IMViC test procedure, Purpose, Result, and Organism List.

IMViC is a mnemonic that stands for four tests used in the identification of enteric bacteria: Indole, Methyl Red, Voges-Proskauer, and Citrate. These tests are commonly used in microbiology laboratories to identify bacterial species in the Enterobacteriaceae family, which includes many common pathogens such as Escherichia coli and Salmonella.

The principle behind the IMViC tests is that different species of enteric bacteria have different metabolic pathways, which can be detected through a series of biochemical tests. Each of the four tests in the IMViC series is designed to identify a specific metabolic pathway, and the results of all four tests can be used to identify the bacterial species present.

The Indole test is used to detect the presence of the enzyme tryptophanase, which breaks down the amino acid tryptophan. The Methyl Red test is used to detect the presence of mixed-acid fermentation, which produces a range of organic acids including lactic acid, acetic acid, and formic acid. The Voges-Proskauer test is used to detect the presence of the enzyme acetylmethylcarbinol acetylase, which is involved in the production of acetoin. The Citrate test is used to detect the ability of the bacteria to utilize citrate as a sole carbon source.

By performing these four tests and examining the results, microbiologists can identify the bacterial species present in a sample and classify it accordingly. This information can be used to diagnose infections, monitor outbreaks, and understand the biology of different bacterial species.

What is IMViC test?

• IMVIC is a group of individual biochemical tests, used for the detection of coliform group.
• The full form of IMViC test is “Indole Methyl red Voges-Proskauer Citrate” test, whereas “I” indicating “Indol”, “M” indicating “Methyl red”, “Vi” indicating a scientist name “Voges-Proskauer”, and “C” indicating “Citrate”.
• IMVIC test is the combination of four tests, such as Indol test, Methyl red test, Voges-Proskauer test, and Citrate utilization test.
• The indole test detects the production of indole from the amino acid tryptophan.
• Methyl red is a pH indicator which determines whether the bacterium carries out mixed acid fermentation.
• Voges-Proskauer (VP) detects the production of acetoin.
• The citrate test determines whether or not the bacterium can use sodium citrate as a sole source of carbon.
• Used to distinguish Escherichia (MR-, VP-, indole+) from Enterobacter (MR-, VP +, indole-) and Klebsiella pneumoniae, (MR- , VP I , indole- ); also used to characterize members of the genus Bacillus

Principle of IMViC Test

The IMViC test is based on the differences between the metabolic requirements and characteristics of several bacterial genera and species. The ‘indole test’ and ‘citrate utilisation test’ in the series measure the ability of bacteria to create and utilise certain enzymes and nutrients, respectively. The ‘MR test’ and ‘VP test’ in the series, on the other hand, detect the final metabolic products created by bacteria consuming certain nutrients. The test microorganisms are cultivated in particular culture media for this purpose; different media are used for each test (expect MR and VP tests which need the same culture media; MR-VP media).

Purpose of imvic test

• Examine the indole production, acid production, acetoin generation, and citrate consumption of unknown isolated bacteria in order to define and identify them.
• To differentiate and identify members of the family Enterobacteriaceae.

IMViC test Procedure

IMViC test performs in four test; each step of 

1. Indole test
2. Methyl red test
3. Voges-Proskauer test
4. Citrate utilization test

Here is a general outline of the procedure for the IMViC test:

1. Indole test: This test is used to determine if the bacterium can produce the enzyme tryptophanase, which breaks down tryptophan into indole and other products. A small amount of the bacterium is added to a tube containing a tryptophan-containing medium and a reagent called Kovac’s reagent. If the bacterium produces tryptophanase, the tryptophan is broken down into indole and the indole is detected by the addition of Kovac’s reagent, which turns pink or red in the presence of indole.
2. Methyl red (MR) test : The methyl red (MR) test is a biochemical test used to identify bacteria that are able to produce large amounts of acidic byproducts during glucose metabolism. The test is performed by adding a small amount of the bacterium to a tube containing a glucose-containing medium and a pH indicator called methyl red. If the bacterium produces large amounts of acidic byproducts, the pH of the medium will drop and the methyl red will change color from yellow to red.
3. Voges-Proskauer (VP) test: This test is used to determine if the bacterium can produce acetylmethylcarbinol (AMC) from glucose through the action of the enzyme acetoin reductase. A small amount of the bacterium is added to a tube containing a glucose-containing medium and a reagent called Barritt’s reagent. If the bacterium produces AMC, it reacts with Barritt’s reagent to produce a pink or red color.
4. Citrate test: This test is used to determine if the bacterium can utilize citrate as a sole carbon source. A small amount of the bacterium is added to a tube containing a citrate-containing medium and a pH indicator. If the bacterium can utilize citrate, it will produce acidic byproducts that lower the pH of the medium and change the color of the indicator.

Each of these tests is performed separately and the results are recorded. The combination of test results can be used to identify the bacterium to the species level.

1. Indole test

What is Indole Test?

The indole test is a biochemical test used to identify bacteria that are able to produce the enzyme tryptophanase, which breaks down the amino acid tryptophan. The test is performed by adding a small amount of the bacterium to a tube containing a tryptophan-containing medium and a reagent called Kovac’s reagent. If the bacterium produces tryptophanase, the tryptophan is broken down into indole and other products. The indole is then detected by the addition of Kovac’s reagent, which turns pink or red in the presence of indole.

The indole test is often used in microbiology laboratories to identify certain types of bacteria, including Escherichia coli, Salmonella, and Proteus. It is a simple and reliable test that can be used to differentiate these bacteria from other types of bacteria that do not produce tryptophanase.

Principle of Indole Test

Some bacteria are capable of producing the enzyme tryptophanase, which allows them to convert the amino acid tryptophan into indole, pyruvic acid, and ammonia.

When the indole reagent is put to a medium containing an indole-producing bacterial culture, the indole interacts with the aldehyde in the reagent to produce a unique colour.

If benzaldehyde is present in the reagent, a pink to violet-red quinoidal compound is generated, resulting in the formation of a pink to red colour ring.

If cinnamaldehyde is present in the reagent, a blue-to-green colour compound is generated, resulting in the formation of a green-to-blue colour ring.

(The indole reagent of Kovac contains amyl alcohol and benzaldehyde. The amyl alcohol is insoluble in water and creates an oily coating, resulting in a cherry or pink-red ring on the surface.)”

After incubating test microorganisms in a medium containing tryptophan for 24 to 48 hours, an indole reagent is added to determine the outcome. Depending on the type of reagent used, a positive result is indicated by the creation of a pink to violet-red or green to blue colour ring. A negative colour is denoted by a lack of colour change or a tiny ring of yellowish colour at the top.

Requirements for Indole test

• New development of gram-negative rods (test organism)
• Sulfide indole kinetics (SIM) Motility indole urea medium (MIU) medium/ peptone water
• Incubator, inoculating wire, and Bunsen burner for Kovac’s reagent

Quality control strains

• Positive control-E. coli (ATCC25922)
• Negative Control-Pseudomonas aeruginosa (ATCC27853)

Procedure of Indole Test

Here is a general outline of the procedure for the indole test:

1. Inoculate a tube of tryptophan broth with a small amount of the bacterium.
2. Incubate the tube at the appropriate temperature for the bacterium for 24-48 hours.
3. Add a few drops of Kovac’s reagent to the tube.
4. Observe the color of the mixture. If the mixture turns pink or red, it is positive for the production of indole. If it remains yellow or amber, it is negative for the production of indole.

Note: It is important to follow proper laboratory techniques and precautions when performing the indole test, including wearing protective clothing and using sterile techniques to avoid contamination.

Result of Indole Test

The result of the indole test is either positive or negative for the production of indole.

A positive result for the indole test indicates that the bacterium is able to produce the enzyme tryptophanase, which breaks down tryptophan into indole and other products. A positive result is indicated by the presence of pink or red color in the mixture after the addition of Kovac’s reagent.

A negative result for the indole test indicates that the bacterium is not able to produce tryptophanase. A negative result is indicated by the absence of pink or red color in the mixture after the addition of Kovac’s reagent. The mixture will remain yellow or amber in color.

The indole test is often used in microbiology laboratories to identify certain types of bacteria, including Escherichia coli, Salmonella, and Proteus. It is a simple and reliable test that can be used to differentiate these bacteria from other types of bacteria that do not produce tryptophanase.

Applications of Indole Test

The indole test has several applications in the field of microbiology:

1. Identification of bacteria: The indole test is often used to identify certain types of bacteria, including Escherichia coli, Salmonella, and Proteus. It is a simple and reliable test that can be used to differentiate these bacteria from other types of bacteria that do not produce tryptophanase.
2. Clinical diagnosis: The indole test can be used in the clinical setting to identify the cause of infections and determine the appropriate treatment. For example, the presence of Escherichia coli in a urine sample may indicate a urinary tract infection, while the presence of Salmonella in a stool sample may indicate food poisoning.
3. Environmental monitoring: The indole test can be used to monitor the presence of bacteria in environmental samples, such as water or soil. This can help to identify sources of contamination and determine the need for treatment or remediation.
4. Industrial applications: The indole test can also be used in industrial settings, such as food processing plants, to monitor the presence of bacteria that could potentially cause food poisoning or other health hazards.

Overall, the indole test is a useful tool for identifying and characterizing bacteria in a variety of settings.

List of Positive and Negative Organism in Indole Test

Microorganism Name	Indole Positive	Indole Negative
Actinobacillus spp.	No	Yes
Aeromonas hydrophila	Yes	No
Aeromonas punctata	Yes	No
Aeromonas salmonicida	No	Yes
Alcaligenes sp.	No	Yes
Bacillus alvei	Yes	No
Bacillus sp.	No	Yes
Bordetella sp.	No	Yes
Edwardsiella sp	Yes	No
Enterobacter sp.	No	Yes
Enterococcus faecalis	Yes	No
Escherichia coli	Yes	No
Flavobacterium sp.	Yes	No
Haemophilus influenzae	Yes	No
Haemophilus sp.	No	Yes
Klebsiella oxytoca	Yes	No
Klebsiella sp.	No	Yes
Lactobacillus reuteri	Yes	No
Mannheimia haemolytica	No	Yes
Neisseria sp.	No	Yes
P. penneri	No	Yes
Pasteurella multocida	Yes	No
Pasteurella pneumotropica	Yes	No
Pasteurella ureae	No	Yes
Plesiomonas shigelloides	Yes	No
Proteus mirabilis	No	Yes
Proteus sp. (not P. mirabilis and P. penneri)	Yes	No
Pseudomonas sp.	No	Yes
Rhizobium sp.	No	Yes
Salmonella sp.	No	Yes
Serratia sp.	No	Yes
Vibrio sp	Yes	No
Yersinia sp.	No	Yes

2. Methyl red test

What is Methyl red test?

The methyl red (MR) test is a biochemical test used to identify bacteria that are able to produce large amounts of acidic byproducts during glucose metabolism. The test is performed by adding a small amount of the bacterium to a tube containing a glucose-containing medium and a pH indicator called methyl red. If the bacterium produces large amounts of acidic byproducts, the pH of the medium will drop and the methyl red will change color from yellow to red.

The MR test is often used in microbiology laboratories to identify certain types of bacteria, including Escherichia coli and Klebsiella pneumoniae. It is a simple and reliable test that can be used to differentiate these bacteria from other types of bacteria that do not produce large amounts of acidic byproducts during glucose metabolism.

The MR test is typically performed as part of a series of biochemical tests called the IMViC (Indole, Motility, Voges-Proskauer, Citrate) test, which is used to differentiate and identify different types of bacteria.

Principle of Methyl red test

The principle of the methyl red (MR) test is based on the ability of certain bacteria to produce large amounts of acidic byproducts during glucose metabolism. These acidic byproducts can lower the pH of the medium, causing it to become more acidic.

During the MR test, a small amount of the bacterium is added to a tube containing a glucose-containing medium and a pH indicator called methyl red. If the bacterium produces large amounts of acidic byproducts, the pH of the medium will drop and the methyl red will change color from yellow to red.

The MR test is based on the observation that certain bacteria, such as Escherichia coli and Klebsiella pneumoniae, are able to produce large amounts of acidic byproducts during glucose metabolism. These bacteria are classified as mixed acid fermenters because they produce a mixture of acids, including lactic acid, acetic acid, and formic acid. The production of these acids lowers the pH of the medium and can be detected by the change in color of the methyl red indicator.

Overall, the MR test is a useful tool for identifying and characterizing bacteria based on their ability to produce acidic byproducts during glucose metabolism.

Composition of MR-VP broth

• Buffered peptone:7.0 g
• Glucose:5.0 g
• Dipotassium phosphate:5.0 g
• Deionized / Distilled water: 1000 ml
• Final pH:6.9

Preparation

1. Dispense roughly 5 ml each tube on average.
2. Use enough broth to cover an upside-down Durham tube, if one is being used.
3. For rapid VP testing, dispense 2 ml of MR-VP broth and for rapid MR testing, dispense 0.5 ml.

Preparation of Methyl red solution

Methyl red solution, 0.02%

1. Dissolve 0.1 g of methyl red in 300 ml of ethyl alcohol containing 95% ethanol.
2. Add enough water distillate to make 500 ml.
3. Store at 4 to 8 degrees Celsius in a brown bottle.
4. The stability of the solution is one year.

Requirement for Methyl red test

1. MR-VP broth
2. Test organism
3. Inoculating wire
4. Bunsen burner
5. Incubator
6. Methyl red indicator
7. Control strains

Procedure of Methyl red test

Here is a general outline of the procedure for the MR test:

1. Inoculate a tube of glucose broth with a small amount of the bacterium.
2. Incubate the tube at the appropriate temperature for the bacterium for 24-48 hours.
3. Add a few drops of methyl red to the tube.
4. Observe the color of the mixture. If the mixture turns red, it is positive for the production of large amounts of acidic byproducts during glucose metabolism. If it remains yellow, it is negative for the production of large amounts of acidic byproducts.

Note: It is important to follow proper laboratory techniques and precautions when performing the MR test, including wearing protective clothing and using sterile techniques to avoid contamination.

The MR test is typically performed as part of a series of biochemical tests called the IMViC (Indole, Motility, Voges-Proskauer, Citrate) test, which is used to differentiate and identify different types of bacteria.

Result of Methyl red test

The result of the methyl red (MR) test is either positive or negative for the production of large amounts of acidic byproducts during glucose metabolism.

A positive result for the MR test indicates that the bacterium is able to produce large amounts of acidic byproducts during glucose metabolism. A positive result is indicated by the presence of red color in the mixture after the addition of methyl red.

A negative result for the MR test indicates that the bacterium is not able to produce large amounts of acidic byproducts during glucose metabolism. A negative result is indicated by the absence of red color in the mixture after the addition of methyl red. The mixture will remain yellow in color.

The MR test is often used in microbiology laboratories to identify certain types of bacteria, including Escherichia coli and Klebsiella pneumoniae. It is a simple and reliable test that can be used to differentiate these bacteria from other types of bacteria that do not produce large amounts of acidic byproducts during glucose metabolism.

Note: The MR test is just one of several tests that may be used to identify a bacterium to the species level. It is important to perform a variety of tests in order to accurately identify the bacterium.

Result of Quality control strains

• Escherichia coli ATCC 25922—MR positive(red),VP negative(no change)
• Klebsiella pneumoniae ATCC 13883—MR negative (yellow), VP positive (red)

List of Positive and Negative Organism in Methyl Red test

Organism Name	Result
Escherichia coli	Positive (MR+)
Klebsiella pneumoniae	Negative (MR-)
Shigella species	Positive (MR+)
Enterobacter species	Negative (MR-)
Salmonella species	Positive (MR+)
Hafnia species	Negative (MR-)
Proteus species	Positive (MR+)
Citrobacter species	Positive (MR+)
Serratia marcescens	Negative (MR-)
Yersinia species	Positive (MR+)

Applications of Methyl red test

• It is used to differentiate between bacteria that produce acid from glucose fermentation (positive result) and those that do not (negative result). The test is performed by adding a small amount of methyl red dye to a culture of the bacterial species being tested. If the bacteria are able to produce acid from glucose fermentation, the pH of the culture will drop, causing the methyl red dye to change color from red to yellow.
• The methyl red test is commonly used in microbiology laboratories to identify and differentiate between different types of bacteria.
• It is often used in conjunction with other biochemical tests, such as the Voges-Proskauer test and the citrate test, to help identify and classify different bacterial species.
• Some examples of bacteria that may produce a positive result on the methyl red test include Escherichia coli, Salmonella, and Proteus vulgaris. Bacteria that do not produce acid from glucose fermentation, such as Staphylococcus aureus and Streptococcus pneumoniae, will produce a negative result on the test.

3. Voges-Proskauer (VP ) Test

What is Voges-Proskauer (VP ) Test?

The Voges-Proskauer (VP) test is a biochemical test used to identify certain bacteria based on their ability to produce acetoin from glucose fermentation. It is used to differentiate between bacteria that produce acetoin (positive result) and those that do not (negative result).

The VP test is performed by adding a small amount of a reagent called alpha-naphthol to a culture of the bacterial species being tested, followed by the addition of a reagent called potassium hydroxide (KOH). If the bacteria are able to produce acetoin, the KOH will convert it to diacetyl, which will then react with the alpha-naphthol to produce a pink to red color.

The VP test is commonly used in microbiology laboratories to identify and differentiate between different types of bacteria. It is often used in conjunction with other biochemical tests, such as the methyl red test and the citrate test, to help identify and classify different bacterial species.

Some examples of bacteria that may produce a positive result on the VP test include Escherichia coli, Salmonella, and Proteus vulgaris. Bacteria that do not produce acetoin, such as Staphylococcus aureus and Streptococcus pneumoniae, will produce a negative result on the test.

Principle of Voges Proskauer test

Using the butylene glycol pathway, organisms make acetylmethylcarbinol (acetoin) and butanediol, which are neutral end products that raise the pH towards neutrality (pH 6) and result in a high ultimate pH. Rarely do both metabolic pathways exist in Enterobacteriaceae, which typically exhibit only one. The Voges-Proskauer (VP) test determines if an organism creates acetylmethylcarbinol from glucose fermentation. In the presence of -naphthol, strong alkali (40% KOH), and ambient oxygen, acetylmethylcarbinol, if present, is transformed to diacetyl. The -naphthol was not originally part of the technique, but it was discovered to be a colour intensifier and must be introduced beforehand. The diacetyl and guanidine-containing chemicals in the broth’s peptones condense to create a pinkish-red polymer.

Composition Of MR-VP Broth

• Buffered peptone:7.0 g
• Glucose:5.0 g
• Dipotassium phosphate:5.0 g
• Deionized / Distilled water: 1000 ml
• Final pH:6.9
• Generally dispense approximately 5 ml per tube.
• Use enough broth to cover an inverted Durham tube, if it is used.

Reagent preparation

Reagent 1:  5% α-Naphthol

Composition: α-Naphthol: 5 g, 95% ethyl alcohol:100 ml

1. Mix 5 grammes of -Naphthol with 100 millilitres of ethyl alcohol at 95% concentration.
2. Store between 4 and 8 degrees Celsius in the dark.
3. The shelf life is two to three weeks.

Reagent 2: 40% Potassium Hydroxide

Composition: Potassium hydroxide( KOH) pellets: 20 gram, Distilled water: up to 100 ml

• Dissolve In a plastic bottle, mix 40 g of potassium hydroxide pellets with 100 ml of distilled water.
• Maintain a bottle in a bath of chilly water while processing.
• KOH is hygroscopic and becomes caustic when it becomes damp. Weigh fast in a beaker with a scale. Avoid storing near acids. Avoid skin exposure.

Requirements for  Voges Proskauer test

• MR-VP broth
• Test organism
• Inoculating wire
• Bunsen burner
• Incubator
• 5%α- naphthol
• 40% KOH
• Control strains
  • Klebsiella pneumoniae ATCC 13883
  • Escherichia coli ATCC 25922

Procedure of Voges Proskauer test

1. Inoculate a colony of the test organism into MR-VP broth and incubate for 18 to 24 hours at 35 degrees Celsius. Avoid tightening caps. Some species, such as Hafnia alvei, Yersinia, and Listeria, can generate acetylmethylcarbinol at ambient temperature instead of 35°C. Inoculate another broth and incubate at room temperature in this instance.
2. If a 5-ml broth culture is utilised, dispense 2.0 ml of broth into a 13 mm by 100 mm test tube. Reserve remaining ingredients for potential reincubation.
3. Add six drops of 5% -naphthol and well mix to aerate.
4. Add 2 drops of 40% potassium hydroxide and well stir to aerate the solution.
5. Observe the surface for a pinkish-red hue within 30 minutes. Shake the tube vigorously for thirty minutes. Note: If the test yields a negative result, the MR-VP broth can be cultured for up to 48 hours and the test repeated.

Result and Interpretation of Voges Proskauer test

A positive result on the Voges-Proskauer (VP) test indicates that the bacterial species being tested is able to produce acetoin from glucose fermentation. This means that the bacteria are able to utilize glucose as a carbon source and produce acetoin as a metabolic byproduct.

A negative result on the VP test indicates that the bacterial species being tested is unable to produce acetoin from glucose fermentation. This may be due to the lack of the enzyme required for acetoin production, or it may be due to the presence of other metabolic pathways that prevent acetoin production.

It’s important to note that the results of the VP test should be interpreted in conjunction with the results of other biochemical tests, as well as other factors such as the appearance and characteristics of the bacterial culture. This will help to accurately identify and classify the bacterial species being tested.

For example, a positive result on the VP test in conjunction with a positive result on the citrate utilization test may indicate that the bacterial species being tested is a member of the genus Escherichia, such as E. coli. A negative result on the VP test in conjunction with a positive result on the methyl red test may indicate that the bacterial species is a member of the genus Proteus, such as P. vulgaris.

• VP Positive Result: A pinkish-red hue on the surface of the Voges Proskauer test sample indicates a positive reaction.
• VP Negative Result: Absence of a pink-red hue indicates a negative response to the Voges-Proskauer test.

List of Positive and Negative Organism in Voges-Proskauer test

Organism Name	Result
Enterobacter	Positive (VP+)
Citrobacter sp.	Negative (VP-)
Klebsiella	Positive (VP+)
Shigella	Negative (VP-)
Serratia marcescens	Positive (VP+)
Yersinia	Negative (VP-)
Hafnia alvei	Positive (VP+)
Vibrio cholera biotype eltor	Positive (VP+)
Edwardsiella	Negative (VP-)
Salmonella	Negative (VP-)
Vibrio furnissii	Negative (VP-)
Vibrio fluvialis	Negative (VP-)
Vibrio vulnificus	Negative (VP-)
Vibrio parahaemolyticus.	Negative (VP-)
Vibrio alginolyticus	Positive (VP+)

Applications of Voges Proskauer test

• The Voges-Proskauer (VP) test is a commonly used biochemical test in microbiology laboratories to identify and differentiate between different types of bacteria.
• It is often used in conjunction with other biochemical tests, such as the methyl red test and the citrate test, to help identify and classify different bacterial species.
• The VP test is useful in identifying bacteria that are able to produce acetoin from glucose fermentation, which can be important in determining the potential pathogenicity of a particular bacterial species. For example, the ability to produce acetoin may be an indicator of the ability of a bacterial species to survive and grow in the human body, making it potentially pathogenic.
• The VP test is also useful in identifying bacteria that are able to utilize glucose as a carbon source in industrial and environmental settings. For example, bacteria that are able to utilize glucose as a carbon source may be used in the production of biofuels or as a means of bioremediation.
• Overall, the VP test is an important tool in microbiology that helps to identify and classify different types of bacteria based on their ability to produce acetoin from glucose fermentation.

Control strains

• Klebsiella pneumoniae ATCC 13883—VP positive (red)
• Escherichia coli ATCC 25922—VP negative (no change)
• Copper should be regarded a negative hue. Rust is a weakly positive colour.
• The majority of species of the family Enterobacteriaceae exhibit opposing MR and VP reactions; however, certain organisms, such as H. alvei and Proteus mirabilis, may exhibit both positive MR and positive VP reactions (often delayed)
• The organisms of the Streptococcus mitis group are VP negative, whereas the organisms of the Streptococcus viridans group are VP positive, with the exception of Streptococcus vestibularis, which is VP variable.
• Listeria organisms are beta-hemolytic, gram-positive, rod-shaped, and VP-positive at 25°C; however, this test is not essential for identification.

4. Citrate utilization test

What is Citrate utilization test?

The citrate utilization test is a biochemical test used to identify certain bacteria based on their ability to use citrate as a sole carbon source. It is used to differentiate between bacteria that can utilize citrate (positive result) and those that cannot (negative result).

The test is performed by adding a small amount of citrate to a culture of the bacterial species being tested and incubating it for a period of time. If the bacteria are able to utilize citrate as a carbon source, they will produce acid, which will lower the pH of the culture and cause a change in the color of the pH indicator.

The citrate utilization test is commonly used in microbiology laboratories to identify and differentiate between different types of bacteria. It is often used in conjunction with other biochemical tests, such as the methyl red test and the Voges-Proskauer test, to help identify and classify different bacterial species.

Some examples of bacteria that may produce a positive result on the citrate utilization test include Escherichia coli, Salmonella, and Proteus vulgaris. Bacteria that are unable to utilize citrate as a carbon source, such as Staphylococcus aureus and Streptococcus pneumoniae, will produce a negative result on the test.

Principle of Citrate utilization test

An organism’s ability to use sodium citrate as its only carbon supply and inorganic ammonium ions as its sole nitrogen source is determined by a citrate utilisation test. When bacteria metabolise citrate, ammonium salts are converted down into ammonia, which raises alkalinity and causes the bromthymol blue indicator to change colour from green to blue.

Requirements for Test

1. Simmons citrate agar slant
2. Cotton plug
3. Sterile Inoculating wire/ sticks
4. Test organism
5. Bunsen burner
6. Incubator
7. Test tube rack

Procedure of citrate utilization test

1. Using a light inoculum taken from the middle of a well-isolated colony, zigzag the slope.
2. Place the top on the tube loosely.
3. 18 to 24 hours of aerobic incubation between 35°C and 37°C.
4. Along the slant, a transition from green to blue is seen.

Result of citrate utilization test

A positive result on the citrate utilization test indicates that the bacterial species being tested is able to use citrate as a sole carbon source. This means that the bacteria are able to utilize citrate for growth and metabolism.

A negative result on the citrate utilization test indicates that the bacterial species being tested is unable to use citrate as a sole carbon source. This means that the bacteria are unable to utilize citrate for growth and metabolism and may require other carbon sources for survival.

It’s important to note that the results of the citrate utilization test should be interpreted in conjunction with the results of other biochemical tests, as well as other factors such as the appearance and characteristics of the bacterial culture. This will help to accurately identify and classify the bacterial species being tested.

• Positive citrate utilisation test: The growth will be apparent on the surface of the slant, and the medium will be a vivid shade of blue. The alkaline carbonates and bicarbonates produced as byproducts of citrate catabolism boost the pH of the medium to greater than 7.6, causing the bromothymol blue to change colour from green to blue.
• Negative citrate utilisation test: traces or no apparent growth would be present. There will be no colour change; the medium will retain its original green tint. On the Simmons citrate medium, only bacteria that can use citrate as their only carbon and energy source will be able to grow; therefore, a citrate utilisation negative test culture will be indistinguishable from an uninoculated slant.
• Uninoculated (UN): There is no growth and no colour change; the slant remains green.
• Negative control: no development and no colour change; the slant does not change colour
• Positive control: growth accompanied by a shift in colour from green to bright blue along a slant.
• Test organism/bacteria: Positive, i.e., growth with a colour change from green to strong blue along the slant.

List of Positive and Negative Organism in Simmons citrate test

Microorganism	Result
Klebsiella pneumoniae	Positive (+)
Salmonella Typhi	Negative (-)
Enterobacter species	Positive (+)
Citrobacter freundii	Positive (+)
Escherichia coli	Negative (-)
Shigella spp	Negative (-)
Serratia marcescens	Positive (+)
Proteus mirabilis	Positive (+)
Salmonella Paratyphi A	Negative (-)
Providencia	Positive (+)
Morganella morganii	Negative (-)
Yersinia enterocolitica	Negative (-)
Salmonella other than Typhi and Paratyphi A	Positive (+)

Quality control

Quality Control strains used in citrate utilization test

• Positive Control (PC)-Klebsiella pneumoniae ATCC 13883
• Negative Control (NC)-Escherichia coli ATCC 25922

Applications of citrate utilization test

• The citrate utilization test is a commonly used biochemical test in microbiology laboratories to identify and differentiate between different types of bacteria.
• It is often used in conjunction with other biochemical tests, such as the methyl red test and the Voges-Proskauer test, to help identify and classify different bacterial species.
• The test is useful in identifying bacteria that are able to utilize citrate as a sole carbon source, which can be important in determining the potential pathogenicity of a particular bacterial species. For example, the ability to utilize citrate may be an indicator of the ability of a bacterial species to survive and grow in the human body, making it potentially pathogenic.
• The citrate utilization test is also useful in identifying bacteria that are able to utilize citrate as a sole carbon source in industrial and environmental settings. For example, bacteria that are able to utilize citrate as a carbon source may be used in the production of bioplastics or as a means of bioremediation.
• Overall, the citrate utilization test is an important tool in microbiology that helps to identify and classify different types of bacteria based on their ability to utilize citrate as a carbon source.

IMVIC test results

S. No	Test Name	Positive result	Negative Result
1.	Indole Test	color changes pink to red (“cherry-red ring”)	no color change occurs
2.	Methyl Red test	a stable red color develops	a yellow color develops
3.	Voges Proskauer Test	a pink-red color develops	a yellow color develops
4.	Citrate Test	develops blue color from green	No color change

E coli imvic test results

Escherichia coli (E. coli) is a gram-negative, rod-shaped bacterium that is commonly found in the human gastrointestinal tract. The results of the IMViC test (Indole production, Methyl red, Voges-Proskauer, Citrate utilization) for E. coli are typically as follows:

1. Indole production test: Positive result
2. Methyl red test: Positive result
3. Voges-Proskauer (VP) test: Negative result
4. Citrate utilization test: Negative result

Uses of IMViC Test

• The IMViC test (Indole production, Methyl red, Voges-Proskauer, Citrate utilization) is a commonly used biochemical test in microbiology laboratories to identify and differentiate between different types of bacteria.
• It is often used in conjunction with other biochemical tests, as well as other factors such as the appearance and characteristics of the bacterial culture, to accurately identify and classify different bacterial species.
• The IMViC test is useful in identifying bacteria based on their metabolism, which can be important in determining the potential pathogenicity of a particular bacterial species. For example, the ability to produce indole, utilize glucose as a carbon source, or utilize citrate as a carbon source may be indicators of the ability of a bacterial species to survive and grow in the human body, making it potentially pathogenic.
• The IMViC test is also useful in identifying bacteria that are able to utilize specific carbon and nitrogen sources in industrial and environmental settings. For example, bacteria that are able to utilize glucose or citrate as a carbon source may be used in the production of biofuels or bioplastics, while bacteria that are able to utilize tryptophan as a nitrogen source may be used in the production of chemicals such as nylon.
• Overall, the IMViC test is an important tool in microbiology that helps to identify and classify different types of bacteria based on their metabolism.

Disadvantages of IMVIC Test

1. Limited scope: The IMViC test is only able to identify certain types of bacteria based on their metabolism, and may not be suitable for identifying all types of bacteria.
2. False positives: There is a risk of false positives with the IMViC test, as some bacteria may produce metabolic byproducts that mimic the results of the test.
3. False negatives: There is also a risk of false negatives with the IMViC test, as some bacteria may not produce the metabolic byproducts that the test is designed to detect.
4. It is not sufficient to identify bacteria to the species level alone. Even for distinguishing Enterobacterales, additional tests are necessary.
5. Different genera produce identical outcomes, resulting in ambiguous outcomes.
6. It is a culture-based test series, which necessitates a longer time frame. Some bacteria require more than 2 days of incubation to produce a VP reaction, and the citrate test may require more than 4 days of incubation.
7. Only microorganisms that can be cultured can be described.
8. Complexity permeates the entire method, which necessitates cultural competence and a variety of resources.

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