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The IMViC tests are a group of individual tests used in microbiology lab testing to identify an organism in the coliform group. A coliform is a gram negative, aerobic, or facultative anaerobic rod, which produces gas from lactose within 48 hours. The presence of some coliforms indicate fecal contamination.
The term "IMViC" is an acronym for each of these tests. "I" is for indole test; "M" is for methyl red test; "V" is for Voges-Proskauer test, and "C" is for citrate test. The lower case "i" is merely for "in" as the Citrate test requires coliform samples to be placed "in Citrate".
These tests are useful in distinguishing members of Enterobacteriaceae.
In this test, the organism under consideration is grown in peptone water broth. It contains tryptophan, which under the action of enzyme tryptophanase is converted to an Indole molecule, pyruvate and ammonium. The indole is then extracted from the broth by means of xylene. The broth is sterilized for 15 minutes at around 121°C. To test the broth for indole production, Kovac's reagent . Kovac's reagent consist of amyl alcohol and para-dimethylaminobenzaldehyde and concentrated hydrochloric acid. Kovac's reagent is actually used to determine ability of an organism to separate indole from amino acid tryptophan and it is added after incubation. A positive result is indicated by a pink/red layer forming on top of the liquid.
These tests both use the same broth for bacterial growth. The broth is called MR-VP broth. After growth, the broth is separated into two different tubes, one for the methyl red (MR) test and one for the Voges-Proskauer (VP) test.
The methyl red test detects production of acids formed during metabolism using mixed acid fermentation pathway using pyruvate as a substrate. The pH indicator Methyl Red is added to one tube and a red color appears at pH's lower than 4.2, indicating a positive test (mixed acid fermentation is used). The solution remaining yellow (pH = 6.2 or above) indicates a negative test, meaning the butanediol fermentation is used.
The VP test uses alpha-naphthol and potassium hydroxide to test for the presence of acetylmethylcarbinol (acetoin), an intermediate of the 2,3-butanediol fermentation pathway. After adding both reagents, the tube is shaken vigorously then allowed to sit for 5–10 minutes. A pinkish-red color indicates a positive test, meaning the 2,3-butanediol fermentation pathway is used.
In the 1930's, S.A. Koser conducted experiments that were used to study bacterial catabolism of organic acids. [1] Koser found that citrate metabolism could be an indicator for bacteria found in natural environments. Additionally, citrate could be used to distinguish bacterial coilforms found in soil, and aquatic environments, such as Enterobacteiacea , and coilforms with fecal contamination. It was found that coilforms without fecal contamination grew, while the coilforms with fecal contamination did not grow.
This test uses Simmon's citrate agar to determine the ability of a microorganism to use citrate as its sole carbon and energy source. The agar contains citrate and ammonium ions (nitrogen source) and bromothymol blue (BTB) as a pH indicator. [2] Bromothymol blue was added in order to reduce false positives. The citrate agar is green before inoculation, and turns blue, because of BTB as a positive test indicator, meaning citrate is utilized. The test is also prepared on a slant to maximize bacterial growth for an even better indication of the use of citrate.
These IMViC tests are useful for differentiating the family Enterobacteriaceae, especially when used alongside the Urease test.
The IMViC results of some important species are shown below. [3]
Species | Indole | Methyl Red | Voges-Proskauer | Citrate |
---|---|---|---|---|
Escherichia coli | Positive | Positive | Negative | Negative |
Staphylococcus aureus | Negative | Positive | Positive | Negative |
Shigella spp. [4] | Negative | Positive | Negative | Negative |
Salmonella spp. | Negative | Positive | Negative | Positive |
Klebsiella spp. | Negative | Negative | Positive | Positive |
Proteus vulgaris [5] | Positive | Positive | Negative | Negative |
Proteus mirabilis | Negative | Positive | Negative | Positive |
Citrobacter freundii | Negative | Positive | Negative | Positive |
Enterobacter aerogenes | Negative | Negative | Positive | Positive |
An agar plate is a Petri dish that contains a growth medium solidified with agar, used to culture microorganisms. Sometimes selective compounds are added to influence growth, such as antibiotics.
Bacteriological water analysis is a method of analysing water to estimate the numbers of bacteria present and, if needed, to find out what sort of bacteria they are. It represents one aspect of water quality. It is a microbiological analytical procedure which uses samples of water and from these samples determines the concentration of bacteria. It is then possible to draw inferences about the suitability of the water for use from these concentrations. This process is used, for example, to routinely confirm that water is safe for human consumption or that bathing and recreational waters are safe to use.
Proteus vulgaris is a rod-shaped, nitrate-reducing, indole-positive and catalase-positive, hydrogen sulfide-producing, Gram-negative bacterium that inhabits the intestinal tracts of humans and animals. It can be found in soil, water, and fecal matter. It is grouped with the Morganellaceae and is an opportunistic pathogen of humans. It is known to cause wound infections and other species of its genera are known to cause urinary tract infections.
Coliform bacteria are defined as either motile or non-motile Gram-negative non-spore forming bacilli that possess β-galactosidase to produce acids and gases under their optimal growth temperature of 35–37 °C. They can be aerobes or facultative aerobes, and are a commonly used indicator of low sanitary quality of foods, milk, and water. Coliforms can be found in the aquatic environment, in soil and on vegetation; they are universally present in large numbers in the feces of warm-blooded animals as they are known to inhabit the gastrointestinal system. While coliform bacteria are not normally causes of serious illness, they are easy to culture, and their presence is used to infer that other pathogenic organisms of fecal origin may be present in a sample, or that said sample is not safe to consume. Such pathogens include disease-causing bacteria, viruses, or protozoa and many multicellular parasites.
Lauryl tryptose broth (LTB) is a selective growth medium (broth) for coliforms.
A growth medium or culture medium is a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation or small plants like the moss Physcomitrella patens. Different types of media are used for growing different types of cells.
A fecal coliform is a facultatively anaerobic, rod-shaped, gram-negative, non-sporulating bacterium. Coliform bacteria generally originate in the intestines of warm-blooded animals. Fecal coliforms are capable of growth in the presence of bile salts or similar surface agents, are oxidase negative, and produce acid and gas from lactose within 48 hours at 44 ± 0.5°C. The term thermotolerant coliform is more correct and is gaining acceptance over "fecal coliform".
Shigella dysenteriae is a species of the rod-shaped bacterial genus Shigella. Shigella species can cause shigellosis. Shigellae are Gram-negative, non-spore-forming, facultatively anaerobic, nonmotile bacteria. S. dysenteriae has the ability to invade and replicate in various species of epithelial cells and enterocytes.
Methyl red (2-(N,N-dimethyl-4-aminophenyl) azobenzenecarboxylic acid), also called C.I. Acid Red 2, is an indicator dye that turns red in acidic solutions. It is an azo dye, and is a dark red crystalline powder. Methyl red is a pH indicator; it is red in pH under 4.4, yellow in pH over 6.2, and orange in between, with a pKa of 5.1. Murexide and methyl red are investigated as promising enhancers of sonochemical destruction of chlorinated hydrocarbon pollutants. Methyl red is classed by the IARC in group 3 - unclassified as to carcinogenic potential in humans.
In biochemistry, mixed acid fermentation is the metabolic process by which a six-carbon sugar is converted into a complex and variable mixture of acids. It is an anaerobic (non-oxygen-requiring) fermentation reaction that is common in bacteria. It is characteristic for members of the Enterobacteriaceae, a large family of Gram-negative bacteria that includes E. coli.
The indole test is a biochemical test performed on bacterial species to determine the ability of the organism to convert tryptophan into indole. This division is performed by a chain of a number of different intracellular enzymes, a system generally referred to as "tryptophanase."
Simmons' citrate agar is used for differentiating gram-negative bacteria on the basis of citrate utilization, especially for distinguishing Gammaproteobacteria of the family Enterobacteriaceae or even between species of the same genus. For example, Salmonella enteritidis would yield a positive (blue) result on Simmons’ agar and thus be distinguished from other Salmonella species like Salmonella typhi, Salmonella pullorum, and Salmonella gallinarum, which would yield a negative (green) result.
The 3M Petrifilm plate is an all-in-one plating system made by the Food Safety Division of the 3M Company. They are heavily used in many microbiology-related industries and fields to culture various micro-organisms and are meant to be a more efficient method for detection and enumeration compared to conventional plating techniques. A majority of its use is for the testing of foodstuffs.
Löwenstein–Jensen medium, more commonly known as LJ medium, is a growth medium specially used for culture of Mycobacterium species, notably Mycobacterium tuberculosis.
Aeromonas salmonicida is a pathogenic bacterium that severely impacts salmonid populations and other species. It was first discovered in a Bavarian brown trout hatchery by Emmerich and Weibel in 1894. Aeromonas salmonicida's ability to infect a variety of hosts, multiply, and adapt, make it a prime virulent bacterium. A. salmonicida is an etiological agent for furunculosis, a disease that causes sepsis, haemorrhages, muscle lesions, inflammation of the lower intestine, spleen enlargement, and death in freshwater fish populations. It is found worldwide with the exception of South America. The major route of contamination is poor water quality; however, it can also be associated stress factors such as overcrowding, high temperatures, and trauma. Spawning and smolting fish are prime victims of furunculosis due to their immunocompromised state of being.
Voges–Proskauer or VP is a test used to detect acetoin in a bacterial broth culture. The test is performed by adding alpha-naphthol and potassium hydroxide to the Voges-Proskauer broth, which is a glucose-phosphate broth that has been inoculated with bacteria. A cherry red color indicates a positive result, while a yellow-brown color indicates a negative result.
Thiosulfate–citrate–bile salts–sucrose agar, or TCBS agar, is a type of selective agar culture plate that is used in microbiology laboratories to isolate Vibrio species. TCBS agar is highly selective for the isolation of V. cholerae and V. parahaemolyticus as well as other Vibrio species. Apart from TCBS agar, other rapid testing dipsticks like immunochromatographic dipstick is also used in endemic areas such as Asia, Africa and Latin America. Though, TCBS agar study is required for confirmation. This becomes immensely important in cases of gastroenteritis caused by campylobacter species, whose symptoms mimic that of cholera. Since no yellow bacterial growth is observed in case of campylobacter species on TCBS agar, chances of incorrect diagnosis can be rectified. TCBS agar contains high concentrations of sodium thiosulfate and sodium citrate to inhibit the growth of Enterobacteriaceae. Inhibition of gram-positive bacteria is achieved by the incorporation of ox gall, which is a naturally occurring substance containing a mixture of bile salts and sodium cholate, a pure bile salt. Sodium thiosulfate also serves as a sulfur source and its presence, in combination with ferric citrate, allows for the easy detection of hydrogen sulfide production. Saccharose (sucrose) is included as a fermentable carbohydrate for metabolism by Vibrio species. The alkaline pH of the medium enhances the recovery of V. cholerae and inhibits the growth of others. Thymol blue and bromothymol blue are included as indicators of pH changes.
The citrate test detects the ability of an organism to use citrate as the sole source of carbon and energy.
Glucose phosphate broth is used to perform methyl red (MR) test and Voges–Proskauer test (VP).
Diagnostic microbiology is the study of microbial identification. Since the discovery of the germ theory of disease, scientists have been finding ways to harvest specific organisms. Using methods such as differential media or genome sequencing, physicians and scientists can observe novel functions in organisms for more effective and accurate diagnosis of organisms. Methods used in diagnostic microbiology are often used to take advantage of a particular difference in organisms and attain information about what species it can be identified as, which is often through a reference of previous studies. New studies provide information that others can reference so that scientists can attain a basic understanding of the organism they are examining.