DEMONSTRATION OF METHYL RED AND VOGES-PROSKAUER TEST

Results of the MR and VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae.
Most members of the family Enterobacteriaceae give either a positive MR test or a positive VP test.
Read the VP test at 48 hours. Increased incubation may produce acid conditions in the broth that will interfere with the readings of the results.

1. DEPARTMENT OF MICROBIOLOGY

2. DEMONSTRATION OF METHYL RED AND VOGES-PROSKAUER TEST

3. INDEX INDEX METHYL RED VOGES-PROSKAUER TEST BROTH PREPARATION MR-VP TEST PRINCIPLE PROCEDURE RESULT LIMITATIONS SAFETY TIP

4. METHYL RED METHYL RED Methyl Red (2-(N,N-Dimethyl-4-aminophenyl) azobenzenecarboxylic acid), also called C.I.Acid Red 2, is an indicator dye that turns red in acidic solution

5. VOGES-PROSKAUER TEST VP is a test used to detect acetoin in a bacterial broth culture. The test is performed by adding alpha-naphthol and potassium hydroxide to the VP broth which has been inoculated with bacteria.

6. Buffered peptone-glucose broth  (commercially available as MR-VP broth) Buffered peptone 7.0 g Dipotassium phosphate 5.0 g  Dextrose 5.0 g Add 1 liter of deionized water for a final pH of 6.9 (± 0.2) at 25°C.  Gently heat to completely dissolve the ingredients and distribute 5 ml aliquots into culture tubes. Sterilize via autoclaving at 121°C and 15 psi for 15 minutes. After the medium cools down it can be store in a refrigerator until use.  

7. This medium is a modification of the original Clark and Lubs medium using the decreased concentration of glucose (0.5%) as well as peptone. It was shown that this amount of glucose allowed for an identification of MR-positive organisms. The reduced amount of peptone results in a less-colored medium, making the interpretation of the color reaction easier.  

8. MR-VP Tests Methyl Red (MR) and Voges-Proskauer (VP) broth is used as a part of the IMViC tests as the medium in which both the Methyl Red and Voges-Proskauer tests can be performed. It is a simple broth that contains peptone, buffers, and dextrose or glucose.

9. IMViC I indicate INDOLE TEST M indicate METHYL RED TEST V indicate VOGES- PROAKAUER TEST i indicate IN C indicate CITRATE TEST

10. Principle MR-positive organisms produce high level of acid during fermentation of dextrose, overcome the phosphate buffer system and produce a red color upon the addition of the methyl red pH indicator. In the VP test, the red color produced by the addition of potassium hydroxide to cultures of certain microbial species is due to the ability of the organisms to produce a neutral end product, acetoin, from the fermentation of dextrose. The acetoin is oxidized in the presence of oxygen and alkali to produce a red color. This is a positive VP reaction. The Voges-Proskauer (VP) test has found wide acceptance in clinical laboratories as a means of classifying strains of Enterobacteriaceae, based on acetoin production.

11. Methyl red solution   Completely dissolve 0.1 g of methyl red in 300 ml of ethanol (95%). Add 200 ml of deionized water to make 500 ml of a 0.05% (wt/vol) solution in 60% (vol/vol) ethanol. Store the prepared methyl red solution at 4°C. 

12. Voges-Proskauer reagents   B arritt’s reagent A: 5% (wt/vol) alpha-naphthol in absolute ethanol  Barritt’s reagent B: 40% (wt/vol) KOH in deionized Water (this might be replaced by a 40% (wt/vol) NaOH solution) Reagents must be prepared fresh. Reagents are also referred to as VP-1 and VP-2 or VP-A and VP-B. 

13. Other materials sterile culture tubes containing 5 ml of MR -VP broth  - sterile culture tubes test and control bacterial strains(commonly used controls are   Escherichia coli  and  Enterobacter cloacae ) from trypticase soy agar or broth - inoculation loop (disposable or have additional equipment available for sterilization of the inoculation loop)  - transfer pipettes

14. PROCEDURE A. Inoculation of medium Prepare MR-VP broth as described above or as directed by the manufacturer. Prior to use, allow the medium to come to room temperature. Inoculate one tube of MR-VP broth from a fresh (18 to 24 hours) pure culture (e.g., grown on trypticase soy agar) of the test culture. Transfer a light inoculum from an isolated colony and resuspend it in the 5 ml MR-VP broth tube

15. B. Incubation   Incubate test and control cultures at 35°C (+/- 2°C) for 48 hours.

16. Methyl red test and results 1. Transfer 2.5 ml of culture into a new sterile culture tube. 2. Add 5 drops of the methyl red reagent.  3. Compare the test organism to the control cultures to immediately interpret the result, MR positive (e.g.,  E. coli ) or MR negative (e.g.,  E. cloacae )   Methyl red reaction.   E. coli  (A) and  E. cloacae  (B) were grown for 48 hours at 35°C in MR-VP medium. After incubation, 5 drops of a 0.05% methyl red solution (per 2.5 ml) were added to each culture. The MR-positive  E. coli  (A) shows a red coloration as a result of high acid production and a decrease in the pH of the culture medium to 4.4. The MR-negative  E. cloacae  culture (B) has a yellow color indicating a less acidic medium.

17.   Voges-Proskauer test and results   1.Use the remaining 2.5 ml of culture grown in MR-VP broth. 2. Add 0.6 ml (or 12 drops) of Barritt’s reagent A.  3. Add 0.2 ml (or 4 drops) of Barritt’s reagent B 4.Carefully shake the tube for 30 seconds to 1 minute to expose the medium to atmospheric oxygen (necessary for oxidation of acetoin to obtain a color reaction). 

18. 5. Allow the tube to stand for at least 30 minutes. 6. Within 1 hour, compare the test result to control cultures to determine if the culture is VP positive like E. cloacae or VP negative like  E. coli  . Delayed reading of the result may lead to an erroneous reading; over time alpha-naphthol and KOH may react to give a copper-like color.    E. cloacae  (A) and  E. coli  (B) were grown in MR-VP-broth for 48 hours at 37°C and Barritt’s reagents A and B were added.  VP-positive  E. cloacae  (A) shows red coloration on top of the culture, whereas VP-negative  E. coli  (B) has a yellowish color. 

19. Limitations Results of the MR and VP tests need to be used in conjunction with other biochemical tests to differentiate genus and species within the Enterobacteriaceae. Most members of the family Enterobacteriaceae give either a positive MR test or a positive VP test. Read the VP test at 48 hours. Increased incubation may produce acid conditions in the broth that will interfere with the readings of the results.

20. VP reagents must be added in the order and the amounts specified or a weak-positive or false-negative reaction may occur. A weak-positive reaction may be masked by a copper-like color which may form due to the reaction of KOH and alpha-naphthol. Read the VP test within 1 hour of adding the reagents. The KOH and alpha-naphthol may react to form a copper-like color, causing a potential false-positive interpretation. Due to the possible presence of acetoin, diacetyl or related substances in certain raw materials, the use of media low in these substances (such as MR-VP media) is recommended for this test.

21. SOME OTHER VP TEST ORGANISM VP POSITIVE/NEGATIVE Klebsiella pneumoniae Positive Proteus mirabilis Negative Enterobacter aerogenes Positive

22. SAFETY Tying long hair back, wearing personal protective equipment(eye protection, coats, gloves, closed shoes; glasses may be preferred to contact lenses) Always using appropriate pipetting device and understanding that mouth pipetting is forbidden Never eating or drinking in the lab. Never apply cosmetics, handling contact lenses or placing objects (fingers, pencils, etc.) in the mouth or touching the face.

23. TIP Use small screw cap tubes for the MR-VP assay to decrease leakage during shaking.

24. GUIDED BY GUIDED BY DR. NEELAM RAWAT JYOTI SHARMA

25. PRESENTEDB BY PRESENTED BY ANAMIKA

26. THANK YOU ANY QUESTION ?