Malonate Test: Result, Principle, Procedure, and Reagents

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Malonate Test

Malonate test is one of the colorimeter tests which detects the ability of the bacteria to utilize the malonate as the source of carbon, which is the end point for the production of the metabolites of the alkaline that helps in inducing the color change. The medium for the malonate test contains the sodium malonate, here malonate is one the enzyme inhibitor which helps in utilization of the succinic acid by the bacteria, thus shutting the process of the Krebs cycle and the glyoxylic cycles. Let us discuss briefly about this test here in detail.

Malonate Test Objective

 The main aim of the test is to test the ability of the organism present in the medium to make use of the malonate as a source of the carbon.

 Malonate test was initially found to originally designed to differentiate the several species of the bacteria from the Escherichia, which does not grow in the medium and the Enterobacter.

 Malonate test is used as the differential medium has broadened to include the other members of the family of Enterobacteriaceae.

Malonate Test Principle

An organism which has the ability to utilize the sodium malonate in the organisms utilizes malonate as its only as a source of carbon and ammonium sulfate as its only nitrogen source and produces an alkalinity as a result of sodium hydroxide formation, this results in the alkaline reaction which a medium containing a malonate which results in change of the color from original green or light blue color into bromothymol blue. Organism that which does not utilize the compounds of malonate and ammonium sulfate that does not ferment dextrose produce no color change. Organisms which are considered as malonate negative does not ferment dextrose no color change. Organisms which are malonate negate but do not ferment the dextrose and results in the development of a yellow color due to the increase acidity in the medium.

In an inoculum that is collected from a pure culture is transferred aseptically to a sterile tube of malonate broth. The inoculated tube is being incubated at a temperature for 24 hours and the results are being determined. Abundant change and growth in the medium are indicates a positive test for growth using a malonate. However, is a microbe can use malonate for carbon and energy it will grow on the malonate broth. Thus, use of the malonate leads to the rise in the pH of a medium and a pH indicator changes the color of the medium.

Malonate Test Reagents

The medium used in the malonate broth is based on the formula by the Leifson, which is in the liquid form containing an ammonium sulfate as the only source of the nitrogen and malonate as the only source of carbon. It also contains minerals salts, sodium citrate for carbon, and the ammonium phosphate for its source of nitrogen, whereas the malonate as the only source of carbon. It contains minerals salts, ammonium phosphate for its source of nitrogen, and sodium citrate as the source of carbon. Here the pH indicator used is bromothymol blue, which results in the formation of a green color at its neutral pH, and yellow at its acidic range in a pH 6.0 and it turns blue at a basic range in an alkaline medium containing a pH of 7.6.


Ammonium sulphate2.0
Dipotassium phosphate0.60
Monopotassium phosphate0.40
Sodium chloride2.0
Sodium malonate3.0
Bromothymol blue0.025
Malonate Test Procedure

 With a help of a light inoculum taken from an 18 to 24-hour pure culture is inoculated into the tube which contains malonate broth.

 Then the inoculated broth is incubated at a temperature of about 35ºC for about 24 to 48 hours. It should also be ensured that caps should be loosened before incubating for a good aeratiation.

 After incubation, the alkalization of medium is noted which results in formation of a blue color after 24 and 48 hours.

Malonate Test Results

Positive Malonate Test Result: In case of positive malonate test it is indicated by the development of a blue color in the medium.

Negative Malonate Test Result: In case of the negative result, a malonate test is usually indicated by change in color of the medium into green . Sometimes it results due to change in color of the yellow due to the fermentation of a dextrose.

Malonate Test Uses

 The malonate test was originally designed to differentiate the species of Escherichia and the Enterobacter.

 It is also being used as a differential medium which is now broadened and it is included along with the other members of the Enterobacteriaceae.

 Malonate reaction is usually used as a todiffe rentiate among the species of Enterobacteriaceae like Klebsiella pneumonia which is one of the positive strained species.

 This test is usually performed to separate the species of Salmonella arizone which is a positive species from the other species of the Salmonella, which is the negative species.

 This test also helps in differentiating the Enterobacteriaceae in the food and diary products.

Malonate Test Citations


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