[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"$fxLN3MUwXCdr5RPjwZYIDpOj8CHyjOmngWTgoKXPtZbg":3,"$fdBo9D_ua7DxONZ2wwBOTAf4la1XcFN0VvhnHQAdXGDw":32,"$f3Ft0rKFJHppdzE-vuveecxx1BUcg9iOlMLtyzf_MJDg":153},[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":61,"related":63},"mixed-acid-fermentation","Mixed Acid Fermentation: The Pathway That Makes E. coli Methyl Red-Positive","Mixed acid fermentation splits glucose into a mixture of strong acids, lactate, acetate, formate, succinate, plus ethanol, in variable proportions. That flood of acid drops the pH below 4.4, which is exactly what the methyl red test detects. Here is the pathway, the enzymes, why \"mixed\" is the whole point, and how it differs from the 2,3-butanediol route that VP detects.",null,"Ashma Shrestha","2023-08-21","2026-07-15",false,"biochemical-tests","## Why It Matters\n\nMost students meet mixed acid fermentation not as biochemistry but as a test result: *E. coli* is methyl red-positive. This article is about *why*, and the why is worth knowing, because it explains a whole family of biochemical tests.\n\nWhen an organism like *E. coli* runs out of oxygen, it ferments glucose. But it does not make a single tidy product. It runs several pathways at once, producing a **mixture** of strong acids, lactic, acetic, formic, and succinic, along with ethanol and gas. That is what \"mixed acid\" means: not one end product in a fixed amount, but several acids in proportions that vary with the organism and conditions.\n\nThe diagnostic consequence is direct. All those acids pour into the medium and drive the pH sharply down, below 4.4. The **methyl red test** is simply a way of asking \"did the pH drop that far?\", and it drops that far only in an organism running mixed acid fermentation. So *E. coli*, a mixed-acid fermenter, turns methyl red red; *Klebsiella* and *Enterobacter*, which take a different route (the 2,3-butanediol pathway, detected by the Voges-Proskauer test), do not.\n\nUnderstanding the pathway turns a memorized test result into a mechanism you can reason from. This article covers the biochemistry of mixed acid fermentation, the enzymes and end products, and how it connects to the methyl red, Voges-Proskauer, and carbohydrate fermentation tests.\n\nFermentation is an anaerobic process of breaking down molecules like glucose and other carbohydrates. The fermentation process is usually helpful in alcohol production. Bacteria follow different fermentation pathways.\n\nAmong them, **mixed acid fermentation** is a characteristic feature of the family **Enterobacteriaceae, especially the genera *Citrobacter, Proteus, Shigella, Salmonella, Escherichia, Aeromonas, Yersinia, Vibrio*,** and **some species of Aeromonas.** Some anaerobic fungi also follow this pathway.\n\nThese microorganisms ferment monosaccharides, disaccharides, polyalcohol, and frequently polysaccharides. The glycolytic pathway of this type of fermentation produces lactic acid, succinic acid, formic acids, acetic acids, and ethanol.\n\n## Reactions Involved in Mixed Acid Fermentation\n\nIn mixed acid fermentation reactions, two stages are present. The first stage of mixed acid fermentation is glycolysis, converting glucose to pyruvate. Here two NADH molecules are produced.\n\nThe second stage of mixed acid fermentation follows the following reactions the conversion of the pyruvate produced after glycolysis to one or more end products. The two NADH molecules produced in the first stage are reoxidized to NAD+. The reactions in the second stage are discussed below.\n\n![ - Mixed acid fermentation inE coli](https:\u002F\u002Fassets.microbeonline.com\u002Fblogs\u002FMixed-Acid-Fermentation-in-E.-coli.jpg)Figure: Mixed acid fermentation in E coli\n\n### Lactate Production\n\nThe enzyme lactate dehydrogenase catalyzes the formation of lactate\u002Flactic acid. Here, [glycolysis](\u002Fglycolysis-enzymes-steps-and-products\u002F) generates two molecules of pyruvate. Each molecule converts to lactate in the presence of a NADH+H+ molecule.\n\nThe overall reaction of lactate production is as follows:\n\n**Pyruvate → Lactate; in presence of lactate dehydrogenase and NADH+H**+**, which converts to NAD**+\n\n### Acetate Production\n\nIn this reaction, pyruvate converts to acetyl CoA with the enzyme pyruvate dehydrogenase and NADH catalysts. The acetyl CoA now converts to acetate, which produces ATP by substrate-level phosphorylation. The conversion of acetyl CoA to acetate is a two-step process requiring two separate enzymes; phosphate acetyltransferase and acetate kinase.\n\nThe overall reaction of this reaction is as follows;\n\n1. **Pyruvate → Acetyl CoA in the presence of pyruvate dehydrogenase**\n2. **Acetyl CoA + Phosphate → Acetyl phosphate + CoA in the presence of phosphate acetyltransferase.**\n3. **Acetyl phosphate + ADP → Acetate + ATP.**\n\n### Ethanol Production\n\nThe reduction of Acetyl CoA with the help of NADH forms the third end product of mixed acid fermentation; ethanol. This ethanol production is a two-step reaction and requires the enzyme alcohol dehydrogenase.\n\nThe overall reaction of this step of fermentation is;\n\n1. **Acetyl CoA + NADH +H**+**→ Acetaldehyde + NAD**++ CoA\n2. **Acetaldehyde + NADH + H**+**→  Ethanol + NAD**+\n\n### Formate Production\n\nThe cleavage of pyruvate helps in the production of Formate. The enzyme pyruvate-formate-lyase catalyzes this production reaction. The enzyme pyruvate-formate-lyase also plays an essential role in regulating anaerobic fermentation in Enterobacteriaceae.\n\nThe overall reaction of the formate production is as follows;\n\n**Pyruvate + CoA  → Acetyl CoA + Formate; catalyzed by pyruvate-formate-lyase.**\n\n### Succinate Production\n\nThe production of succinate is a multi-step process. The glycolytic pathway intermediate, phosphoenol pyruvate, is the first substrate for carboxylation to form oxaloacetate, with the enzyme phosphoenol pyruvate carboxylase catalyzing this step.\n\nIn the second step, the oxaloacetate converts to malate in the presence of malate dehydrogenase. The third step is the formation of fumarate from the dehydration of malate in the fact of fumarate hydratase.\n\n**Phosphoenol pyruvate + HCO3→ Oxaloacetate +Phosphate**\n\n**Oxaloacetate + NADH + H**+**→ Malate + NAD**+\n\n**Malate → Fumarate + H2O**\n\nThe final step is succinate production by reducing formate catalyzed by the enzyme fumarate reductase.\n\n**Fumarate + NADH + NAD**+**→  Succinate + NAD**+\n\nThe reduction is the anaerobic respiration reaction that uses electrons in NADH dehydrogenase and the electron transport chain. ATP is produced using electrochemical balance and ATP synthetase—this step of fermentation produces ATP, not through substrate-level phosphorylation.\n\n### Hydrogen and Carbon Dioxide Production\n\nThe enzyme formate hydrogen lyase catalyzes the conversion of formate to hydrogen carbon dioxide gas. The production of these gases helps in preventing acidic condition inside the cells.\n\n## End products of mixed acid fermentation\n\n| End product | Formed from | Key enzyme | Note |\n| --- | --- | --- | --- |\n| Lactic acid | Pyruvate | Lactate dehydrogenase | Reduces pyruvate, regenerates NAD⁺ |\n| Formic acid | Pyruvate | Pyruvate-formate-lyase | May be split further into CO₂ + H₂ |\n| Acetic acid | Acetyl-CoA | Via acetyl-phosphate | Yields extra ATP |\n| Ethanol | Acetyl-CoA | Alcohol dehydrogenase (2 steps) | Regenerates NAD⁺ |\n| Succinic acid | PEP → oxaloacetate | PEP carboxylase, then reduction | Multi-step; consumes CO₂ |\n| CO₂ + H₂ (gas) | Formic acid | Formate hydrogen-lyase | The gas seen in Durham tubes |\n\nThe defining feature is in the plural: **several** of these are produced **at once**, in proportions that vary by species. *E. coli* makes two-to-threefold more lactic, succinic, and acetic acid than *Enterobacter* does. This variable mixture, rather than a single fixed product, is what distinguishes mixed acid fermentation from pathways like homolactic (lactate only) or alcoholic (ethanol + CO₂) fermentation.\n\n## Why \"mixed\" is the whole point\n\nThe name is doing real work. Many fermentations produce **one** product in a **fixed** amount: homolactic fermentation makes only lactate; alcoholic fermentation makes ethanol and CO₂. Mixed acid fermentation is different: it runs **several terminal pathways simultaneously**, producing a mixture of acids whose proportions shift with the organism and the growth conditions.\n\nThis matters for two reasons:\n\n1. **It is why the pH crashes.** A single weak acid might not lower the pH much. But several strong acids accumulating together drive the pH below 4.4, low enough for methyl red (which changes color at pH 4.4) to register. A butanediol fermenter, making mostly neutral products, never gets there.\n2. **It is why the products are useful industrially.** Because *E. coli* can be pushed toward one product or another, strains have been engineered to over-produce ethanol (biofuel), succinate, lactate, or acetate. The same flexibility that makes the pathway \"mixed\" makes it a target for metabolic engineering.\n\nSo the \"mixed\" in mixed acid fermentation is not incidental. It is the reason the test works and the reason the pathway is industrially valuable.\n\n## The fork: mixed acid vs 2,3-butanediol\n\nAfter glucose is fermented to pyruvate, enteric bacteria take one of two roads, and which road they take is the basis of two classic tests:\n\n- **The mixed acid road** produces the strong acids described above, drops the pH below 4.4, and is detected by the [**methyl red test** ](https:\u002F\u002Fmicrobeonline.com\u002Fmethyl-red-mr-test-principle-procedure-results\u002F)(positive: red). *E. coli*, *Salmonella*, *Shigella*, *Proteus* take this road.\n- **The 2,3-butanediol road** produces mostly neutral acetoin and 2,3-butanediol, spares the pH, and is detected by the [**Voges-Proskauer test**](https:\u002F\u002Fmicrobeonline.com\u002Fvoges-proskauer-test-principle-procedure-results\u002F) (positive: red with the addition of reagents that detect acetoin). *Klebsiella*, *Enterobacter*, *Serratia* take this road.\n\nThe two roads are largely mutually exclusive, which is why methyl red and VP usually give opposite results in the same organism, and why they are run from the same MR-VP broth. Mixed acid fermentation is the biochemistry behind the \"MR\" half of that pairing.\n\nFor the tests themselves, see the **methyl red test** (which detects mixed acid fermentation) and the **Voges-Proskauer test** (which detects the butanediol alternative). For the general reading of sugar fermentation and gas production, see the [**carbohydrate fermentation test**](https:\u002F\u002Fmicrobeonline.com\u002Fcarbohydrate-fermentation-test-uses-principle-procedure-results\u002F).\n\n## Application of Mixed Acid Fermentation\n\nMixed Acid fermentation is applicable in various fields of science, especially for producing many helpful end products. The applications of mixed acid fermentation are as follows:\n\n1. The use of single bacteria can help produce various products in biotechnology and the food industry.\n2. Likewise, many different strains of bacteria have been metabolically engineered in the laboratory to increase the yield of the specific end product.\n3. This fermentation method is applied to identify bacteria in the laboratory. Methyl red test is standard for detecting the bacteria following the mixed acid fermentation reaction. Here, the test solution turns red if the pH drops below 4.4 in the presence of those microorganisms that follows the mixed acid fermentation pathway.\n\n## How to remember\n\n**\"Mixed\" = many acids at once = low pH = methyl red red.** The logic chains cleanly. The organism makes a mixture of strong acids simultaneously; the acids crash the pH below 4.4; methyl red turns red at 4.4. So mixed acid fermentation is *why* an organism is methyl-red-positive. *E. coli* is the poster child.\n\n**Mixed acid vs butanediol is MR vs VP.** Two roads from pyruvate: the acid road (methyl red-positive, *E. coli*) and the neutral butanediol road (VP-positive, *Klebsiella*). Same MR-VP broth, opposite results, because the two roads are mutually exclusive.\n\n**Formate is the gas source.** Formic acid can be split by formate hydrogen-lyase into CO₂ and H₂, the gas you see in a Durham tube. So a mixed-acid fermenter that has this enzyme is also gas-positive; one that lacks it (like *Shigella*) is anaerogenic.\n\n**Variable, not fixed.** Unlike homolactic (only lactate) or alcoholic (only ethanol + CO₂) fermentation, mixed acid makes several products in proportions that shift. That variability is the defining trait and the reason the pathway is engineered industrially.\n\n## Key exam facts in one table\n\n| Question | Answer | The reason behind it |\n| --- | --- | --- |\n| What is mixed acid fermentation? | Fermentation producing a mixture of acids | Several terminal pathways run at once |\n| End products | Lactic, acetic, formic, succinic acids + ethanol + gas | Multiple, in variable proportions |\n| Key distinguishing feature | Multiple products, variable amounts | Unlike single-product fermentations |\n| Classic organism | *Escherichia coli* | The model mixed-acid fermenter |\n| Detected by | Methyl red test | The acids drop pH below 4.4 |\n| Why pH drops so low | Several strong acids accumulate together | Enough to reach the MR threshold (4.4) |\n| Alternative pathway | 2,3-butanediol (acetoin) | Neutral products; detected by VP |\n| Organisms taking the butanediol road | *Klebsiella*, *Enterobacter*, *Serratia* | MR-negative, VP-positive |\n| Formate → gas | Formate hydrogen-lyase → CO₂ + H₂ | The Durham-tube gas |\n| Anaerogenic mixed-acid fermenter | *Shigella*, *S.* Typhi (no gas) | Lack\u002Flimited formate hydrogen-lyase |\n| Succinate formation | PEP → oxaloacetate → succinate | PEP carboxylase; consumes CO₂ |\n| Ethanol formation | Acetyl-CoA → acetaldehyde → ethanol | Alcohol dehydrogenase; regenerates NAD⁺ |\n| Formate formation | Pyruvate → acetyl-CoA + formate | Pyruvate-formate-lyase |\n| Industrial relevance | Engineered for ethanol, succinate, lactate | The variable pathway is tunable |\n| Relationship to MR\u002FVP | MR detects this pathway; VP detects the alternative | Two roads from pyruvate |\n\n## Where students get confused\n\n**Thinking mixed acid fermentation makes one product.** The defining feature is the opposite: it makes *several* acids at once, in variable proportions. \"Mixed\" is literal. Homolactic fermentation makes only lactate; mixed acid makes lactate *and* acetate *and* formate *and* succinate, plus ethanol.\n\n**Separating the pathway from the test.** Mixed acid fermentation is the *biochemistry*; the methyl red test is how you *detect* it. They are two views of the same thing. An organism is methyl-red-positive *because* it runs mixed acid fermentation and crashes the pH. Do not treat them as unrelated facts.\n\n**Confusing it with the VP pathway.** Mixed acid (MR-positive) and 2,3-butanediol (VP-positive) are the two roads from pyruvate, and they are largely mutually exclusive. An organism is usually one or the other, which is why MR and VP typically give opposite results. Mixing them up is a classic error.\n\n**Assuming all mixed-acid fermenters make gas.** Gas (CO₂ + H₂) comes from splitting formate via formate hydrogen-lyase. A mixed-acid fermenter with this enzyme is gas-positive; one without it, like *Shigella* or *S.* Typhi, ferments to acid but makes no gas (anaerogenic). Acid production and gas production are separate.\n\n**Overstating the fixed proportions.** The amounts of each acid vary with the organism and the conditions. *E. coli* and *Enterobacter* both can do mixed-acid chemistry but in different proportions. The pathway is defined by variability, not a fixed recipe.\n\n**References**\n\n1. Thakker C, Martínez I, San KY, Bennett GN. Succinate production in *Escherichia coli*. *Biotechnol J.* 2012;7(2):213-224. doi:10.1002\u002Fbiot.201100061\n2. Vuoristo KS, Mars AE, Sangra JV, Springer J, Eggink G, Sanders JP, Weusthuis RA. Metabolic engineering of the mixed-acid fermentation pathway of *Escherichia coli* for anaerobic production of glutamate and itaconate. *AMB Express.* 2015;5:61. doi:10.1186\u002Fs13568-015-0147-y\n3. Madigan MT, Bender KS, Buckley DH, Sattley WM, Stahl DA. *Brock Biology of Microorganisms.* 15th ed. Boston: Pearson; 2018.\n4. Tille PM. *Bailey and Scott's Diagnostic Microbiology.* 15th ed. St. Louis: Elsevier; 2022.",[46,49,52,55,58],{"question":47,"answer":48},"Why is E. coli methyl red-positive?","Because E. coli carries out mixed acid fermentation. When it ferments glucose anaerobically, it produces a mixture of strong acids, lactic, acetic, formic, and succinic, along with ethanol, all at once. These acids accumulate and drive the pH of the medium below 4.4. The methyl red test detects exactly this: methyl red turns red at pH 4.4 or below, so a mixed-acid fermenter like E. coli gives a positive (red) methyl red result. The test is essentially a readout of whether the organism ran mixed acid fermentation.",{"question":50,"answer":51},"What makes mixed acid fermentation different from other fermentations?","It produces several end products simultaneously in variable proportions, rather than one product in a fixed amount. Homolactic fermentation makes only lactate; alcoholic fermentation makes ethanol and CO2. Mixed acid fermentation makes a mixture, lactic, acetic, formic, and succinic acids plus ethanol and gas, and the exact proportions vary with the organism and conditions. This variable mixture is what the name refers to and is why it drives the pH low enough to be detected by the methyl red test.",{"question":53,"answer":54},"What is the difference between mixed acid fermentation and the 2,3-butanediol pathway?","They are the two roads enteric bacteria take from pyruvate. Mixed acid fermentation produces strong acids that lower the pH and is detected by the methyl red test; E. coli, Salmonella, Shigella, and Proteus take this road. The 2,3-butanediol pathway produces mostly neutral products (acetoin and 2,3-butanediol), spares the pH, and is detected by the Voges-Proskauer test; Klebsiella, Enterobacter, and Serratia take this road. The two are largely mutually exclusive, which is why the methyl red and VP tests usually give opposite results.",{"question":56,"answer":57},"Why do some mixed acid fermenters produce gas and others do not?","Gas production depends on a specific enzyme, formate hydrogen-lyase, which splits the formic acid made during mixed acid fermentation into carbon dioxide and hydrogen. A mixed-acid fermenter that has this enzyme produces gas, seen as a bubble in a Durham tube. One that lacks or has limited formate hydrogen-lyase, such as Shigella or Salmonella Typhi, still ferments glucose to acid but produces no gas, making it anaerogenic. So acid production and gas production are separate features.",{"question":59,"answer":60},"Why is mixed acid fermentation important in biotechnology?","Because its end products, ethanol, succinate, lactate, and acetate, are commercially valuable, and the pathway's flexibility makes it tunable. Since E. coli can be directed toward one product or another, strains have been metabolically engineered to over-produce specific end products, for example ethanol as a biofuel or succinate as a chemical feedstock. The same feature that makes the pathway produce a variable mixture makes it a useful target for metabolic engineering, with redox balance being the main constraint.",[62],"enterobacteriaceae",[64,72,80,97,104,134,140,147],{"slug":65,"title":66,"description":66,"seoTitle":38,"seoDescription":38,"author":39,"createdDate":67,"lastUpdatedDate":68,"draft":42,"category":69,"image":38,"faq":70,"tags":71},"klebsiella-oxytoca-properties-and-pathogenesis","Klebsiella oxytoca: Properties and Pathogenesis","2022-07-12","2026-07-05","bacteriology",[],[62],{"slug":73,"title":74,"description":74,"seoTitle":38,"seoDescription":38,"author":75,"createdDate":76,"lastUpdatedDate":77,"draft":42,"category":69,"image":38,"faq":78,"tags":79},"yersinia-pestis-properties-disease-diagnosis","Yersinia pestis: Properties, Disease, Lab Diagnosis","Acharya Tankeshwar","2020-04-24","2026-07-19",[],[62],{"slug":81,"title":82,"description":83,"seoTitle":38,"seoDescription":38,"author":75,"createdDate":84,"lastUpdatedDate":85,"draft":42,"category":43,"image":38,"faq":86,"tags":96},"kliglers-iron-agar-kia-principle-procedure-and-results","Kligler’s Iron Agar (KIA): Principle, Procedure, Results","A faint black line at the slant-butt junction is easy to overlook, and it's exactly the reaction that can point toward Salmonella Typhi. Full KIA principle, tube-reading rules, and the KIA\u002FTSI distinction explained.","2019-04-30","2026-07-12",[87,90,93],{"question":88,"answer":89},"Why does KIA give an alkaline slant\u002Facid butt (K\u002FA) result for glucose-only fermenters?","KIA contains glucose (0.1%) and lactose (1.0%) at a 10:1 concentration ratio. A glucose-only fermenter initially produces acid throughout the tube, turning both slant and butt yellow. However, glucose is quickly exhausted. The organism then shifts to aerobic oxidative metabolism on the slant surface, oxidatively deaminating peptone to produce alkaline amine compounds — the slant reverts to red\u002Falkaline. The anaerobic butt retains the acid from initial glucose fermentation. Result: K\u002FA. Organisms that ferment lactose (10x more concentrated) maintain sustained acid production throughout the tube, giving A\u002FA.",{"question":91,"answer":92},"What is the critical difference between Vibrio cholerae results on KIA vs TSI?","Vibrio cholerae ferments glucose and sucrose but NOT lactose. On KIA (which contains only glucose and lactose): the organism ferments glucose only, giving a K\u002FA result — identical to Salmonella or Shigella. On TSI (which contains glucose, lactose, AND sucrose): V. cholerae also ferments sucrose, giving an A\u002FA result. This distinction is diagnostically critical — in a cholera outbreak setting, a K\u002FA result on KIA alone could lead to V. cholerae being worked up as Salmonella. Always use TSI rather than KIA alone when V. cholerae is clinically suspected, and confirm with oxidase test, string test, and serology.",{"question":94,"answer":95},"If a KIA tube shows heavy black precipitate throughout, how should the butt colour be interpreted?","A heavily H₂S-positive KIA tube where black iron sulphide precipitate obscures the butt colour should always be interpreted as acid (A) in the butt — regardless of whether yellow colour is visible. H₂S is only produced in an acidic environment, so the presence of H₂S itself confirms the butt is acid. Never report an H₂S-positive tube as K\u002FK. The blackening in the butt indicates glucose fermentation (acid) plus H₂S production, making the full reading: K\u002FA + H₂S positive — consistent with Salmonella (non-Typhi) or Citrobacter.",[62],{"slug":98,"title":99,"description":99,"seoTitle":38,"seoDescription":38,"author":75,"createdDate":100,"lastUpdatedDate":101,"draft":42,"category":69,"image":38,"faq":102,"tags":103},"klebsiella-pneumoniae-properties-virulence-diseases-diagnosis","Klebsiella pneumoniae: Properties, Diseases, Lab Diagnosis","2019-03-26","2026-07-18",[],[62],{"slug":105,"title":106,"description":107,"seoTitle":108,"seoDescription":109,"author":75,"createdDate":110,"lastUpdatedDate":101,"draft":42,"category":43,"image":38,"faq":111,"tags":133},"api-20e-test-system-introduction-procedure-results-interpretations","API 20E Test: Procedure, Reading the 21 Reactions, and the 7-Digit Profile Code","How to set up, incubate, and read the API 20E strip: which wells need oil, which need reagents, how to run the 21st test (oxidase), and how to build the 7-digit profile number for identification.","API 20E: Inoculation, Reading, Profile Number, and Identification","Prepare and inoculate an API 20E strip, add reagents, read biochemical reactions, calculate the profile number, and interpret organism identification.","2015-05-06",[112,115,118,121,124,127,130],{"question":113,"answer":114},"How many tests are in the API 20E, 20 or 21?","The strip has 20 wells, but a complete identification uses 21 reactions. The oxidase test is performed separately, off the strip, and fills the last position in the profile code.",{"question":116,"answer":117},"Which API 20E wells need a mineral oil overlay?","Five: ADH, LDC, ODC, URE, and H₂S. The oil creates the anaerobic conditions these reactions need. Without it, they read falsely.",{"question":119,"answer":120},"Which wells need reagents added after incubation?","Three: TDA (ferric chloride), IND (Kovács' reagent), and VP (KOH followed by α-naphthol). Add these only after reading every self-developing well.",{"question":122,"answer":123},"Why does the VP well take longer to read?","The pink-red color from acetoin detection can take up to 10 minutes to develop. Do not call VP negative before then. TDA and IND, by contrast, are read almost immediately.",{"question":125,"answer":126},"How is the 7-digit profile number generated?","The 21 reactions are grouped into seven triplets. Within each triplet the wells score 1, 2, and 4 from top to bottom; you add up only the positives, giving a digit from 0 to 7. The seven digits form the profile, which you look up in apiweb or the API catalog.",{"question":128,"answer":129},"What do I do if the profile gives a doubtful or low-confidence identification?","apiweb reports a %ID and a T-value; a low or non-discriminating result means you need supplementary tests (such as oxidase, nitrate reduction, or motility) or a repeat run, rather than accepting the closest match.",{"question":131,"answer":132},"Can API 20E identify organisms other than Enterobacteriaceae?","It is designed for Enterobacteriaceae and other non-fastidious Gram-negative rods. It is not suitable for fastidious organisms or non-fermenters outside its database scope, which need different panels.",[62],{"slug":135,"title":136,"description":136,"seoTitle":38,"seoDescription":38,"author":75,"createdDate":137,"lastUpdatedDate":101,"draft":42,"category":69,"image":38,"faq":138,"tags":139},"shigella-disease-properties-pathogenesis-and-laboratory-diagnosis","Shigella: Disease, Properties, Pathogenesis, Lab Diagnosis","2013-05-18",[],[62],{"slug":141,"title":142,"description":142,"seoTitle":38,"seoDescription":38,"author":75,"createdDate":143,"lastUpdatedDate":68,"draft":42,"category":144,"image":38,"faq":145,"tags":146},"e-coli-only-bacteria-that-wins-record-number-of-nobel-prizes","E. coli: Only bacterium that wins records number of Nobel Prizes","2013-05-04","general-microbiology",[],[62],{"slug":148,"title":149,"description":149,"seoTitle":38,"seoDescription":38,"author":75,"createdDate":150,"lastUpdatedDate":101,"draft":42,"category":69,"image":38,"faq":151,"tags":152},"e-coli-disease-properties-pathogenesis-and-laboratory-diagnosis","Escherichia coli: Properties and Identification","2013-04-27",[],[62],[154,160,166,171,175,179,184,189,193,197],{"slug":155,"name":75,"description":156,"image":157,"body":158,"postCount":159},"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.*",432,{"slug":161,"name":39,"description":162,"image":163,"body":164,"postCount":165},"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":167,"name":168,"description":169,"image":38,"body":38,"postCount":170},"sushmita-baniya","Sushmita Baniya","Author \u002F Contributor",32,{"slug":172,"name":173,"description":169,"image":38,"body":38,"postCount":174},"samikshya-acharya","Samikshya Acharya",20,{"slug":176,"name":177,"description":169,"image":38,"body":38,"postCount":178},"alisha-tripathi","Alisha Tripathi",6,{"slug":180,"name":181,"description":182,"image":38,"body":38,"postCount":183},"aastha-shrestha","Aastha Shrestha"," Author \u002F Contributor",10,{"slug":185,"name":186,"description":187,"image":38,"body":38,"postCount":188},"guest-author","Guest Author","Guest Author \u002F Contributor",2,{"slug":190,"name":191,"description":169,"image":38,"body":38,"postCount":192},"srijana-khanal","Srijana Khanal",18,{"slug":194,"name":195,"description":187,"image":38,"body":38,"postCount":196},"dr-poonam-acharya","Dr. Poonam Acharya",1,{"slug":198,"name":199,"description":169,"image":38,"body":200,"postCount":201},"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]