Malonate Test- Objectives, Principle, Procedure and Results

The malonate test is a biochemical test that can be used to identify bacteria based on their ability to use malonate as a sole source of carbon.

The objectives of the malonate test are:

• To test the ability of the organism to utilize malonate as a sole source of carbon
• To differentiate between Escherichia and Enterobacter

The malonate test is based on the ability of some bacteria to use sodium malonate as a sole source of carbon and energy.

The principle of the malonate test involves:

• Utilization of sodium malonate and ammonium sulfate by the bacteria
• Production of sodium hydroxide as a by-product
• Increase in the pH of the medium, turning it blue

The medium used for the malonate test is malonate broth.

The malonate broth contains:

• Sodium malonate as the sole source of carbon
• Ammonium sulfate as the sole source of nitrogen
• Yeast extract and glucose to stimulate the growth of some Salmonella organisms
• Bromothymol blue as the pH indicator
• Distilled water to dissolve and dilute the other ingredients

The procedure of the malonate test involves the following steps:

1. Obtain a tube containing malonate broth
2. Inoculate the tube with a small amount of 18-24 hours pure culture of the organism
3. Loosen the cap of the tube and incubate it at 35ºC for 24-48 hours
4. Observe the color change in the medium after incubation

The result of the malonate test is based on the color change of the bromothymol blue indicator in the malonate broth.

The interpretation of the result is as follows:

• A positive test is indicated by the development of a blue color in the medium
• A negative test is indicated by the media remaining green or turning yellow

The malonate test has some limitations:

• The test organisms must be in pure culture and 18-24 hours old
• Some malonate-positive organisms produce only a slight alkalinity, which renders difficulty in interpretation
• Yeast extract and glucose are needed to stimulate the growth of some Salmonella organisms, making the test unreliable for differentiating Salmonella from other enteric bacteria
• Some organisms may produce acid from other sources in the medium, which may mask the alkaline reaction of malonate utilization
• Some organisms may produce pigments that interfere with the color change of the indicator

Some examples of bacteria that can be differentiated by the malonate test are:

• Escherichia coli is malonate negative
• Enterobacter aerogenes is malonate positive
• Klebsiella pneumoniae is malonate positive, while Klebsiella ozaenae is malonate negative
• Citrobacter koseri is malonate positive, while Citrobacter amalonaticus is malonate negative
• Salmonella typhimurium is malonate negative, while Salmonella enteritidis is malonate positive

The malonate test can also be used as part of the identification of other members of Enterobacteriaceae.

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