[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fxi6aQK9h-NAH0E4Fz_7zVisEMi4-cKUrdKzYqEqsDLg":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":66},[4,8,12,16,20,24,28],{"title":5,"slug":6,"path":7},"About Microbeonline.com","about-microbeonline-com","\u002Fabout-microbeonline-com\u002F",{"title":9,"slug":10,"path":11},"About Me","about-me","\u002Fabout-microbeonline-com\u002Fabout-me\u002F",{"title":13,"slug":14,"path":15},"Advertise with Us","advertise-us","\u002Fadvertise-us\u002F",{"title":17,"slug":18,"path":19},"Privacy Policy","privacy-policy","\u002Fprivacy-policy\u002F",{"title":21,"slug":22,"path":23},"Abbreviations","abbreviations","\u002Fabbreviations\u002F",{"title":25,"slug":26,"path":27},"Microbes","microbes","\u002Fmicrobes\u002F",{"title":29,"slug":30,"path":31},"Books","recommended-books","\u002Frecommended-books\u002F",{"type":33,"data":34},"blog",{"slug":35,"title":36,"description":37,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":40,"lastUpdatedDate":41,"draft":42,"category":43,"image":38,"body":44,"faq":45,"tags":64,"related":65},"salt-tolerance-test-enterococcus-species-principle-procedure-results","Salt Tolerance Test for Enterococcus species","Salt tolerance test principle, procedure, and interpretation for presumptively identifying Enterococcus species using 6.5% NaCl broth.",null,"Acharya Tankeshwar","2014-01-21","2026-07-12",false,"biochemical-tests","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](\u002Fviridans-streptococci-pathogenesis-and-lab-diagnosis\u002F).\n\n**Principle**\n\n*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\n\nThis 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](\u002Fbile-esculin-test-enterococcus-species-principle-procedure-results\u002F), 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.\n\n[Brain Heart infusion (BHI) broth](\u002Fbrain-heart-infusion-bhi-broth-composition-preparation-and-uses\u002F) 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.\n\n### How to Remember\n\nThis is the same \"survives everything\" story from the [*Enterococcus faecalis* article. ](https:\u002F\u002Fmicrobeonline.com\u002Fenterococcus-faecalis-pathogenesis-diagnosis\u002F)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.\n\n## Procedure of the Salt Tolerance Test\n\n1. 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.\n2. Loosen the cap and incubate aerobically for 24 hours at 37°C.\n3. Continue incubation up to 72 hours if you get a negative result at 24 hours.\n\n![](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FSalt-Tolerance-Test.jpg)**Results and Interpretation**\n\n1. 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.\n2. A negative result is indicated by no growth after 72 hours. *Enterococcus* spp typically changes the media color within 24 hours.\n\n**Organisms and their results:**\n\n1. Positive growth: *Enterococcus faecalis*\n2. Negative growth: *Streptococcus gallolyticus* (*S. bovis* group), the non-enterococcal group D streptococci\n\n| Test | *Enterococcus* | *S. gallolyticus* group |\n| --- | --- | --- |\n| Bile esculin | Positive | Positive |\n| **6.5% NaCl (this test)** | **Positive** | **Negative** |\n| PYR | Positive | Negative |\n\nThis test is the one that separates the two group D organisms; the others confirm.\n\n### Where students actually get confused\n\n- **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.\n- ***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\u002FLAP 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).\n- **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.\n- **Heavy inoculation manufactures a false positive.** The inoculum itself can be mistaken for turbidity if you don't inoculate lightly, 2-3 colonies only.\n- **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.\n- **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.\n\n### Key exam facts in one table\n\n| Feature | Detail | Memory hook |\n| --- | --- | --- |\n| Tests for | Growth in 6.5% NaCl | Same \"survives everything\" story |\n| Mechanism | Glycine betaine osmoprotectant transport | Internal counterweight to external salt |\n| Medium | BHI broth + 6.5% NaCl + bromocresol purple | Purple to yellow on fermentation |\n| Read time | 24h, extend to 72h if negative | Different timescale than most tests |\n| Prerequisite | Catalase test (must be negative first) | Staph tolerates salt too, different reason |\n| Pair with | Bile esculin test | Neither alone is conclusive |\n| False-positive risk | Heavy inoculum, up to 80% of GBS |  |\n| False-negative risk | Settling, not waiting the full 72h | Agitate before reading |\n\n**References**\n\n1. 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. \u003Chttps:\u002F\u002Fdoi.org\u002F10.4014\u002Fjmb.1908.08015>\n2. Facklam R. R. (1973). Comparison of several laboratory media for presumptive identification of enterococci and group D streptococci. *Applied microbiology*, *26*(2), 138–145. \u003Chttps:\u002F\u002Fdoi.org\u002F10.1128\u002Fam.26.2.138-145.1973>\n3. Said MS, Tirthani E, Lesho E. Enterococcus Infections. \\[Updated 2022 May 2\\]. In: StatPearls \\[Internet\\]. Treasure Island (FL): StatPearls Publishing; 2023 Jan-. Available from: \u003Chttps:\u002F\u002Fwww.ncbi.nlm.nih.gov\u002Fbooks\u002FNBK567759\u002F>",[46,49,52,55,58,61],{"question":47,"answer":48},"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.",{"question":50,"answer":51},"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.",{"question":53,"answer":54},"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.",{"question":56,"answer":57},"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.",{"question":59,"answer":60},"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.",{"question":62,"answer":63},"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.",[],[],[67,73,80,85,89,93,98,103,107,111],{"slug":68,"name":39,"description":69,"image":70,"body":71,"postCount":72},"acharya-tankeshwar","Editor-in-chief","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Ftankeshwar-acharya-author-microbeonline.jpg","***Tankeshwar Acharya, MSc (Medical Microbiology)***\n\n*Tankeshwar Acharya is an Assistant Professor in the Department of Microbiology at Patan Academy of Health Sciences (PAHS), Nepal, where he has been teaching and practicing clinical microbiology for over 14 years. He is the founder of Microbe Online, one of the leading free microbiology education resources on the web, covering bacteriology, mycology, parasitology, immunology, and clinical laboratory diagnostics written from direct experience in both the classroom and the diagnostic laboratory.*",433,{"slug":74,"name":75,"description":76,"image":77,"body":78,"postCount":79},"ashma-shrestha","Ashma Shrestha","SEO Copywriter and Science Communicator\nKathmandu, Nepal","https:\u002F\u002Fassets.microbeonline.com\u002Fauthors\u002Fashma-shrestha.png","Ashma Shrestha holds a Master of Science in Medical Microbiology from the Institute of Science and Technology (IOST), Tribhuvan University, Nepal, where she developed a strong foundation in virology, molecular biology, and diagnostic microbiology.\n\nShe now works as an SEO Copywriter at Resolution Digital, where she combines her scientific training with research-driven content strategy. She is certified in Google Analytics and Google Business Profile (GBP), and brings a data-informed approach to science communication writing content that is not only accurate but structured to reach and serve the students who need it most.\n\nAt microbeonline, Ashma contributes articles primarily in virology and molecular biology, areas she finds most compelling for their mechanistic depth and their growing clinical relevance. Her writing reflects the same standard the site is built on: factual rigor, clear explanation of the *why* behind microbiology concepts, and content that helps students move from memorization to genuine understanding.\n\nShe is passionate about making complex microbiological concepts accessible without sacrificing accuracy; a skill that sits at the intersection of her scientific training and her professional work in content and SEO.",81,{"slug":81,"name":82,"description":83,"image":38,"body":38,"postCount":84},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":86,"name":87,"description":83,"image":38,"body":38,"postCount":88},"samikshya-acharya","Samikshya Acharya",20,{"slug":90,"name":91,"description":83,"image":38,"body":38,"postCount":92},"alisha-tripathi","Alisha Tripathi",6,{"slug":94,"name":95,"description":96,"image":38,"body":38,"postCount":97},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":99,"name":100,"description":101,"image":38,"body":38,"postCount":102},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":104,"name":105,"description":83,"image":38,"body":38,"postCount":106},"srijana-khanal","Srijana Khanal",18,{"slug":108,"name":109,"description":101,"image":38,"body":38,"postCount":110},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":112,"name":113,"description":83,"image":38,"body":114,"postCount":115},"nisha-rijal","Nisha Rijal","**Nisha Rijal** is a microbiologist and quality assurance specialist. She served for nearly 12 years as a microbiologist at the National Public Health Laboratory (NPHL), Nepal's national reference laboratory, and continues to work as a consultant microbiologist in international public health organization. ",51]