IMViC and Hydrogen Sulfide Experiment
IMViC and Hydrogen Sulfide Experiment
IMVIC is an acronym for four tests: indol test, methyl red test, Voges-Prokauer test and citrate test. i is added in English for ease of pronunciation. These four tests are mainly used to identify E. coli and Enterobacter aerogenes (Enterobacter aerogenes), mostly used for bacteriological examination of water. Although E. coli is not a pathogen, but if E. coli exceeds a certain number in drinking water, it means that it is contaminated by fecal matter, and Enterobacter aerogenes is also widely found in nature, so it is important to distinguish the two when examining water. The hydrogen sulfide test is also a biochemical test to check for Enterobacteriaceae. Source: Laboratory Microbiology (Third Edition)
Operation method
basic program
Principle
1. Indole test. The indole test is used to detect the production of indole. Some bacteria produce tryptophanase, which breaks down tryptophan in peptone to produce indole and pyruvic acid.Indole combines with p-dimethylaminobenzaldehyde to form red rose indole.However,not all microorganisms have the ability to break down tryptophan to produce indole.Therefore,the indole test can be used as a biochemical assay.The indole test is used to detect the production of indole in E. coli.The indole test is used to detect the production of indole in E. coli. Tryptophan hydrolysis: indole reacts with p-dimethylaminobenzaldehyde: E. coli is positive for indole and Enterobacter aerogenes is negative. 2. The methyl red test is used to detect organic acids produced from glucose such as formic acid, acetic acid, lactic acid and so on. When bacteria metabolize sugar to produce acid, the medium becomes acidic, causing the methyl red indicator added to the medium to change from orange (pH 6.3) to red (pH 4.2), i.e. the methyl red reaction. Although all gut microorganisms can ferment glucose to produce organic acids, this test is still valuable in distinguishing between E. coli and Enterobacter aerogenes. Both bacteria produce organic acids early in the culture, but E. coli maintains an acidic pH of 4 late in the culture, whereas E. aerogenes converts the organic acids to non-acidic end products such as ethanol, pyruvate, etc., which raises the pH to about 6. Thus, the reaction is positive for E. coli, and negative for E. aerogenes. 3. The volt-prandial test is used to determine the ability of certain bacteria to use glucose to produce non-acidic or neutral end products, such as propionic acid, or to produce glucose from glucose. The volt-prandial test is used to determine the ability of certain bacteria to utilize glucose to produce non-acidic or neutral end products, such as pyruvic acid, which undergoes condensation and decarboxylation to produce acetylmethylmethanol, a compound that can be oxidized to diacetyl by oxygen in the air under alkaline conditions. Diacetyl and peptone guanidino acid guanidino group, generate red compounds, that is, the volt-prandial reaction is positive; does not produce red compounds for the negative reaction, sometimes in order to make the reaction more pronounced, can be added to less guanidino group-containing compounds, such as creatine and so on. The chemical reaction is as follows: Enterobacter aerogenes is positive and E. coli is negative. 4. The citrate test is used to detect whether citrate is being utilized. Some bacteria are able to utilize sodium citrate as a carbon source, such as Enterobacter aerogenes, while others are unable to utilize citrate, such as E. coli. When the bacteria break down the citrate and the ammonium phosphate in the medium, they produce an alkaline compound that raises the pH of the medium, which changes from green to a dark blue color when 1% bromo muscovanol blue indicator is added. Bromomuskohol blue has a range of pH less than 6.0 (yellow), pH 6.0-7.0 (green), and pH greater than 7.6 (blue). 5. Hydrogen Sulfide Test is a test for the production of hydrogen sulfide and is a common biochemical test used for intestinal bacterial investigations. Some bacteria can decompose sulfur-containing organic matter, such as cystine, cysteine, methionine, etc. to produce hydrogen sulfide, which, when encountered with lead or iron salts in the medium, forms black lead sulfide or iron sparing precipitates. Taking cysteine as an example, the chemical reaction process is as follows: E. coli is negative and Enterobacter aerogenes is positive.
Materials and Instruments
Escherichia coli Enterobacter aerogenes Move 1. Puncture E. coli and E. aerogenes into 2 lead acetate medium (hydrogen sulfide test) with an inoculating needle and incubate at 37℃ for 48 h. 2. Inoculate the above two bacteria into 2 peptone water medium (indole test) and 2 glucose peptone water medium (methyl red test and vol-pe test). 2. Inoculate the above two bacteria into 2 peptone water medium (indole test), 2 glucose peptone water medium (methyl red test and vorp test), 2 citrate slant medium and 2 lead acetate medium, and incubate at 37℃ for 2d. Caveat 1. When preparing citric acid slant medium, its pH should not deviate, and light green color is preferred. 2. The peptone water medium used in the indole test is suitable for proteins with high tryptophan content, such as peptones obtained from tryptic protein hydrolysate. 3. Do not add too much methyl red reagent to avoid false positive reaction. Common Problems "Observation of results" 1. 1) Hydrogen sulfide test: Observe the production of black lead sulfide after 48 hours of incubation. 2. Indole test: Add 3-4 drops of ether to the peptone water medium after 2 days of incubation, shake several times, and let it stand for 1-3 min. After the ether has risen, add 2 drops of indole reagent along the wall of the test tube, and the production of a red ring between the ether and the culture will be considered as a positive reaction. Preparation of peptone water medium, the peptones used are preferably high in tryptophan, such as trypsin hydrolysis of tyrosine peptones with a high content of tryptophan. 3. 3. Methyl red test after 2 d of incubation, add 2 drops of methyl red reagent to 1 stick of glucose peptone water culture, the medium becomes red as positive, and yellow as negative. 4. 4. After 2d incubation, add 5-10 drops of 40% KOH into another glucose peptone water culture, then add equal 5% α-naphthol solution, shake vigorously, and then put it into a warm box to keep warm for 15-30 min to speed up the reaction. If the culture shows red color, the reaction is positive. 5. 5. Observe whether there is any bacterial growth on the citrate slant medium and whether it changes color after 48 hours of incubation in citrate test. Blue color is positive and green color is negative. For more product details, please visit Aladdin Scientific website.
Peptone water medium Glucose peptone water medium Citrate slant medium Lead acetate medium
Methyl red indicator 40% KOH 5% α-naphthol ether Indole reagent