IMViC Test – 4 Test | Principle, Procedure, and Result | Biologyideas.com

IMViC Test Full Form

IMViC Test stands for Indole, Methyl red, Voges-Proskauer, Citrate Test. This as a set of four biochemical tests used to differentiate between Escherichia coli, Klebsiella Enterobacter, and Enterobacteriaceae family organisms.

Indole Test

Organisms produce indole from the amino acid tryptophan by means of the enzyme tryptophanase. The source of tryptophan in the medium can be peptone or tryptone (which has an especially high content of tryptophan). The reaction of breakdown of tryptophan to indole is as shown in the figure below:

Figure – Enzymatic degradation of tryptophan

Indole reaction with Kovac’s/Ehrlich’s reagent:

The chemical reaction for detection of indole is based on the fact that when a pyrrole (indole = benzo-pyrrole) and a weakly acid alcoholic solution of p-dimethylaminobenzalaldehyde – HCL are mixed (in the presence of heat), a red-violet color develops. The reaction will occur without heat if the reagent is made with conc. HCL.

The indole produced by the organism can be detected with several different reagents, but the basic chemical reaction in the detection is the same.

The Ehrlich-Boehme reagent consists of two parts, p-dimethylaminobenzalaldehyde HCL in alcohol and potassium persulfate; each reagent is added, and a red colour (rose-indole dye) develops if indole is present.

Indole Test Definition

1. An alternative procedure is to add a solvent, such as xylene, ether or chloroform, to extract and concentrate the indole: to this is then added the p-dimethylaminobenzalaldehyde reagent, and red colour is formed in the solvent layer, if indole is present.
2. The latter procedure with xylene is the one usually used in the Ehrlich test.
3. Kovac’s modified the Ehrlich-Boehme reagent by using amyl alcohol in place of ethyl alcohol and found that extraction and concentrate of indole occurred with addition of one reagent. Kovac’s reagent is probably the most widely used today.
4. A more rapid test used by taking heavy inoculums from growth on a solid medium and inoculating a small amount (0.3-0.5ml) of tryptone broth.
5. The tryptophanase already present in the organisms will break down the tryptophan in the broth within 4 hours, and indole may be detected in this short amount of time.         
6. A rapid spot for indole production has also been used.
7. In this test, a piece of filter paper moistened with p-dimethylaminobenzalaldehyde- reagent is placed in a petri dish.
8. A loopful of growth from a medium containing tryptophan is placed on the filter paper; development of a red color within a few seconds indicates the presence of indole.

Requirements of Indole Test

• Sterile tryptone water medium (0.5-1.0 ml in each tube)

Constituent	Quantity
Tryptone	10.0g
Sodium chloride	5.0g
Distilled water	1000 ml
pH	7.4

• The medium is sterilized by autoclaving at 110^o C for 10 minutes. 
• Test culture suspension. 
• Kovac’s reagent

Dissolve 10g of p-dimethylaminobenzalaldehyde in 150 ml of amyl, isoamyl or butyl alcohol. Heat it in a 56^oC water bath until dissolved. Cool. Slowly add 50 ml of conc. HCL. Store it in a glass-stoppered brown bottle in the refrigerator. This reagent should be light yellow in color.

• Ehrlich’s reagent

Dissolve 1 g of p-dimethylaminobenzalaldehyde in 95 ml of 95% ethyl alcohol. Slowly add 20 ml of conc. HCL acid. Store in a glass stoppered brown bottle in the refrigerator. This reagent should be light yellow in color.

Procedure of Indole Test

1. Inoculate the tryptone broth (or 2% peptone broth) with the test culture. 
2. Incubate at 37^oC for 24 hours. 
3. Add 1ml of xylene in the test tube and shake vigorously. 
4. Let the tube stand for 1-2 minutes. 
5. Slowly add about 0.5 ml of Kovac’s or Ehrlich’s reagent down of the side of the tube. Do not shake. 
6. A reddish pink color imparted to the xylene layer indicates a positive test.

Indole Test Results

Indole Positive Bacteria	Indole Negative Bacteria
Escherichia coli	Klebsiella pneumoniae
Klebsiella oxytoca	Proteus mirabilis
V. cholerae	Salmonella spp.
Proteus vulgaris	Shigella spp.
Porphyromonas asaccharolytica	Pseudomonas aeruginosa
Vibrio spp.	Bacteroides fragilis
Flavobacterium spp.	Staphylococcus aureus
Haemophilus influenzae	Citrobacter freundii

Methyl Red (MR) Test

• MR Test was first described by Clark and Lubs in 1915. They found it useful in differentiating between the E. coli and Enterobacteraerogenes group of enteric bacteria. Previously, these two groups were divided on the basis of CO2 _/ H₂ ratios produced when the organisms were grown in presence of glucose.
• When grown in vacuum one group produced a ratio of 1.06 (‘low ratio’) and the other a ratio of 1.9 -3.0 (‘high ratio’). These gas ratio differences were attributed to the basic difference in metabolism between the coli-aerogenes groups, and could be an accurate means of differentiation.
• However, determination of gas ratios is not a convenient method for identifying large numbers of cultures.
• Therefore, Clark and Lubs Developed a simple method correlated with the differences in gas ratios. They noted that when the coli organisms (“low ratio”) were  grown in glucose broth,acid was formed until a pH between 4 and 5 and then remained stable.
• At this point the organisms ceased activity and no pH change was seen on further metabolize, with the medium then becoming more alkaline (pH 6-7).
• The explanation for this was that the aerogenes organisms attack the acids that cannot utilize their acid end products.
• The higher pH obtained with the aerogenes group was also attributed to the acetoin formed in its fermentation of glucose. Acetoin is neutral, and thus the pH is higher thanin the cultures of the coli organisms where the major end products of fermentation are acids.
• Under aerobic conditions, both E. coli and Enterobacter aerogenes have the potential to attack their respective fermentation products.
• However E. coli produces about 130 moles of acid per 100 moles of glucose fermented, whereas E. aerogenes produces only 20 moles of acid from the same amount of glucose. Therefore, E.coli would have to neutralize by oxidation five times the amount of acid as E. aerogenes in order to raise the pH above 6.0.        
• The concentration of glucose in the medium (0.5%) is critical as it should not be completely used up with the result that under aerobic conditions of the tests, the medium might revert to alkaline. This medium is also the recommended medium for the Voges Proskauer test which detects the presence of the butylene glycol pathways.
• To determine the pH of the medium after incubation, methyl red indicator is used because this indicator is red at pH 4.5-5.0 & yellow at pH 6.0-7.0. A red color after addition of the indicator indicates a positive methyl red test (mixed acid fermentation), and yellow indicates a negative methyl red test (butylene glycol fermentation).  
• This test may not be very clear at all times where colours are not always clear cut. This is because the recommended incubation time for the test (prescribed by Clark & Lubs) is 5 days at 30^oC.
• This can be overcome by using the heavy inoculums in a small volume (0.5ml) of the medium. Also, inoculated medium can now be incubated at 37^oC because of the reduction in broth volume.

Principle of Methyl Red Test

The principle of the test is based on the ability of bacteria to produce organic acids during glucose fermentation, which lowers the pH of the medium.

In the methyl red test, a pH indicator called methyl red is added to the culture medium. Methyl red is yellow at a pH above 6.0, but it turns red at a pH below 4.4. After inoculating the bacteria into the medium, they ferment glucose and produce organic acids. If the bacteria produce enough acid to lower the pH of the medium below 4.4, the methyl red indicator will turn red.

Requirements of MR Test

1. Sterile glucose phosphate broth (GPB) medium (0.5ml in each tube)
2. Glucose phosphate broth (GPB) 
3. Test culture suspension
4. Methyl red indicator
5. Dissolve 0.1 g methyl red in 300 ml 95% ethanol. Add distilled water to make up the volume to 500ml.

Procedure of MR Test

1. Inoculate the GPB medium with culture suspension.
2. Incubate at 37^oC for 24 hours. 
3. Add 5-6 drops of methyl red indicator. 
4. Positive test is indicated by a bright red color of the medium.
5. A negative test indicated by the medium remaining yellow or turning orange.

Results of Methyl Red (MR) Test

Positive MR Test	Negative MR Test
E. coli	Enterobacter aerogenes
Yersinia sps	Klebsiella pneumoniae

Uses of MR Test

1. Identification of bacteria: The methyl red test is used to differentiate between different types of bacteria based on their ability to produce stable acid end products during glucose fermentation. It is commonly used in the identification of enteric bacteria, such as Escherichia coli and Enterobacter aerogenes.
2. Quality control: The methyl red test is also used as a quality control measure for microbiological media. It is a simple and reliable method to ensure that the culture media is free from contamination and that the pH of the medium is within the acceptable range for growth of microorganisms.
3. Research: The methyl red test can be used in research to study bacterial metabolism and the pathways of glucose fermentation. It is a useful tool to investigate the effects of various treatments or mutations on bacterial metabolism.

Voges-Proskauer (VP) Test

Voges-Proskauer (VP) Test confirms the butylene glycol type of the fermentation by the organism. In general, two pathways of glucose fermentation exist among the Enterobacteriaceae.       

Mixed acid fermentation resulting in large amount of formic, acetic, lactic and succinic acids and ethanol. 

Butylene glycol fermentation resulting in little or nor of the acids, but a large amount of a polar butylene glycol and neutral ethanol. Acetyl methyl carbinol or acetoin, is an intermediate in the production of butylene glycol. 

The VP test detects the presence of acetoin in the culture medium, thus confirming the butylene glycol pathway.    

Voges and Proskauer in 1898 grew the organisms in a medium containing sugar and then added potash to the culture. After 24 hours or longer at room temperature, a red colour developed in the medium, indicating what they called a positive reaction.

Hearden in 1906 showed that the red colour developed was due to the production of acetylmethylcarbinol. In the presence of potash and air, this compound is oxidized diacetyl, which reacts with some constituent of the medium to form a red colour (Diacetyl alone in the presence of potash does not yield red colour). This medium constituent was identified to be free NH₂ group on a guanidine moiety, such as that available from arginine present in the peptone.

In 1931 O’Meara found that the colour was intensified by the addition of creatinine, which provides a greater amount of the guanidine groups. With this modification, the red colour developed in 15 minutes.

In  1936,  Barritt  made  the  test  more  sensitive  by  adding  α-napthol.  With  this  the sensitivity of the test was increased 50-fold (without napthol, at least 10 ppm of diacetyl was detectable). The exact role of α-napthol is not identified.

Some precautions necessary for the VP test are:

1. Meat infusion broth should not be used to grow the culture because the presence of acetoin, diacetyl and related substances in the muscle extract would give a false positive test. 
2. α-napthol should be added before the KOH.

Voges Proskauer VP Test Principle

The principle of the VP test is based on the ability of these bacteria to convert glucose into acetoin via a series of enzymatic reactions.

In the VP test, the bacteria are inoculated into a glucose-containing medium and allowed to ferment the glucose. After incubation, the culture is treated with a solution containing alpha-naphthol and potassium hydroxide (KOH), which react with the acetoin to produce a reddish-brown color. The intensity of the color is proportional to the amount of acetoin produced, and thus indicates the presence of the fermenting bacteria.

Rapid VP tests, in general, consist of inoculation of heavy growth of organism from a solid medium into a small amount (0.3-0.5ml) of GPB medium. After incubation of 4 hours at 37^oC sufficient acetoin can be produced to be detectable. If the organism has already been grown on a medium containing glucose, acetoin produced in that medium may be carried over and can result in a red color immediately. Another rapid test utilizes reagent impregnated strips.

2 pyruvate = acetoin + 2CO2
acetoin + NADH + H+ = 2,3-butanediol + NAD+

Requirements VP Test

1. Sterile glucose phosphate broth (GPB) medium (0.5-1.0ml in each tube). 
2. Test culture suspension (Enterobacter / Klebsiella). 
3. 5%- α-napthol in absolute ethanol. 
4. 40% KOH solution (containing 0.5% creatinine, if desired). 

Procedure of VP Test

1. Inoculate GPB medium with the culture suspension. 
2. Incubate at 37^oC for 24 hours. 
3. Add 0.6 ml of 5% α-napthol and mix well. 
4. Add 0.2 ml of 40% KOH solution, shake well. 
5. Positive VP test is indicated by a red color of the medium, within 5 minutes. A negative VP test is indicated by the medium remaining brown.

VP Test Results

VP Test Positive	VP Test Negative
Viridans group streptococci	Streptococcus mitis
Listeria	Citrobacter Spp.
Enterobacter	Shigella Spp.
Klebsiella	Yersinia
Serratia marcescens	Edwardsiella
Hafnia alvei	Salmonella
Vibrio eltor	Vibrio fluvialis
Vibrio alginolyticus	Vibrio vulnificus

Uses of VP Test

1. The VP test is often used to differentiate between members of the Enterobacteriaceae family, which includes important human pathogens such as Escherichia coli, Salmonella, and Shigella. This test helps in the identification of these organisms in clinical settings.
2. The VP test is used in the production of fermented beverages, such as beer and wine, to determine the presence of acetoin-producing bacteria. It is also used in the food industry to detect certain spoilage organisms.
3. The VP test can be used to identify acetoin-producing bacteria in environmental samples, such as soil and water. This can be useful in monitoring the quality of water and soil for potential bacterial contamination.
4. The VP test is commonly used in microbiological research to investigate the metabolic pathways and physiology of bacteria. It is often used in conjunction with other tests to identify bacterial strains and study their characteristics.

Citrate utilization Test

Citrate Utilization Test is chiefly used in the differentiation of Gram positive rods, by checking their ability to utilize the carbon of sodium citrate as the sole source of carbon.

Some bacteria can convert salts of organic acids to alkaline carbonates. This was first demonstrated in milk where an alkaline reaction was further confirmed in a medium where nitrogen was supplied containing sodium ammonium phosphate as nitrogen source (peptone free nitrogen base).

It was also found that the growth of certain organisms was enhanced by sodium citrate, whereas the growth of other was completely inhibited.  

In 1923, Koser devised a liquid basal broth medium in which ammonium phosphate supplied as the source of nitrogen, to which he added each organic acid in a concentration of 0.2%. He studied 18 organic acids and evaluated the ability of various organisms to utilize these acids as carbon sources.

The most striking result of this work was the recognition that the aerogenes group of organisms could utilize citrate for growth and the coli group of organisms was completely unable to grow in this medium. This was the introduction of carbon utilization as a diagnostic aid.          

Since determining growth on solid medium is easier than interpreting turbidity in broth, Simon’s added agar to Koser’s medium and incorporated an indicator, bromothymol blue, which was green at a pH of 6.8 and blue at pH greater than 7.6.

The alkaline reaction involved in citrate utilization is not due to release of ammonia. The alkaline reaction mostly occurs because an excess of carbon dioxide is generated as citrate is cleaved to oxaloacetate, which is then decarboxylated to pyruvate and carbon dioxide.

The excess of carbon dioxide may combine with sodium and water to form sodium carbonate, which is sufficiently alkaline to change the indicator from green to deep blue.

It is also possible that the sodium ion may remain in excess in this reaction and may attract the hydroxyl group from the available water in the medium and form sodium hydroxide.  

The enzyme that catalyzedthe cleavage of citrate has been given a number of names: citrate lyase, citratase, citrase, citrate aldolase, citridesmolase.

The primary products of cleavage are oxaloacetate and acetate which are subsequently converted to pyruvate and carbon dioxide by an oxaloacetate decarboxylase.

E. coli has been shown to possess citrate lyase although it cannot utilize citrate in Koser’s or Simmon’s media. It apparently lacks the transport system or permease that would permit the citrate to enter the cell, to be used as a carbon source. The permease for citrate utilization is inducible.

When performing this test, it is important to use a light inoculum and minimize carryover of any nutritive constituents of the culture medium from which the inoculums is taken.

Requirements of citrate utilization Test

1. Test culture suspension. 
2. Sterile Koser’s citrate medium (1.0 ml in each tube) / Simon’s citrate medium (agar slant) 
3. Koser’s citrate medium: 

Constituent	Amount
Sodium chloride	0.5g
Tri-sodium citrate	0.5g
Ammonium dihydrogen phosphate	1.0g
Magnesium sulfate	0.02g
Potassium dihydrogen phosphate	0.1g
Distilled water	100ml
pH	6.5-6.8

Medium is autoclaved at 110^oC for 10 minutes.

Citrate Utilization Test Procedure

1. Inoculate the medium (Koser’s broth or Simon’s agar) with the culture suspension. 
2. Incubate at 37^oC for 24 hours. 
3. Check for turbidity (indicating positive test) in Koser’s medium; growth and change in colour of indicator to blue on Simon’s citrate agar (positive test). 

Further Readings

1. Indole Test
2. Methyl Red Test
3. Voges Proskauer Test
4. Acid fast staining of bacteria
5. Algae
6. Aseptic Transfer Technique
7. Bacterial Flagella, Fimbriae and Pili
8. Bacterial Growth and Nutrition
9. Extremophiles
10. Fimbriae vs Flagella
11. Fimbriae vs Pili
12. Fundamental Microscopy
13. Growth Curve of Bacteria
14. Instruments used in Microbiology Laboratory
15. MacConkey agar
16. McFarland Standards
17. Negative Staining
18. Nutritional Requirements of Micro-Organisms
19. Preparation and Sterilization of Culture Media
20. Serial Dilution in Microbiology
21. Spread Plate Technique
22. Streak Plate Technique

FAQs on IMViC Test