BacterTube™ Methyl Red-Voges Proskauer (MR-VP)

Test medium used for the identification of members of the Enterobacteriaceae family
Code: 08033
Packaging: 50 tubes/ box or as per customer request.
Storage Conditions: 2–8°C.
Transportation: Ambient temperature.
Shelf Life: 6 months from the date of manufacture.

INTENDED USE

BacterTube™ Methyl Red-Voges Proskauer (MR-VP) is recommended for use in the performance of the Voges-Proskauer and Methyl Red tests as an aid in the identification of enteric gram-negative bacilli.

PRINCIPLES

Voges-Proskauer (VP) Test
- Certain bacteria produce neutral-reacting end products when cultivated in specific media. Particular enteric bacteria that ferment glucose, further metabolize pyruvic acid to form acetyl-methyl carbinol (acetoin). This end product, in the presence of atmospheric oxygen and 40% potassium hydroxide is converted to diacetyl. Diacetyl, under the catalytic action of alpha-naphthol and creatine, is converted into a red complex. This is a positive Voges – Proskauer (VP) test reaction. The VP test is used primarily to separate Escherichia coli (VP – negative) from the Klebsiella – Enterobacter groups (VP – positive)
Methyl Red (MR) Test
- The Methyl Red (MR) test is based on the use of methyl red, a pH indicator, to detect acidity when an organism ferments glucose. Because all Enterobacteriaceae ferment glucose, acidic metabolic byproducts are initially formed. However, with further incubation (2 – 5 days), MR-positive organisms continue to produce more acids. The increased acidity overcomes the phosphate buffer, thus resulting in a low pH and development of a red color.
- MR – negative organisms, such as Klebsiella pneumoniae and Enterobacter aerogenes, further metabolize the fermentation products by decarboxylation. The result is an alkaline reaction (no red color) due to the production of acetoin which neutralizes the pH and results in no red color development.

Prior to inoculation, allow medium to equilibrate to room temperature
Using organisms taken from an 18 – 24 hour pure culture, lightly inoculate the medium
Incubate aerobically at 35ºC for 24 hours.
Following 24 hours of incubation, aliquot 1ml of the broth to a clean test tube.
Reincubate the remaining broth for an additional 24 hours.

Voges – Proskauer Test
- Add 0,6mL of 5% alpha-naphthol to the aliquot. Next, add 0,2mL of 40% KOH.
- Gently shake the tube to expose the medium to atmospheric oxygen.
- Allow the tube to remain undisturbed for 10 – 15 minutes.
- Observe the medium for a pink – red color development. The test may be read for up to, but not beyond , one hour following addition of the reagents.
- Note: If test reactions are negative (no red color produced) or questionable, the test can be repeated using the reincubated broth (without reagents. Reincubation and repeat testing can be performed for up to 5 days.
Methyl Red Test
- Following 48 hours of incubation, aliquot 2,5mL of the broth to a clean test tube.
- Add five drops of Methyl Red reagent
- Observe the medium for the immediate development of a red color

RESULTS

Voges – Proskauer Test
- A positive VP test is demonstrated by the development of a pink-red color on the surface of the medium 15 minutes to one hour after the addition of the reagents.
- A negative VP test is demonstrated by the appearance of a yellow color on the surface of the medium. Development of a copper – like color is also interpreted as negative.
Methyl Red Test
- A positive MR test is demonstrated by the development of a stable red color on the surface of the medium after the addition of methyl red indicator.
- A negative MR test is demonstrated by the development of a yellow color on the surface of the medium

BacterLab ensures the quality of each product batch by testing with ATCC reference strains.

Reference strains	Incubation conditions	Expected results
Escherichia coli ATCC 25922	18 – 24 hours, 35 – 37^oC	Growth: MR positive (red); VP negative (yellow)
Enterobacter cloacae ATCC 23355	18 – 24 hours, 35 – 37^oC	Growth: MR negative (yellow); VP positive (red)