The MRVP test is a standard microbiological procedure used to differentiate types of bacteria based on their unique metabolic pathways for fermenting glucose. This assessment combines two biochemical tests—the Methyl Red (MR) test and the Voges-Proskauer (VP) test—performed simultaneously in the same nutrient-rich medium, MRVP broth. The broth contains glucose and a phosphate buffer. The test classifies bacteria, particularly those in the Enterobacteriaceae family, by examining the stable end products of their glucose metabolism. It determines whether an organism produces large quantities of stable acids or converts those acids into neutral compounds, providing clues for identification in clinical and environmental laboratories.
The Methyl Red Pathway and Its Detection
The Methyl Red (MR) test detects organisms that perform mixed acid fermentation of glucose. Bacteria using this pathway produce significant and stable quantities of strong organic acids, including lactic, acetic, succinic, and formic acids.
The MRVP broth contains a buffer, dipotassium phosphate, which resists pH changes. A positive MR organism produces such a high concentration of stable acids that they overwhelm the buffer. This acid production results in a sustained drop in the culture’s pH to 4.4 or lower.
The test is performed by adding the pH indicator Methyl Red to the incubated culture. Methyl Red changes color based on acidity. When the medium’s pH is 4.4 or below, the indicator turns a visible, stable red color, signifying a positive result.
A negative MR result is indicated by the medium remaining yellow, which occurs at a pH of 5.8 or higher. This yellow color suggests the organism either did not ferment glucose or metabolized the initial acids further. Organisms that produce less stable acids often quickly convert them into neutral products, preventing the pH from dropping low enough for the red color change.
This ability to maintain an extremely acidic environment is a specific metabolic trait. It is a direct consequence of the specific enzymes bacteria possess for processing pyruvic acid, the intermediate product of glucose metabolism.
The Voges-Proskauer Pathway and Its Detection
The Voges-Proskauer (VP) test identifies bacteria utilizing butanediol fermentation. These organisms initially produce pyruvic acid but quickly convert it into neutral end products. The primary neutral compound is 2,3-butanediol, formed from the precursor molecule acetoin.
Because the organisms rapidly convert acidic products into neutral compounds, the medium’s acidity does not decrease substantially. The culture’s pH remains relatively high, usually around 6.0 or above. This higher pH explains why VP-positive organisms are typically negative for the MR test.
The VP test targets the intermediate compound, acetoin (acetylmethylcarbinol), rather than the final product, 2,3-butanediol. The test requires adding two chemical reagents: alpha-naphthol and potassium hydroxide (KOH), often called Barritt’s reagents. Potassium hydroxide oxidizes any acetoin present in the medium to diacetyl.
Alpha-naphthol acts as a color intensifier. The diacetyl then reacts with guanidine-containing compounds in the broth’s peptone. This chemical reaction produces a visible pink-red color at the surface of the medium, indicating a positive VP result. Since the reaction requires oxygen, the color change first appears at the liquid’s surface.
Using MRVP Results for Bacterial Identification
The MRVP test differentiates organisms based on the divergence of their glucose fermentation pathways. Since most bacteria exhibit only one pathway, the results are typically reciprocal: one test is positive while the other is negative. This inverse relationship provides a clear metabolic signature for identification.
Organisms producing stable acid concentrations result in a positive MR test and a negative VP test (MR+/VP-). These bacteria follow the mixed acid fermentation pathway. Examples include Escherichia coli, a common intestinal inhabitant, and many species of Salmonella.
Conversely, organisms that convert initial acid production into neutral products yield a negative MR test and a positive VP test (MR-/VP+). These bacteria utilize the butanediol fermentation pathway. Examples exhibiting this pattern include Klebsiella pneumoniae and many species within the genus Enterobacter.
This separation of metabolic types is valuable for classifying Gram-negative rods within the Enterobacteriaceae family. This group contains many human pathogens and environmental indicators that are morphologically similar. The MRVP test is one of four components in the larger IMViC battery of tests, which is a rapid method for narrowing down the identity of an unknown bacterial isolate.
By demonstrating a specific metabolic capability, the MRVP results significantly reduce the number of potential species. This allows microbiologists to quickly move toward a definitive identification. This rapid differentiation between species, such as E. coli and Klebsiella, is an important step in patient care and public health monitoring.