoculated with bacterial cultures. The inoculated tubes are incubated aerobically at 35±2°C for 24 to 72 hours and observed for visible growth (turbidity) or color change.

The culture media used in salt tolerance test are important for differentiating salt-tolerant bacteria such as Enterococcus spp., Staphylococcus aureus and Streptococcus agalactiae from non-salt-tolerant bacteria such as Streptococcus bovis, Stomatococcus spp. and Helococcus spp.

Modified salt broth is a culture medium that contains 6.5% sodium chloride (NaCl) and is used to test the ability of bacteria to tolerate high salt concentration. The broth also contains peptone, glucose, and bromocresol purple as a pH indicator. The composition and preparation of modified salt broth are as follows:

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
• Bromocresol purple- 0.016 grams
• Final pH 7.2 ±0.2 at 25°C

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 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.

Brain Heart Infusion (BHI) broth is a general-purpose liquid medium that contains infusions from calf brain and beef heart, proteose peptone, dextrose, sodium chloride and disodium phosphate. It is used for the cultivation and maintenance of a wide variety of fastidious and non-fastidious microorganisms, including aerobic and anaerobic bacteria, yeast, and molds .

Modified BHI broth is a variation of BHI broth that is supplemented with 6.5% sodium chloride and 0.1% bromocresol purple indicator. It is used for the salt tolerance test to differentiate Enterococcus spp. from other non-hemolytic Streptococci/Group D Streptococci (non-enterococci).

The composition of modified BHI broth per 1000 mL is as follows :

• Calf brain, infusion from: 200 g
• Beef heart, infusion from: 250 g
• Proteose peptone: 10 g
• Dextrose: 2 g
• Sodium chloride: 65 g
• Disodium phosphate: 2.5 g
• Bromocresol purple: 0.016 g
• Final pH: 7.4 ±0.2 at 25°C

The procedure for preparation of modified BHI broth is as follows :

• Suspend 37 g of BHI broth powder (or the media components) in a conical flask (or glass bottle) with 1000 mL distilled water.
• Add 60 g of sodium chloride and 0.016 g of bromocresol purple per 1000 mL 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 on the cap or cotton plug.
• Sterilize by autoclaving at 121°C and 15 lbs pressure for 15 minutes and let them cool to around 40 – 45°C before inoculation.

The reagents and equipment required for performing the salt tolerance test are as follows:

• Reagents:
  • Sodium chloride (NaCl): This is the main ingredient of the test medium, which creates a high salt concentration of 6.5% and acts as a selective agent for salt-tolerant bacteria.
  • Bromocresol purple: This is a pH indicator that changes color from purple to yellow when the medium becomes acidic due to bacterial fermentation of dextrose.
  • Dextrose: This is a fermentable carbohydrate that serves as a source of energy and carbon for bacteria and also helps to detect acid production by color change of the indicator.
  • Brain-heart infusion broth (BHI) or peptone water: These are the base media that provide nutrients and support bacterial growth. BHI broth is preferred over peptone water as it enhances the growth of enterococci and other fastidious bacteria.
• Equipment:
  • Test tubes: These are used to hold the test medium and inoculate the bacteria. The test tubes should be sterilized before use and have a loose cap or cotton plug to allow air exchange.
  • Inoculating loop or wire: These are used to transfer bacterial colonies from solid culture media to the test medium. The loop or wire should be sterilized by flaming before and after each inoculation.
  • Incubator: This is used to provide optimal temperature and environment for bacterial growth. The incubator should be set at 35±2°C and have a timer to monitor the incubation period.
  • Bunsen burner: This is used to provide a flame for sterilizing the loop or wire and also to prevent contamination by creating an upward air current around the work area.
  • Test tube rack: This is used to hold the test tubes in an upright position and facilitate observation of turbidity and color change. The rack should be labeled with the name of the test organism and the date of inoculation.
  • Quality control strains: These are reference bacteria that are used to verify the validity and accuracy of the test results. The quality control strains for salt tolerance test are Enterococcus faecalis ATCC 29212 (positive control) and Streptococcus bovis ATCC 33317 (negative control).

The salt tolerance test is mainly used to differentiate enterococci (positive) from non-enterococci (negative) among the catalase-negative, gram-positive cocci. Enterococci are a group of bacteria that can cause various infections in humans and animals, such as urinary tract infections, endocarditis, wound infections, and septicemia. They are also important indicators of fecal contamination in water and food.

Some examples of enterococci that are salt tolerant are:

• Enterococcus faecalis
• Enterococcus faecium
• Enterococcus durans
• Enterococcus avium
• Enterococcus casseliflavus

Some examples of non-enterococci that are salt intolerant are:

• Streptococcus bovis
• Streptococcus equinus
• Streptococcus gallolyticus
• Streptococcus infantarius
• Streptococcus lutetiensis

These bacteria are usually associated with animal infections, especially in ruminants. They can also cause human infections such as bacteremia, endocarditis, and meningitis.

Besides enterococci and non-enterococci, some other bacteria that can grow in 6.5% NaCl broth are:

• Aerococcus spp. (e.g., Aerococcus viridans, Aerococcus urinae)
• Pediococcus spp. (e.g., Pediococcus acidilactici, Pediococcus pentosaceus)
• Leuconostoc spp. (e.g., Leuconostoc mesenteroides, Leuconostoc lactis)
• Beta-hemolytic streptococci (e.g., Streptococcus agalactiae, Streptococcus pyogenes)

These bacteria are usually found in dairy products, fermented foods, and human or animal mucous membranes. They can also cause opportunistic infections in immunocompromised hosts.

Therefore, it is important to use other biochemical tests along with the salt tolerance test to confirm the identity of the bacteria.

• 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.)
• Incubate the tubes aerobically for 24 hours at 35±2°C and observe for visible growth (turbidity) or color change.
• 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.

The procedure of salt tolerance test is simple and straightforward. It involves inoculating the sample bacteria into the modified salt broth or modified BHI broth and incubating them under aerobic conditions. The growth and acid production of the bacteria are then observed by checking the turbidity and color change of the medium. The test can be performed on pure cultures or clinical specimens such as blood, urine, cerebrospinal fluid, etc. The test can also be performed on agar plates by streaking the bacteria on the surface of the agar supplemented with 6.5% NaCl and incubating them for 24 to 72 hours.

The salt tolerance test is based on the principle that only salt-tolerant or halophilic bacteria can grow and produce acid in a medium with a high salt concentration of 6.5%. The acid production lowers the pH of the medium and causes a color change from purple to yellow due to the bromocresol purple indicator. The test is useful for differentiating Enterococcus spp. from other non-hemolytic Streptococci/Group D Streptococci (non-enterococci) and Aerococcus spp. from other similar characters showing cocci like Stomatococcus and Helococcus.

The procedure of salt tolerance test is summarized in the following table:

• A positive test is indicated by bacterial 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 after 72 hours of incubation.
• A positive test indicates that the bacteria are salt-tolerant or halophilic, which is a characteristic of Enterococcus spp., Staphylococcus aureus, Streptococcus agalactiae, Aerococcus spp., and some other bacteria .
• A negative test indicates that the bacteria are salt-sensitive or non-halophilic, which is a characteristic of most Streptococcus spp., Lactococcus spp., Leuconostoc spp., Pediococcus spp., and some other bacteria .
• The color change of the medium is due to the fermentation of glucose by the bacteria, which produces acidic byproducts that lower the pH and turn the indicator bromocresol purple from purple to yellow .
• The turbidity of the medium is due to the multiplication of bacteria in the high salt concentration, which indicates their ability to adapt and survive in such environments .

Quality control (QC) is the process of ensuring that the laboratory results are accurate, reliable and reproducible. QC involves monitoring the performance of the test methods, the equipment, the reagents and the personnel involved in the testing process. QC also involves taking corrective actions when any deviation from the expected standards is detected.

QC is essential for any microbiology test, including the salt tolerance test, because it helps to:

• Detect and prevent errors that may affect the test results
• Ensure the validity and comparability of the test results
• Maintain confidence and trust in the laboratory services
• Comply with the regulatory and accreditation requirements

QC Measures for Salt Tolerance Test

Some of the QC measures that should be followed for performing the salt tolerance test are:

• Use only sterile and labeled tubes and media for inoculation and incubation
• Use only fresh and well-isolated colonies of sample bacteria for inoculation
• Use appropriate positive and negative control organisms for each batch of testing
• Use light inoculum to avoid false positive results due to heavy turbidity
• Incubate the tubes at the optimal temperature and time as per the test protocol
• Observe the tubes for growth (turbidity) and color change at 24, 48 and 72 hours of incubation
• Record and report the results as per the standard criteria
• Compare the results with the expected outcomes of the control organisms
• Investigate and troubleshoot any discrepancies or unexpected results
• Document all the QC activities and results in a logbook or a database

QC Organisms for Salt Tolerance Test

The QC organisms that are commonly used for salt tolerance test are:

• Enterococcus faecalis ATCC 29212: This is a positive control organism that can grow and produce acid in 6.5% NaCl broth, resulting in turbidity and yellow color change of the medium within 24 hours of incubation.
• Streptococcus bovis ATCC 33317: This is a negative control organism that cannot grow in 6.5% NaCl broth, resulting in no turbidity and no color change of the medium even after 72 hours of incubation.

These QC organisms should be tested along with each batch of sample bacteria to ensure that the test method, media and reagents are working properly. The QC organisms should also be checked for their viability, purity and identity before use.

QC Criteria for Salt Tolerance Test

The QC criteria for salt tolerance test are:

• The positive control organism (Enterococcus faecalis ATCC 29212) should show growth (turbidity) and acid production (yellow color change) in 6.5% NaCl broth within 24 hours of incubation.
• The negative control organism (Streptococcus bovis ATCC 33317) should show no growth (turbidity) and no acid production (no color change) in 6.5% NaCl broth even after 72 hours of incubation.
• The sample bacteria should show either positive or negative results as per the test interpretation.
• If any of these criteria are not met, the test should be repeated or investigated for possible sources of error.

Some cocci bacteria can grow and survive in high salt concentration environments, such as 6.5% NaCl broth or agar. These bacteria are called salt tolerant or halophilic bacteria. Salt tolerance is a useful characteristic for the identification and differentiation of some pathogenic cocci bacteria from other non-pathogenic or less virulent ones. Some of the common salt tolerant pathogenic cocci bacteria are:

• Enterococcus spp.: These are gram-positive cocci that are arranged in pairs or short chains. They are normal inhabitants of the gastrointestinal tract, but they can also cause infections of the urinary tract, bloodstream, heart valves, and wounds. They are resistant to many antibiotics and can survive in harsh conditions, such as high temperature, low pH, and high salt concentration. Enterococcus spp. can ferment mannitol and produce acid, which changes the color of the bromocresol purple indicator in the modified BHI broth from purple to yellow .

• Staphylococcus aureus: This is a gram-positive coccus that is arranged in grape-like clusters. It is a common colonizer of the skin and mucous membranes, but it can also cause serious infections of the skin, soft tissues, bones, joints, lungs, heart, and blood. It produces various toxins and enzymes that contribute to its virulence and invasiveness. It is also resistant to many antibiotics and can grow in high salt concentration, such as 10% NaCl agar.

• Streptococcus agalactiae: This is a gram-positive coccus that is arranged in chains. It is a part of the normal flora of the female genital tract, but it can also cause infections of the newborns, such as sepsis, meningitis, and pneumonia. It is beta-hemolytic, meaning that it can lyse red blood cells on blood agar plates. It can also grow in 6.5% NaCl broth, which distinguishes it from other beta-hemolytic streptococci.

Precautions to be taken while performing Salt Tolerance Test

• Before proceeding with the salt tolerance test, perform a catalase test to rule out staphylococci, which can grow in media containing even 10% salt.
• Use a light inoculum (one or two colonies) to avoid false-positive results due to heavy inoculation.
• Do not report negative results after 24 hours; re-incubate for up to 72 hours before reporting negative.
• While supplementing ready-made culture medium with salt, check the amount of NaCl already present in the medium and add accordingly to make a final NaCl concentration of 6.5%.

• Be aware of the limitations of the salt tolerance test, such as the possibility of slow reactions, false-positive results with some beta-hemolytic streptococci and pediococci, and false-negative results with some enterococci . Confirm the identification of the bacteria with other biochemical, immunological, molecular, or mass spectrometry tests.

  Applications of Salt Tolerance Test

• The salt tolerance test is mainly used to differentiate enterococci (positive) from non-enterococci (negative) . Enterococci are a group of gram-positive cocci that can cause infections such as endocarditis, urinary tract infections, wound infections, and bacteremia.

• The salt tolerance test can also help to differentiate non-beta-hemolytic strains of catalase-negative, gram-positive cocci (such as Enterococcus and Aerococcus) based on their ability to grow in a 6.5% sodium chloride broth. Aerococcus species such as A. viridans and A. urinae can also grow in 6.5% NaCl, therefore salt tolerance broth can also be used to differentiate Aerococcus species from other similar organisms such as Stomatococcus and Helcococcus.
• The salt tolerance test can also be used to identify some halophilic bacteria that can grow in high salt concentrations, such as Mycobacterium spp. and Actinobacteria. These bacteria may have ecological and biotechnological importance, as they can survive in extreme environments and produce useful metabolites.

Limitations of Salt Tolerance Test

• The salt tolerance test is not a confirmatory test for the identification of bacteria. It only indicates the ability of bacteria to grow in a high salt concentration environment. Therefore, it should be used in conjunction with other biochemical tests to confirm the bacterial genera and species.
• Some β-hemolytic Streptococci (such as Streptococcus agalactiae) also grow in 6.5% NaCl concentration and may create confusion with Enterococci identification. Thus, the salt tolerance test should be performed along with other tests such as bile esculin hydrolysis test, pyrrolidonyl arylamidase (PYR) test, and Lancefield grouping to differentiate between Enterococci and other β-hemolytic Streptococci.
• Some bacteria may show little growth without acid production in the salt tolerance test. This may create difficulty in reading the turbidity and color change of the medium. For example, some strains of Enterococcus faecium may grow slowly and weakly in 6.5% NaCl broth without producing acid. In such cases, it is advisable to use a spectrophotometer to measure the optical density of the broth or subculture the broth onto blood agar plates to check for colony growth.
• If bacteria grow without producing acid in the salt tolerance test, the color of the medium does not change from purple to yellow. This may make it difficult to interpret the result visually. For example, some strains of Staphylococcus aureus may grow in 6.5% NaCl broth without producing acid. In such cases, it is recommended to use an indicator paper or a pH meter to measure the pH of the broth or subculture the broth onto mannitol salt agar plates to check for acid production.
• The salt tolerance test requires at least 72 hours for reporting a negative result. This may delay the diagnosis and treatment of bacterial infections caused by salt-tolerant pathogens. Moreover, some bacteria may take longer than 72 hours to grow in 6.5% NaCl broth, such as Mycobacterium spp. and Actinobacteria. In such cases, it is suggested to use alternative methods such as molecular techniques or MALDI-TOF mass spectrometry for rapid and accurate identification of bacteria.

• The salt tolerance test is not a confirmatory test for the identification of bacteria. It only indicates the ability of bacteria to grow in a high salt concentration environment. Therefore, it should be used in conjunction with other biochemical tests to confirm the bacterial genera and species.
• Some β-hemolytic Streptococci (such as Streptococcus agalactiae) also grow in 6.5% NaCl concentration and may create confusion with Enterococci identification. Thus, the salt tolerance test should be performed along with other tests such as bile esculin hydrolysis test, pyrrolidonyl arylamidase (PYR) test, and Lancefield grouping to differentiate between Enterococci and other β-hemolytic Streptococci.
• Some bacteria may show little growth without acid production in the salt tolerance test. This may create difficulty in reading the turbidity and color change of the medium. For example, some strains of Enterococcus faecium may grow slowly and weakly in 6.5% NaCl broth without producing acid. In such cases, it is advisable to use a spectrophotometer to measure the optical density of the broth or subculture the broth onto blood agar plates to check for colony growth.
• If bacteria grow without producing acid in the salt tolerance test, the color of the medium does not change from purple to yellow. This may make it difficult to interpret the result visually. For example, some strains of Staphylococcus aureus may grow in 6.5% NaCl broth without producing acid. In such cases, it is recommended to use an indicator paper or a pH meter to measure the pH of the broth or subculture the broth onto mannitol salt agar plates to check for acid production.
• The salt tolerance test requires at least 72 hours for reporting a negative result. This may delay the diagnosis and treatment of bacterial infections caused by salt-tolerant pathogens. Moreover, some bacteria may take longer than 72 hours to grow in 6.5% NaCl broth, such as Mycobacterium spp. and Actinobacteria. In such cases, it is suggested to use alternative methods such as molecular techniques or MALDI-TOF mass spectrometry for rapid and accurate identification of bacteria.

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