Why must the catalase test be performed before the salt tolerance test?

Salt tolerance alone doesn't distinguish Enterococcus from Staphylococcus, both can grow in high-salt media, but through entirely different mechanisms. The salt tolerance test is only meaningful once catalase has confirmed the organism is catalase-negative, ruling out Staphylococcus first.

Why does Enterococcus survive in 6.5% NaCl broth when many other Gram-positive cocci can't?

Enterococcus faecalis and E. faecium import glycine betaine, a compatible solute, through dedicated membrane transporters. This molecule balances internal osmotic pressure against the high external salt concentration, preventing the water loss that would otherwise kill a less salt-tolerant organism.

Is a clear salt tolerance broth at 24 hours enough to call the result negative?

Not yet. A true negative requires extending incubation to 72 hours before reporting. The tube should also be gently agitated before reading, since growth can settle out during incubation and be mistaken for no growth at all.

How do the bile esculin and salt tolerance tests work together to identify Enterococcus?

Bile esculin narrows an organism to either Enterococcus or a non-enterococcal group D streptococcus (the S. gallolyticus group), because both are bile esculin positive. Salt tolerance then splits them: Enterococcus grows in 6.5% NaCl, while the S. gallolyticus group does not. So bile esculin positive plus salt tolerance positive indicates Enterococcus, and bile esculin positive plus salt tolerance negative indicates a group D streptococcus. PYR confirms the call, positive for Enterococcus and negative for S. gallolyticus.

Why can the salt tolerance test give a false positive?

The most common cause is a heavy inoculum: the inoculum itself can cloud the broth and be mistaken for growth. Inoculate lightly, with only 2 to 3 colonies. Biologically, some organisms other than Enterococcus also tolerate 6.5% NaCl, including up to 80% of Group B streptococci, occasional Group A strains, and Aerococcus species. This is why salt tolerance must be paired with bile esculin and read after a negative catalase, never used alone to call Enterococcus.

Why should a negative salt tolerance result not be reported at 24 hours?

Because the test runs on a longer timescale than many others. A true negative requires incubation out to 72 hours; reading once at 24 hours and reporting negative can miss slower growth. Also, growth can settle to the bottom of the tube and make the broth look clear, so the tube should be gently agitated before reading to resuspend any sediment. Enterococcus typically shows growth and a color change within 24 hours, but a negative call needs the full 72.

Salt Tolerance Test for Enterococcus species

Salt tolerance test is used in the identification of enterococcal group D Streptococcus on the basis of their salt tolerance. The ability of the bacteria to grow in the presence of a variable amount of sodium chloride (NaCl) has been used to characterize several bacteria, including viridans streptococci.

Principle

Enterococcus faecalis and E. faecium tolerate this concentration of salt because they import glycine betaine, a compatible solute, through dedicated membrane transporters. This molecule balances the internal osmotic pressure against the high salt concentration outside the cell, preventing the water loss that would otherwise kill a less salt-tolerant organism

This test is particularly useful for presumptive identification of the enterococcal group D organisms, which have the specific ability to grow in the presence of 6.5% NaCl incorporated into either a broth or an agar medium. This test, along with the bile-esculin test, is used in many laboratories to distinguish Enterococcus species from the non-enterococcal group D streptococci, chiefly the Streptococcus bovis group (now Streptococcus gallolyticus). Enterococci is a significant cause of endocarditis with a high degree of mortality.

Brain Heart infusion (BHI) broth supplemented with 6.5% sodium chloride and bromocresol purple as a pH indicator is used for this purpose. The indicator is included to make reading the test results easier. The broth also includes dextrose. The fermentation of dextrose (glucose) results in the production of acid. This changes the pH of the media causing the media to turn from purple to yellow.

How to Remember

This is the same "survives everything" story from the Enterococcus faecalis article. The salt tolerance test isn't testing some unrelated quirky trait, it's testing the exact survival machinery already described there, here aimed specifically at osmotic stress. The organism imports its own internal counterweight (glycine betaine) to balance the salt outside, so it simply doesn't shrivel the way a less hardy organism would.

Procedure of the Salt Tolerance Test

Select no more than 2-3 colonies (preferably from an overnight culture) to inoculate a tube of salt tolerance broth Note: It is important to lightly inoculate the tube otherwise you may get a false positive.
Loosen the cap and incubate aerobically for 24 hours at 37°C.
Continue incubation up to 72 hours if you get a negative result at 24 hours.

Salt Tolerance Test Results and Interpretation

A positive test is the presence of obvious bacterial growth (turbidity in liquid medium) in the medium, with or without a color change in the indicator. If the organism is bile-esculin-positive and grows in the 6.5% NaCl broth, the organisms are an Enterococcus species. If the organism is bile-esculin-positive and fails to grow in the 6.5% NaCl broth, the organism is a group D streptococcus.
A negative result is indicated by no growth after 72 hours. Enterococcus spp typically changes the media color within 24 hours.

Organisms and their results:

Positive growth: Enterococcus faecalis
Negative growth: Streptococcus gallolyticus (S. bovis group), the non-enterococcal group D streptococci

Test	Enterococcus	S. gallolyticus group
Bile esculin	Positive	Positive
6.5% NaCl (this test)	Positive	Negative
PYR	Positive	Negative

This test is the one that separates the two group D organisms; the others confirm.

Where students actually get confused

Catalase must be run first, every time. Salt tolerance alone doesn't distinguish Enterococcus from Staphylococcus, many staphylococci also tolerate high salt, but through an entirely different, unrelated mechanism. A salt-tolerant, catalase-positive coccus is Staph, not Enterococcus. This test only means something once catalase has already ruled Staph out.
Aerococcus passes both tests. Unlike the other mimics, Aerococcus (A. viridans, A. urinae) grows in 6.5% NaCl and is bile-esculin-positive, so it clears both hurdles of the group D pairing and can be mistaken for Enterococcus. This is the one mimic the two-test pairing does not settle. Colony morphology, arrangement (Aerococcus forms tetrads and clusters, not chains), and PYR/LAP or definitive testing separate them. In practice, salt tolerance is also used specifically to tell Aerococcus (salt-tolerant) from lookalikes such as Stomatococcus and Helcococcus (not salt-tolerant).
Salt tolerance alone doesn't confirm Enterococcus either. Up to 80% of Group B streptococci can be salt-tolerant, and occasional Group A isolates too. Pair this result with bile esculin before calling it. The full presumptive workup is three tests read together: bile esculin (positive for both Enterococcus and group D strep), 6.5% NaCl (this test: positive for Enterococcus, negative for the S. gallolyticus group), and PYR (positive for Enterococcus, negative for S. gallolyticus). Salt tolerance is the test that actually splits the two; PYR confirms the call. See the bile esculin article for the full differentiation grid.
Heavy inoculation manufactures a false positive. The inoculum itself can be mistaken for turbidity if you don't inoculate lightly, 2-3 colonies only.
A negative result at 24 hours isn't final. This test runs on a different timescale than most others in this batch, a true negative requires waiting out to 72 hours, not reading once and reporting.
Settling during incubation can fake a negative. Gently agitate the tube before reading, growth can sediment out and look like clear, ungrown broth if left undisturbed.

Key exam facts in one table

Feature	Detail	Memory hook
Tests for	Growth in 6.5% NaCl	Same "survives everything" story
Mechanism	Glycine betaine osmoprotectant transport	Internal counterweight to external salt
Medium	BHI broth + 6.5% NaCl + bromocresol purple	Purple to yellow on fermentation
Read time	24h, extend to 72h if negative	Different timescale than most tests
Prerequisite	Catalase test (must be negative first)	Staph tolerates salt too, different reason
Pair with	Bile esculin test	Neither alone is conclusive
False-positive risk	Heavy inoculum, up to 80% of GBS
False-negative risk	Settling, not waiting the full 72h	Agitate before reading

References

Heo, S., Lee, J., Lee, J. H., & Jeong, D. W. (2019). Genomic Insight into the Salt Tolerance of Enterococcus faecium, Enterococcus faecalis and Tetragenococcus halophilus. Journal of microbiology and biotechnology, 29(10), 1591–1602. https://doi.org/10.4014/jmb.1908.08015
Facklam R. R. (1973). Comparison of several laboratory media for presumptive identification of enterococci and group D streptococci. Applied microbiology, 26(2), 138–145. https://doi.org/10.1128/am.26.2.138-145.1973
Said MS, Tirthani E, Lesho E. Enterococcus Infections. [Updated 2022 May 2]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK567759/

How to Remember

Procedure of the Salt Tolerance Test

Where students actually get confused

Key exam facts in one table

Frequently Asked Questions