IMViC Test
The IMViC test is a collection of four distinct biochemical assays used to detect and distinguish bacteria, particularly those belonging to the Enterobacteriaceae family. It may (and is) used to identify any sort of bacterium, however, it is primarily employed to identify Gram-negative bacteria. It is essential for distinguishing and classifying members of the Enterobacteriaceae family.
The abbreviation IMViC stands for four separate biochemical tests, with the exception of “I,” each letter standing for a different test in this series. The following biochemical tests are included in the IMViC series
“I” for Indole Test
“M” for Methyl Red (MR) Test
“Vi” for Voges-Proskauer (VP) Test
“C” for Citrate Utilization Test
We can examine six biochemical properties by making a slight alteration, such as using SIM (Sulfide-Indole-Motility) agar medium in place of 4. The ability to assess bacteria’s “motility” and “sulfide production” (H2S production) ability is revealed by the usage of SIM medium. As a result, it is a set of biochemical testing that is universally accepted.
The most popular primary biochemical test series is IMViC. Although crucial for distinguishing Enterobacterales, it is also utilized to characterize and identify a number of Gram-positive bacteria. It is frequently applied for both teaching and research objectives in clinical laboratories. Each test in the series is simple to administer and provides results in 24 to 48 hours. employed as a major screening method as result.
IMViC test objectives
- To characterize and identify isolated unknown bacteria by looking at some of their biochemical traits, such as indole generation, acid production, acetylmethylcarbinol (acetoin) production, and citrate consumption.
- To distinguish and recognize specific members of the Enterobacteriaceae family.
IMViC Test Principle
The IMViC test is based on variances in the metabolic needs and characteristics of various bacterial genera and species. The series’ “indole test” and “citrate utilization test” look for bacteria’s capacity to make and use particular enzymes and nutrients, respectively. The ‘MR test’ and ‘VP test’ in the series, on the other hand, identify the ultimate metabolic byproducts created by the bacteria using a given food. The test bacteria are cultivated in a specific culture medium for this purpose; several media are used for various tests (except MR and VP tests which need the same culture media; MR-VP media).
IMViC Test Requirements
The IMViC test is a collection of various biochemical assessments that call for various culture mediums and reagents. Broths were the only media utilized in the past, however diverse solid media are now highly recommended for their simplicity and versatility in testing various qualities.
Indole Test prerequisites
Culture media
Historically, tryptophan broth was employed.
Sulfide – Indole – Motility (SIM) medium has recently gained a lot of popularity (because it gives the result of H2S production and motility also).Also suggested is the Motility, Indole, and Urea (MIU) medium (because it gives the result of urease production and motility also).
Reagents
- The most commonly used is Kovac’s Indole Reagent, which is a 4-(dimethylamino) benzaldehyde and hydrochloric acid solution in n-butanol or amyl alcohol. Aerobic organisms prefer it.
- For anaerobic and weak indole-producing organisms, Ehrlich’s Reagent (a mixture of p-dimethylaminobenzaldehydre and hydrochloric acid in ethyl alcohol) is preferable.
- For the spot indole test, use 5% p-dimethylaminobenzaldehyde or 1% p-dimethylaminocinnamaldehyde in 10% (v/v) concentrated HCl.
Requirements for the MR test include
Culture Media MR-VP broth
Reagents Methyl red indicator
Requirements for the VP Test include
Culture Media MR-VP broth
Reagents VP Reagent I (5% – -Naphthol Solution) or Barritt’s A Solution
VP Reagent II (40% KOH solution) or Barritt’s B solution
Requirements for the Citrate Test include
Culture Media Simmon’s Citrate Agar
Reagents Simmon’s citrate medium already contains the bromothymol blue indicator.
Although it is developed, the streamlined IMViC agar plate with modified media that contains all four IMVic test media is not generally used.
Describe the Indole Test
The IMViC test series includes a biochemical test called the indole test that looks for bacteria’s capacity to create indole as a byproduct of tryptophan metabolism. The IMViC’s letter “I” serves as a visual cue.
Basics of the Indole Test
Tryptophan is an amino acid that some bacteria may generate the enzyme “tryptophanase” to assist them to break down into “indole, pyruvic acid, and ammonia.”
When added to a medium containing a bacterial culture that has produced indole, the indole reacts with the aldehyde in the indole reagent to produce a distinctive color.
A pink-to-red quinoidal compound occurs, giving rise to a pink-to-red color ring if the reagent contains benzaldehyde.
When cinnamaldehyde is present in the reagent, a green-to-blue color ring develops, which results in the creation of a blue-to-green molecule.
Benzaldehyde and amyl alcohol are both used in Kovac’s indole reagent. The top develops a cherry-red or pink-red ring due to the presence of amyl alcohol, which leaves an oily coating and is insoluble in water.
Tryptophan-containing culture media is used to cultivate test microorganisms for 24 to 48 hours, after which the result is read using an indole reagent. Depending on the reagent type utilized, the development of a pink-to-violet-red or green-to-blue color ring indicates a favorable outcome. A lack of color change or the emergence of a faintly yellowish color ring at the top are indicators of a negative color.
Indole Positive Bacteria
Escherichia coli, Klebsiella oxytoca, V. cholerae, Proteus vulgaris, Porphyromonas asaccharolytica, Vibrio spp., Flavobacterium spp., Providencia spp., Enterococcus faecalis, Haemophilus influenzae, Morganella morganii, Aeromonas spp., Citrobacter koseri
Indole Negative Bacteria
Klebsiella pneumoniae, Proteus mirabilis, Salmonella spp., Shigella spp., Citrobacter freundii, Pseudomonas aeruginosa, Bacteroides fragilis, Staphylococcus aureus
Methyl Red (MR) Test: What Is It?
A biochemical test called the Methyl Red (MR) Test can be used to identify bacteria’s capacity to create stable mixed acids as metabolic byproducts of glucose metabolism. The IMViC’s letter “M” serves as a clue.
Basics of MR Testing
The mixed acid fermentation pathway is used by several bacterial species to metabolize glucose. They transform pyruvate into stable mixed acids by using this metabolic process.
When grown in a medium containing glucose (or another carbohydrate), these acid-fermenting bacteria will release acids, lowering the pH of the medium to 4.4 or below. A methyl red indicator added to a medium containing such acid fermenters will cause the medium to turn red.
The 24-hour incubation period is followed by the addition of a methyl red indicator to the MR-VP broth. A favorable outcome is indicated by the increase of red color, whilst an unfavorable outcome is indicated by the emergence of a yellowish hue.
MR Positive Bacteria
Escherichia coli, Salmonella spp., Shigella spp., Citrobacter spp., Proteus spp., Yersinia spp., Edwardsiella spp., Staphylococcus aureus
MR Negative Bacteria
Klebsiella pneumoniae, Enterobacter spp., Hafnia spp., Serratia marcescens
What is the VP Test (Voges-Proskauer)?
The IMViC test series includes the biochemical Voges-Proskauer (VP) Test, which identifies the presence of bacteria that can convert pyruvate into the neutral intermediate product acetylmethylcarbinol or acetoin. The IMViC’s letter “V” serves as a visual cue.
Fundamentals of VP Test
During the butanediol pathway of 2,3-butanediol synthesis, pyruvate can be converted into the neutral intermediate product acetyl methyl carbinol, also known as acetoin.
If acetoin is present in the medium, air and KOH will quickly oxidize it to diacetyl. In the presence of -naphthol, the guanidine component of peptone reacts with the resulting diacetyl to yield a pink to the red-colored product. VP reagents I and II are introduced after the 48-hour aerobic incubation on MR-VP broth, and the color change is visible within 30 minutes. The appearance of pink or red color at the top of the soup right away, within 30 minutes, but no later than an hour, is a sign of a successful outcome. A negative VP test is represented by no change in color.
VP Positive Bacteria
Klebsiella spp., Enterobacter spp., Viridans Streptococci (except S. mitis, and S. vestibularis), Proteus mirabilis, Hafnia spp., Serratia spp., Staphylococcus aureus
VP Negative Bacteria
Escherichia spp., Proteus vulgaris, Citrobacter freundii
Citrate Utilization Test: What is it?
The IMViC test series includes a biochemical test called the Citrate Utilization Test that measures an organism’s capacity to use citrate as its only source of energy. The IMViC’s letter “C” serves as a clue.
Citrate Utilization Test Principle
Citrate can be the only source of carbon for some bacteria. Citrate is broken down into acetic acid and oxaloacetic acid by the citrase enzymes produced by these bacteria. Following the decarboxylation of the oxaloacetic acid, pyruvate and CO2 will be produced.
Alkaline “sodium carbonate” will be created when released CO2 combines with water and extra sodium from sodium citrate. The pH of the medium will rise thanks to the sodium carbonate.
CO2 + H2O + more sodium from sodium citrate → Na2CO3 (alkaline)
Furthermore, CO2 emissions will start the metabolism of ammonium salts. Ammonia will be produced if the ammonium salts are used as a source of nitrogen (or ammonium hydroxide).
Ammonium salt → Ammonium hydroxide (alkaline)
Ammonium hydroxide and sodium carbonate working together will raise the media’s pH above 7.6. The pH indicator bromothymol blue in the media will change from deep forest green (at a neutral pH) to Prussian blue as a result of the increase in pH.
After 24 to 48 hours of incubation (up to 4 days for some), bacterial growth and color change are seen in the slanted part. Growth and a switch from green to a deep blue hue of the slant are signs of a successful outcome. If the color of the slant does not change, the outcome is negative.
Citrate Positive Bacteria
Klebsiella spp., Citrobacter spp., Serratia marcescens, Proteus mirabilis, Enterobacter spp., Salmonella spp. (except Salmonella Typhi and Paratyphi A), Edwardsiella spp., Providencia spp.
Citrate Variable Bacteria
Proteus vulgaris, V. cholera, V. parahaemolyticus.
Citrate Negative Bacteria
Escherichia coli, Shigella spp., Salmonella Typhi and Paratyphi A, Yersinia spp. Morganella morganii, Staphylococcus aureus, etc.
The IMViC Test’s Uses
- It is frequently used to describe and identify unidentified isolated bacteria up to the genus level in clinical, research, and teaching laboratories.
- The test can distinguish between and identify the members of the Enterobacteriaceae family, in addition to the Urease test and the TSI (Triple Sugar Iron) test.
- To distinguish between species within a genus, some tests are performed. Examples include distinguishing between K. oxytoca and K. pneumoniae, C. koseri and C. freundii, P. vulgaris and P. mirabilis, etc. using the indole test.
