IMViC Test: Introduction, Principle, Test Procedure and Result Interpretation

IMViC test

IMViC Test

IMViC tests stands for Indole test , Methyl red test , Voges-Proskauer test and Citrate  test. These tests are useful for differentiating the family Enterobacteriaceae as shown above image.

Various test strips are commercially available for these four tests in a single form but here we discuss singly as shown below-

# 1. Indole test for bacteria#

This indole test is useful for both aerobic and anaerobic gram-negative rods and really very useful in the routine identification of bacteria in Microbiology.

Principle of Indole test

The ability of bacteria to split indole from the amino acid tryptophan is due to the presence of the enzyme, tryptophanase.  Indole,  if present combines with an aldehyde in the reagent to produce a pink to red-violet quinoidal compound if using benzaldehyde reagent or blue to green when using cinnamaldehyde reagent.

Requirements for Indole test

Quality control strains

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

 Test Procedure

  1. Inoculate liquid tube medium or stab agar medium with the colony.
  2. Incubate for 18 to 24 hours in a BOD incubator.
  3. Add 3 drops of Kovac’s reagent down the side of the tube and observe color change at the meniscus.
  4. If the test is negative, repeat the test after additional 24 hours of incubation if desired.

Result Interpretation

  • Development of a brown-red to purple-red color:  Test  Positive (presence of indole)
  • Colorless or slightly yellow: Negative test
  • Tests positive (left)
  • Test negative (Right) as shown above picture.

Indole test positive organisms are-

  • Escherichia coli
  • Klebsiella oxytoca
  • Proteus vulgaris
  • Citrobacter koseri
  • Morgenella morganii
  • Vibrio cholerae
  • Providencia species
  • Aeromonas species
  • Plesiomonas species
  • Pasteurella species
  • Cardiobacterium hominis
  • Propionibacterium acenes

While indole test negative organisms are-

  • Klebsiella pneumoniae
  • Enterobacter species
  • Proteus mirabilis
  • Citrobacter freundii

Limitation of indole test

  • Do not use media that contain dyes (e.g. Eosin methylene blue, MacConkey agar).

#2. Methyl Red (MR) test#

Methyl Red test is for enteric gram-negative rods, as part of identification to species level.

Principle of methyl red test

The methyl red (MR) test uses to determine if an organism is able to produce stable acid end products from glucose fermentation Methyl red indicator (red color below pH 4.4; yellow color at pH 5.8) uses to determine the pH after an enteric gram-negative rod has fermented glucose to completion. All members of the Enterobacteriaceae give a positive methyl red reaction when tested up to 24 h due to conversion of glucose to pyruvic acid by the Embden-Meyerhof pathway. After further incubation (2 to 5 days) those organisms that are MR positive continue to metabolize pyruvic acid to lactic, acetic, and formic acids by the mixed acid pathway and are able to maintain the acid pH (4.4).

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
  1. Generally dispense approximately 5 ml per tube.
  2. Use enough broth to cover an inverted Durham tube, if it is used.
  3. Dispense 2 ml of MR-VP broth for rapid VP testing and 0.5 ml for rapid MR testing.

Preparation of Methyl red solution

Methyl red solution, 0.02%

  1.  Dissolve 0.1 g of methyl red in 300 ml of 95% ethyl alcohol.
  2. Add sufficient distilled water to make 500 ml.
  3. Store at 4 to 8°C in a brown bottle.
  4. The solution is stable for 1 year.

Requirements 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

  1. Inoculate test organism and incubate at 35C for at least 48 hours. Note: If the center of one colony is inoculated to a 0.5-ml volume of MR-VP broth, the test can be read at 18 to 24 hours.
  2. Remove approximately 1 ml of the 48-hours broth to a 13-by 100-mm tube. (The remainder should be reserved for testing at 3 to 5 days if necessary.)
  3. Add 3 to 6 drops (or 1 drop to 0.5 ml) of methyl red indicator to the aliquot.
  4.  Observe for red color immediately.

Result and Interpretation of Methyl red test

The development of a stable red color on the surface of the medium indicates sufficient acid production to lower the pH to 4.4 and constitutes a positive test. Because other organisms may produce smaller quantities of acid from the test substrate, an intermediate orange color between yellow and red may develop. This does not indicate a positive test. The yellow color indicates a negative test.

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 methyl red  test positive and negative bacteria

Methyl red test  positive bacteria are as follows:

  • Escherichia coli
  • Klebsiella ozanae
  • Klebsiella rhinoscleromatis
  • Klebsiella ornithiolytica
  • Citrobacter
  • Proteus
  • Yersinia
  • Edwardsiella
  • Salmonella

Methyl red test negative bacteria

  • Klebsiella pneumoniae
  • Enterobacter species

#M R(Methyl Red) Test/ bacteria-

#3. Voges-Proskauer (VP ) Test#

Voges Proskauer  test for identification to the species level of the following groups of organisms

  • Enteric gram-negative rods, Aeromonas, and Vibrio
  • Viridans group streptococci
  • Staphylococci

Principle  of Voges Proskauer test

Organisms utilizing the butylene glycol pathway produce acetylmethylcarbinol (acetoin) and butanediol, neutral end products that raise the pH towards neutrality (pH 6) and result in a high final pH. Most Enterobacteriaceae demonstrate one or the other metabolic pathway but rarely both. The Voges-Proskauer (VP) test is used to determine if an organism produces acetylmethylcarbinol from glucose fermentation. If present, acetylmethylcarbinol is converted to diacetyl in the presence of α-naphthol, strong alkali (40% KOH), and atmospheric oxygen. The α-naphthol was not part of the original procedure but was found to act as a color intensifier and must be added first. The diacetyl and guanidine-containing compounds found in the peptones of the broth then condense to form 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
  1. Generally dispense approximately 5 ml per tube.
  2. Use enough broth to cover an inverted Durham tube, if it is used.

Reagent preparation

Reagent 1:  5% α-Naphthol

α-Naphthol: 5 g

95% ethyl alcohol:100 ml

  1. Take 5-gram α-Naphthol in 100 ml of  95% ethyl alcohol and mix properly.
  2. Store at 4 to 8°C in the dark.
  3. Shelf life: 2 to 3 weeks.Reagent 2: 40% Potassium HydroxidePotassium hydroxide( KOH) pellets: 20 gramDistilled water: up to 100 ml

Dissolve 40 g of potassium hydroxide pellets in 100 ml of distilled water in a polyethylene bottle

Keep a bottle in a cool water bath during preparation.

Caution: KOH is hygroscopic and becomes caustic when moist. Weigh quickly in a tared beaker. Store away from acids. Avoid exposure to skin.

Requirements for  Voges Proskauer test

MR-VP broth

Test organism

Inoculating wire

Bunsen burner

Incubator

5%α- naphthol

40% KOH

Control strains

  1. Klebsiella pneumoniae ATCC 13883
  2. Escherichia coli ATCC 25922

Procedure of Voges Proskauer test

  1. Inoculate a colony of test organism in MR-VP broth and incubate at 35°C for 18 to 24 hours. Do not tighten caps. Note: Some organisms may produce acetylmethylcarbinol at room temperature and not 35°C e.g. Hafnia alvei, Yersinia, Listeria. In this case, inoculate another broth and incubate at room temperature.
  2.  If a 5-ml broth culture is used, aliquot 2.0 ml of broth into  13 by 100-mm test tube. Hold the remainder for possible reincubation.
  3. Add 6 drops of 5% α-naphthol, and mix well to aerate.
  4. Add 2 drops of 40% potassium hydroxide, and mix well to aerate.
  5. Observe for a pink-red color at the surface within 30 min. Shake the tube vigorously during the 30-min period. Note: If the result is negative, MR-VP broth can be incubated for up 48 h and the test repeated.

Result and Interpretation of Voges Proskauer test

  1. Voges Proskauer  test  positive: A pink-red color at the surface is a positive reaction
  2. Voges Proskauer test negative: A lack of a pink-red color is a negative reaction.

Control strains

  • Klebsiella pneumoniae ATCC 13883—VP positive (red)
  • Escherichia coli ATCC 25922—VP negative (no change)
  • A copper color should be considered negative. Rust color is a weak positive
  • Most members of the family Enterobacteriaceae give opposite MR and VP reactions; however, certain organisms, like H. alvei and Proteus mirabilis, may give both a positive MR reaction and a positive VP reaction (often delayed)
  • Streptococcus mitis group organisms are VP negative, whereas the other viridans group streptococci are VP positive, except Streptococcus vestibularis, which is a VP variable.
  • Listeria organisms are beta-hemolytic, gram-positive rods that are VP positive at 25°C, but this test is not a key test in the identification.

#4. Citrate Test#

Principle of Citrate Utilization Test

A citrate utilization test is used to determine the ability of an organism to utilize sodium citrate as its only carbon source and inorganic ammonium salts as its only nitrogen source. When the bacteria metabolize citrate, the ammonium salts are broken down to ammonia, which increases alkalinity turning the bromthymol blue indicator from green to blue.

Principle of Citrate Utilization Test
Principle of Citrate Utilization Test

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. Streak the slant back and forth with a light inoculum picked from the center of a well-isolated colony.
  2. Place cap loosely on the tube.
  3. Incubate aerobically at 35°C to 37°C for 18 to 24 hours.
  4. Observe a color change from green to blue along the slant.

Quality control

Quality Control strains used in citrate utilization test

Positive Control (PC)-Klebsiella pneumoniae ATCC 13883

Negative Control (NC)-Escherichia coli ATCC 25922

Observation

  • Positive: Growth on the medium with color change from green to intense blue.
  • Negative: Absence of growth and color change

Result and interpretation in citrate utilization test

  • Citrate utilization test positive:  The growth will be visible on the slant surface and the medium will be an intense blue. The alkaline carbonates and bicarbonates produced as by-products of citrate catabolism raise the pH of the medium to above 7.6, causing the bromothymol blue to change from the original green color to blue.
  • Citrate utilization test negative:  trace or no growth will be visible.  No color change will occur; the medium will remain the green color of the uninoculated agar.  Only bacteria that can utilize citrate as the sole carbon and energy source will be able to grow on the Simmons citrate medium, thus a citrate utilization negative test culture will be virtually indistinguishable from an uninoculated slant.
  • Uninoculated (UN): No growth and no color change; slant remains green
  • Negative control: No growth and no color change; slant remains green
  • Positive control: Growth with color change from green to intense blue along the slant
  • Test organism/bacteria: Positive i.e. growth with color change from green to intense blue along the slant as shown above figure.

 List of Bacteria which gives positive citrate utilization test

  1. Klebsiella pneumoniae
  2. Citrobacter freundii
  3. Enterobacter cloacae (a minority of strains gives negative results)
  4. Salmonella other than Typhi and Paratyphi A
  5. Serratia marcescens
  6. Proteus mirabilis(a minority of strains gives negative results)
  7. Providencia alcalifaeciens
  8. Vibrio vulnificus
  9. Euringella Americana
  10.  Achromobacter oxylosoxidans

 

Citrate Test: variable (different strains give different results)

  •  Proteus vulgaris
  • Vibrio cholerae
  • Vibrio parahaemolyticus

Citrate test: negative

  • Escherichia coli
  • Shigella species
  • SalmonellaTyphi
  •  SalmonellaParatyphi A
  • Morganella morganii
  • Yersinia enterocolitica
  • Edwardsiella tarda
  • Vibrio holisae

Keynotes on Citrate utilization Test

  1. Generally, incubate aerobically at 35°C to 37°C for 18 to 24 hours but if negative Some organisms may require up to 4 days of incubation due to their limited rate of growth on citrate medium.
  2. Other methods of citrate utilization test available are-IMViC the test kit method and API test kit method
  3.  As you know, Escherichia coli is citrate utilization test negative; although uncommon, natural E. coli variants that are citrate positive have been isolated.  Citrate-negative strains of E. aerogenes have also been found.
  4. Other media used for citrate utilization test are-

Koser’s Citrate Medium

  • It is a liquid medium lacking agar.
  • It does not contain any indicator.
  • A positive test is shown by the presence of turbidity in the medium.

Christen’s citrate medium

  • Same as Simmons citrate medium in which bromothymol blue indicator is replaced by phenol red.

Limitations of Citrate utilization test 

The limitations of this test are as follow:

  1. Luxuriant growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with less inoculum.
  2. Do not stab the slant, since the test requires an aerobic environment.
  3. Do not inoculate from the broth culture, due to carryover of media.
  4. Tests with equivocal results should be repeated.
  5. To avoid false-positive reactions, use a light inoculum to prevent the carryover of t substances from the previous medium.
  6. The reaction of this medium alone is not sufficient for identification at the species level.

Bibliography

  1. Cowan & Steel’s Manual for identification of Medical Bacteria. Editors: G.I. Barron & R.K. Felthani, 3rd ed 1993, Publisher Cambridge University Press.
  2. Bailey & Scott’s Diagnostic Microbiology. Editors: Bettey A. Forbes, Daniel F. Sahm & Alice S. Weissfeld, 12th ed 2007, Publisher Elsevier.
  3. Clinical Microbiology Procedure Handbook, Chief in editor H.D. Isenberg, Albert Einstein College of Medicine, New York, Publisher ASM (American Society for Microbiology), Washington DC.
  4. Colour Atlas and Textbook of Diagnostic Microbiology. Editors: Koneman E.W., Allen D.D., Dowell V.R. Jr, and Sommers H.M.
  5. Jawetz, Melnick and Adelberg’s Medical Microbiology. Editors: Geo. F. Brook, Janet S. Butel & Stephen A. Morse, 21st ed 1998, Publisher Appleton & Lance, Co Stamford Connecticut.
  6. Mackie and Mc Cartney Practical Medical Microbiology. Editors: J.G. Colle, A.G. Fraser, B.P. Marmion, A. Simmous, 4th ed, Publisher Churchill Living Stone, New York, Melborne, Sans Franscisco 1996.
  7. Textbook of Diagnostic Microbiology. Editors: Connie R. Mahon, Donald G. Lehman & George Manuselis, 3rd edition2007, Publisher Elsevier.
  8. Jean F. Mac Faddin Biochemical tests for Identification of Medical bacteria
  9. Monica Cheesbrough Distinct Laboratory Practice in Tropical Countries…2nd Edition
  10.  Textbook of practical Microbiology – Subhash Chandra Parija
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