[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fHHx5LSZCRY-eJqyeuRN4bgwdKg8cJ9MtKcN3CXsIkxs":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":48},[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":46,"related":47},"hektoen-enteric-agar-composition-principle-uses","Hektoen Enteric (HE) Agar: Composition, Principle, Colony Characteristics, and Uses","Hektoen Enteric (HE) Agar is a selective and differential medium for Salmonella and Shigella with better Shigella recovery than SS agar. Learn its green-medium principle, three-carbohydrate differentiation, H2S indicator system, and colony colours.",null,"Acharya Tankeshwar","2015-01-27","2026-06-23",false,"culture-media","In 1968, microbiologists at the Hektoen Institute in Chicago were trying to solve a specific problem: *Shigella* strains were being missed in stool cultures because the highly selective media available at the time — particularly SS agar — were too inhibitory. Brilliant green dye, the primary selective agent in SS agar, suppressed not just coliforms but also the *Shigella* the medium was supposed to isolate.\n\nSylvia King and William Metzger's solution was to reformulate the medium from the ground up: use lower-toxicity dyes (bromthymol blue and acid fuchsin instead of brilliant green), add extra carbohydrates and peptones to offset bile salt inhibition, and build in a three-carbohydrate differentiation system that could distinguish *Salmonella* and *Shigella* from each other and from coliforms in a single incubation.\n\nThe result was HE agar — a medium specifically engineered for better enteric pathogen recovery, with *Shigella* isolation in mind from the outset.\n\nHektoen Enteric (HE) Agar is currently applicable as both direct and indirect plating medium for fecal specimens to enhance the recovery of species of Salmonella and Shigella from heavy numbers of mixed normal fecal flora.\n\n## Uses of Hektoen Enteric Agar\n\n**1. Primary plating of stool specimens for enteric pathogen isolation** HE agar is used as a primary selective\u002Fdifferential plating medium for fecal specimens from:\n\n- Acute diarrhoeal illness (watery or bloody)\n- Suspected typhoid fever or non-typhoidal salmonellosis\n- Suspected bacillary dysentery — particularly where *Shigella* recovery is a priority\n- Gastroenteritis outbreak investigation\n\n**2. Subculture from enrichment broth** After overnight incubation in selenite broth or GN (gram-negative) broth, subculture onto HE agar provides selective solid medium particularly suited for low-count *Salmonella* specimens or carrier detection, where the combination of enrichment and HE plating maximises recovery.\n\n**3. Food and water microbiology** HE agar is used for *Salmonella* and *Shigella* detection in food samples (particularly poultry, eggs, raw vegetables) and water samples during public health investigations.\n\n**4. Preferred medium when *Shigella* recovery is the clinical priority** Because HE agar uses lower-toxicity dyes than SS agar, it is the preferred enteric selective medium when *Shigella dysenteriae* or other *Shigella* species are clinically suspected — for example, in paediatric bloody diarrhoea, during epidemic dysentery outbreaks, or in regions where *Shigella* is endemic.\n\n## Principle\n\nHektoen enteric agar is a selective as well as differential media for the isolation and differentiation of enteric pathogens from clinical specimens.\n\nThe presence of the bile salts and dyes inhibits most gram-positive organisms allowing only gram-negative rods to grow on HE agar. The high concentration of bile salts partially or fully inhibits most of the nonpathogenic coliform flora of the intestinal tract.  Since the enteric pathogens, *Salmonella* and *Shigella* can tolerate these inhibitory substances they generally grow faster and larger than the coliforms.\n\n**Why HE agar is green — and what colour changes mean:**\n\nHE agar appears **deep green** when freshly prepared. This colour comes from bromthymol blue at its neutral-to-alkaline pH range (green at pH 6.0–7.6). Organisms that ferment any of the three carbohydrates (lactose, sucrose, salicin) produce acid, lowering the pH and shifting bromthymol blue from green toward yellow, while acid fuchsin contributes orange-red tones — resulting in the characteristic **orange-yellow to salmon-pink** colonies of fermenters. Organisms that do not ferment any carbohydrate maintain the alkaline or neutral local environment, producing **blue-green to teal** colonies that stand out against the orange background of coliform growth.\n\n**Why lower-toxicity dyes improve *Shigella* recovery:**\n\nSS agar uses brilliant green as its primary selective dye. Brilliant green is highly effective at suppressing coliforms but also inhibits most *Shigella* strains because they share a susceptibility to this compound. HE agar replaces brilliant green with bromthymol blue and acid fuchsin — dyes with selective activity against Gram-positive organisms but significantly less inhibitory effect on *Shigella*. The result is that HE agar recovers *Shigella dysenteriae*, *S. sonnei*, and *S. flexneri* more reliably than SS agar, while maintaining good selectivity against the normal coliform flora.\n\n## Composition of Hektoen Enteric Agar\n\n| Ingredient | Amount (g\u002FL) | Function |\n| --- | --- | --- |\n| Protease peptone | 12.0 | Nitrogen, amino acids, growth factors |\n| Yeast extract | 3.0 | Vitamins, nitrogen, carbon |\n| Lactose | 12.0 | Fermentable carbohydrate — most coliforms ferment this |\n| Sucrose | 12.0 | Fermentable carbohydrate — additional fermenter discrimination |\n| Salicin | 2.0 | Fermentable carbohydrate — some organisms ferment salicin but not lactose\u002Fsucrose; helps discriminate further |\n| Sodium chloride | 5.0 | Osmotic balance |\n| Bile salts | 9.0 | Selective agent — inhibits all Gram-positive organisms; partially inhibits coliforms |\n| Sodium thiosulfate | 5.0 | Sulphur source; H2S indicator (reacts with ferric ammonium citrate) |\n| Ferric ammonium citrate | 1.5 | H2S indicator — reacts with H2S → black iron sulfide precipitate |\n| Bromthymol blue | 0.064 | pH indicator — green at neutral\u002Falkaline; yellow-orange when acid |\n| Acid fuchsin | 0.1 | pH indicator — adds red-orange component to fermenter colonies; low toxicity (cf. brilliant green in SS agar) |\n| Agar | 13.5 | Solidifying agent |\n\n**Final pH:** 7.5 ± 0.2 at 25°C — medium appears **deep green** at baseline\n\n> **Three carbohydrates — why this matters:** Most coliforms ferment at least one of lactose, sucrose, or salicin, producing orange-yellow colonies on HE agar. *Salmonella* and *Shigella* ferment none of these three, maintaining blue-green colonies. The triple-carbohydrate system gives HE agar more discriminating power than media using only lactose: organisms that are lactose-negative but sucrose- or salicin-positive (e.g., some *Proteus* strains) are still identified as fermenters and produce orange-yellow rather than blue-green colonies.\n>\n> ##\n\n![Hektoen enteric agar colonies - Stool culture on Hektoen enteric agar: mixed flora including Escherichia coli (red arrow), Salmonella (blue arrow), and Proteus vulgaris (yellow arrow).](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FHektoen-enteric-agar-colonies-300x288.jpg)Figure: Stool culture on Hektoen enteric agar: mixed flora including *Escherichia coli* (red arrow), Salmonella (blue arrow), and *Proteus vulgaris* (yellow arrow).\n\n## Preparation of HE Agar\n\n1. Suspend 76 grams of dehydrated HE agar powder in one liter of distilled or deionised water. (Follow manufacturer instructions — formulations vary between Oxoid, BD Difco, and HiMedia.)\n2. Mix well and heat with frequent agitation, bringing to the boil.\n3. **Do not autoclave.** Autoclaving degrades the carbohydrates, dyes, and bile salts, reducing both selectivity and differential capacity. Boiling for one minute only is sufficient for sterilization of this medium.\n4. Cool to 50°C and pour into sterile Petri dishes (approximately 20 mL per plate).\n5. Allow to solidify on a level surface. The set medium should appear deep blue-green.\n6. Store inverted at 2–8°C. Use within two weeks.\n7. Plates should be used immediately after warming to room temperature — do not allow plates to remain at room temperature for extended periods before inoculation, as the medium continues to acidify slightly with time.\n\n> **Quality check:** Uninoculated HE agar should be deep green to blue-green after solidification. A yellow or orange tint before inoculation indicates acid degradation during preparation — discard and remake. A black precipitate forming before inoculation indicates thiosulfate-ferric citrate reaction has already occurred — discard the batch.\n\n## Colony Characteristics on HE Agar\n\nThe deep green background of HE agar makes colony colours particularly vivid. Reading the plate requires noting both colony colour and the presence or absence of a black centre.\n\n| Organism | Colony colour | Black centre | Mechanism | Notes |\n| --- | --- | --- | --- | --- |\n| *Salmonella typhi* | Blue-green to teal | Yes (variable; may be small) | No carbohydrate fermentation; H2S positive | Small black centre can be faint — always pick blue-green colonies regardless |\n| *Salmonella typhimurium* (non-typhi) | Blue-green to teal | Yes (prominent) | No carbohydrate fermentation; H2S positive | Classic HE agar *Salmonella* appearance |\n| *Salmonella paratyphi A* | Blue-green | **No black centre** | No carbohydrate fermentation; H2S **negative** | Critical: mimics *Shigella* — differentiate by serology and TSI |\n| *Shigella sonnei* | **Greener** than *Salmonella*; blue-green fading at colony edge | No black centre | No carbohydrate fermentation; H2S negative | Colour fades toward periphery — useful distinguishing feature from *Salmonella* |\n| *Shigella flexneri* | Blue-green | No black centre | No carbohydrate fermentation; H2S negative | Similar to *S. sonnei*; serology for speciation |\n| *Shigella dysenteriae* | Blue-green (grows better on HE than on SS agar) | No | No carbohydrate fermentation | Better recovery on HE than SS agar — key advantage of this medium |\n| *Escherichia coli* | **Orange to salmon-pink** | No | Rapid lactose fermenter; acid production | Moderately inhibited but may produce visible orange colonies in direct plating |\n| *Klebsiella pneumoniae* | **Orange-yellow, mucoid** | No | Lactose + sucrose fermenter; mucoid capsule | Very mucoid; may be hard to pick individual colonies |\n| *Proteus mirabilis* | Small, transparent, glistening; **orange-yellow with black centre (variable)** | Variable | H2S positive; may ferment salicin | **Proteus mimicry warning:** orange-yellow colony + black centre; distinguish from *Salmonella* (blue-green + black centre) by colony colour and urease test |\n| *Citrobacter freundii* | **Orange-yellow with black centre** | Yes | H2S positive; lactose fermenter (variable) | Another H2S-positive mimic; bile salts usually suppress it; if it grows, orange-yellow colour distinguishes it from *Salmonella* |\n| *Yersinia enterocolitica* | Small, blue-green, 0.5–1 mm | No | Non-fermenter; H2S negative | Small colony size; better recovered at 25°C; important in paediatric diarrhoea |\n\n> **The key visual rule for HE agar:** Blue-green = non-fermenter = potential pathogen (*Salmonella* or *Shigella*). Orange-yellow = fermenter = likely coliform. The exception — orange-yellow with a black centre — usually means *Proteus* or *Citrobacter*, not *Salmonella* (which would be blue-green with a black centre). When in doubt: urease test immediately differentiates *Proteus* (strongly positive) from *Salmonella* (negative).\n\n![ yellow-orange colonies of Proteus vulgaris in Hektoen Enteric Agar - Yellow-orange colonies ofProteus vulgarisin Hektoen Enteric Agar](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FProteus-vulgaris-in-hektoen-enteric-agar-300x216.jpg)Figure: Yellow-orange colonies of *Proteus vulgaris* in Hektoen Enteric Agar\n\n## HE Agar vs Other Enteric Media\n\n| Feature | HE Agar | [XLD Agar](https:\u002F\u002Fmicrobeonline.com\u002Fxylose-lysine-deoxycholate-xld-agar-composition-preparation-results-uses\u002F) | [SS Agar](https:\u002F\u002Fmicrobeonline.com\u002Fsalmonella-shigella-ss-agar-composition-principle-procedure-results\u002F) | [DCA](https:\u002F\u002Fmicrobeonline.com\u002Fdeoxycholate-citrate-agar-dca-preparation-uses-colony\u002F) |\n| --- | --- | --- | --- | --- |\n| Primary selective agents | Bile salts + bromthymol blue + acid fuchsin | Sodium deoxycholate | Bile salts + sodium citrate + **brilliant green** | Sodium deoxycholate + sodium citrate |\n| Selectivity level | Moderate-high | Moderate-high | High | Moderate |\n| *Shigella dysenteriae* recovery | **Good** | Good | Poor (inhibited by brilliant green) | Moderate |\n| *Salmonella* colony appearance | Blue-green with black centre | Red-pink with black centre | Colourless with black centre | Colourless with black centre |\n| *Shigella* colony appearance | Blue-green, fading at edge, no black centre | Red-pink, no black centre | Colourless (if grows) | Colourless |\n| Coliform appearance | Orange-yellow | Yellow | Pink-red | Pink |\n| Baseline medium colour | **Deep green** | Bright red | Pale | Pale pink |\n| H2S indicator system | Thiosulfate + ferric ammonium citrate | Thiosulfate + ferric ammonium citrate | Thiosulfate + ferric citrate | Thiosulfate + ferric ammonium citrate |\n| Do not autoclave | Yes | Yes | Yes | Yes |\n| Best for | *Shigella* recovery; when brilliant green toxicity a concern | Three-step *Salmonella* differentiation; best single enteric medium | Maximum coliform suppression; *Salmonella* specialist | Standard stool culture; *Salmonella* + *Shigella* |\n\n> **Practical recommendation:** For the most complete enteric pathogen recovery, use **XLD + MacConkey** as the standard two-medium battery, with HE agar substituted for XLD when *Shigella* epidemic dysentery is the primary clinical concern. HE agar and XLD together in a two-plate battery provides excellent coverage of both *Salmonella* and *Shigella* across all species.\n\n## How to Remember\n\n**HE agar was designed to fix a specific problem — and the name of that problem is brilliant green.**\n\nSS agar uses brilliant green, which works brilliantly for suppressing coliforms but also kills *Shigella*. HE agar replaced brilliant green with two milder dyes (bromthymol blue + acid fuchsin) and added extra carbohydrates to compensate. The result: better *Shigella* recovery without losing *Salmonella* selectivity.\n\n**The green medium rule:**\n\n> Green plate → blue-green colony = non-fermenter = **pick it** (*Salmonella* or *Shigella* suspect) Green plate → orange-yellow colony = fermenter = **ignore it** (coliform) Orange-yellow + black centre = **Proteus\u002FCitrobacter**, not *Salmonella* (which is blue-green + black centre)\n\n**Three carbohydrates, one rule:** Lactose, sucrose, salicin — *Salmonella* and *Shigella* ferment none of them. Any organism that ferments even one of these three turns orange-yellow. This three-carbohydrate system reduces false negatives from organisms that might escape detection with lactose alone.\n\n**Historical anchor:** King and Metzger, Hektoen Institute, 1968. The medium is named after the institute, not a person. The goal was to recover more *Shigella* — and the key innovation was replacing brilliant green. This historical context helps remember *why* HE agar exists in a world that already had SS agar, XLD, and DCA: it was a deliberate improvement in *Shigella* sensitivity.\n\n**References**\n\n1. Goo, V. Y., Ching, G. Q., & Gooch, J. M. (1973). Comparison of brilliant green agar and Hektoen enteric agar media in the isolation of salmonellae from food products. *Applied microbiology*, *26*(3), 288–292. \u003Chttps:\u002F\u002Fdoi.org\u002F10.1128\u002Fam.26.3.288-292.1973>\n2. King, S., & Metzger, W. I. (1968). A new plating medium for the isolation of enteric pathogens. I. hektoen enteric agar. *Applied microbiology*, *16*(4), 577–578. \u003Chttps:\u002F\u002Fdoi.org\u002F10.1128\u002Fam.16.4.577-578.1968>\n3. Tille, P. M. (2017). *Bailey and Scott's Diagnostic Microbiology* (14th ed.). Elsevier.\n4. Cheesbrough, M. (2006). *District Laboratory Practice in Tropical Countries, Part 2* (2nd ed.). Cambridge University Press.",[],[],[],[49,55,62,67,71,75,80,85,89,93],{"slug":50,"name":39,"description":51,"image":52,"body":53,"postCount":54},"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":56,"name":57,"description":58,"image":59,"body":60,"postCount":61},"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":63,"name":64,"description":65,"image":38,"body":38,"postCount":66},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":68,"name":69,"description":65,"image":38,"body":38,"postCount":70},"samikshya-acharya","Samikshya Acharya",20,{"slug":72,"name":73,"description":65,"image":38,"body":38,"postCount":74},"alisha-tripathi","Alisha Tripathi",6,{"slug":76,"name":77,"description":78,"image":38,"body":38,"postCount":79},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",9,{"slug":81,"name":82,"description":83,"image":38,"body":38,"postCount":84},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":86,"name":87,"description":65,"image":38,"body":38,"postCount":88},"srijana-khanal","Srijana Khanal",18,{"slug":90,"name":91,"description":83,"image":38,"body":38,"postCount":92},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":94,"name":95,"description":65,"image":38,"body":96,"postCount":97},"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]