MRVP MEDIUM

Code: CM0043

A medium recommended for the Methyl-red and Voges-Proskauer tests for the differentiation of the coli-aerogenes group.

Typical Formula*	gm/litre
Peptone	7.0
Glucose	5.0
Phosphate buffer	5.0
pH 6.9 ± 0.2

* Adjusted as required to meet performance standards

Directions
Add 17g to 1 litre of distilled water. Mix well, distribute into final containers and sterilise by autoclaving at 121°C for 15 minutes.

Description
This glucose-phosphate medium is recommended for the Methyl-red and Voges-Proskauer tests, for the differentiation of the coli-aerogenes group¹.

Smith² noted the low acid production of Enterobacter aerogenes cultures as compared with those of Escherichia coli. Clark & Lubs ³ employed methyl-red as a hydrogen-ion concentration indicator in order to differentiate glucose phosphate peptone water cultures of members of the coli-typhoid group. This test, now known as the Methyl-red test, distinguishes those organisms able to form large amounts of acid from glucose so that the pH falls below 4.4 and those organisms which cannot produce a low pH level.

The difference in pH value is visualised by adding methyl-red to the culture, (< pH 4.4 red: pH 5.0-5.8 orange: > pH 6.0 yellow).

Methyl-red reaction

Colour	Organism
Orange to red (MR positive)	Escherichia coli, Citrobacter spp. and others
Orange to yellow (MR negative)	Enterobacter spp., Klebsiella pneumoniae and others

Voges & Proskauer ⁴ described a red fluorescent coloration which appeared after the addition of potassium hydroxide to cultures of certain organisms in glucose medium. The coloration was shown to be due to the oxidation of the acetylmethyl- carbinol producing diacetyl which reacts with the peptone of the medium to give a red colour^5,6. Durham⁷ noted that Enterobacter aerogenes gave a positive reaction but that Escherichia coli produce no coloration, and it later became clear that there was a negative correlation between the Methyl-red and Voges-Proskauer tests^8,9 for lactose-fermenting coliform organisms.

Voges-Proskauer reaction

Red (Positive)	Enterobacter spp. and others
No colour (Negative)	Escherichia coli and others

Technique
Inoculate a 10ml tube of MRVP Medium with two loopfuls of a pure, 4-6 hours old, Peptone Water CM0009 culture of the organism under test.
Incubate not less than 48 hours at 35°C for the MR test but more usually 3-5 days at 30°C. A heavy inoculum and 18-24 hours incubation at 35°C may give a rapid result ¹⁰. A rapid VP test may be carried out from a heavy inoculum and incubation in a water bath at 35°C for 4-5 hours. Some organisms (Hafnia alvei) require incubation at 25°C to give a positive VP test.

After incubation, test one portion of the broth with 5 drops of 0.4% w/v methyl-red solution and read the colour on the surface of the medium immediately.

The second portion of the broth is used for the VP reaction by one of the following methods:
1. Add 3ml of 5% w/v alcoholic a-naphthol solution and 3 ml of 40% w/v KOH solution (Barritt’s method¹²).
2. Add a trace amount of creatine (2 drops of a 0.3% w/v solution) and 5ml of 40% KOH solution (O’Meara’s method¹³).
A bright pink or eosin red colour will appear after gentle shaking for 30 seconds. A pink colour is positive; no colour is negative.
Barry & Feeney ¹⁴ obtained rapid results by adding creatine to Barritt’s reagents.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared medium at 2-6°C.

Appearance
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured solution