MR-VP Broth TESTS:
Atlas pages MR=63 and VP=82
Methyl Red Broth (Atlas page 63) or Mixed Acid Fermentation TESTS:
The MR and VP tests are done with using the same test broth. Inoculation is with a loop. The methyl red (MR) and Voges-Proskauer (VP) tests are read from two inoculated tubes of MR-VP broth. One tube is used for the MR test; the other is used for the VP test. Methyl Red indicator is added (1/2 dropper-full) to the incubated tube and mixed. A red or pink tube is positive for mixed acid fermentation. The MR tube is read after 2-3 labs of incubation.
MR-VP media contains glucose and peptone. All enterics oxidize glucose for energy; however the end products vary depending on bacterial enzymes. Both the MR and VP tests are used to determine what end products result when the test organism degrades glucose. E. coli is one of the bacteria that produces acids, causing the pH to drop below 4.4. When the pH indicator methyl red is added to this acidic broth it will be cherry red (a positive MR test).
Klebsiella and Enterobacter produce more neutral products from glucose (e.g. ethyl alcohol, acetyl methyl carbinol). In this neutral pH the growth of the bacteria is not inhibited. The bacteria thus begin to attack the peptone in the broth, causing the pH to rise above 6.2. At this pH, methyl red indicator is a yellow color (a negative MR test).
CAUTIONS: take 2 tubes... label one MR and the other VP. Incubate MR 7 days and VP 14 days (emergency could be 10 days). Make sure you record the COLORS of the results as sometimes these are misread as being POSITIVE when YELLOW IS NEGATIVE ON THE MR TEST!!!
The VP, Voges-Proskauer, Acetoin, or 2,3 Butanediol TEST: Atlas page 82
The reagents used for the VP test are Barritt's A (alpha-napthol) and Barritt's B (potassium hydroxide). When these reagents are added to a broth in which acetyl methyl carbinol is present, they turn a pink-burgundy color (a positive VP test). This color may take 20 to 30 minutes to develop. E. coli does not produce acetyl methyl carbinol, but Enterobacter and Klebsiella do.
The MR and VP tests are done with using the same test broth. The methyl red (MR) and Voges-Proskauer (VP) tests are read from two loop inoculated tubes of MR-VP broth. One tube is used for the MR test; the other is used for the VP test. After no less than 4 labs of incubation a 1/2 dropper full of both Barritt's Reagents are added to the VP tube and the tube is mixed and read against white paper over a period of 2 DAYS. A red or pink tube is positive for ACETOIN production (the second to last step in producing 2,3 butanediol from pyruvate) - AFTER ADDING THE REAGENTS AND MIXING THE VP TEST, LEAVE YOUR TEST WITH THE INSTRUCTOR OVERNIGHT FOR LATER DETERMINATION!!!!
CAUTIONS: take 2 tubes... label one MR and the other VP. Incubate MR 7 days and VP 14 days (emergency could be 10 days). After incubating 14 days and adding the REAGENTS and mixing for 2 hours... you may have to let the VP test sit overnight...
The MR & VP TESTS are a part of the important IMViC which separates E. coli from similar microbes. I=INDOLE TEST; M=METHYL RED TEST; V=VOGES PROSKUER TEST; C=CITRATE TESTS.... or click on the IMViC TESTS link button below...
In the photos below, note the two photos of the Methyl Red Tests on the first row (each have a yellow negative and a pink/redish positive) & on the second row, the more difficult to read negative and slightly pinkish positive VP Tests.
|The VP TEST should be incubated longer and the results are less definitive as the pink layered positive color is diffuse...|
Click on the BLUE buttons below to follow that link...