Malonate Test: Principle, Procedure, and Results

Malonate is an ionized form of malonic acid CH2(COOH)2, and its ester and salts. The medium for the malonate test contains sodium malonate.

Malonate is a reversible inhibitor of succinate dehydrogenase. Due to its structural similarity with succinate, it competitively inhibits the utilization of succinic acid by bacteria, shutting down the Krebs (tricarboxylic acid cycle) and the glyoxylate cycle. Malonate test is recommended to differentiate among the Enterobacteriaceae family especially species of Klebsiella and Salmonella.

The attachment of malonate to the enzyme succinate dehydrogenase prevents the attachment of succinate and, thus, the conversion of succinate to fumarate.


The malonate utilization test is a single substrate utilization test containing sodium malonate as a single carbon source. Apart from sodium malonate, this test medium also contains a trace amount of glucose to stimulate the growth of microorganisms. Organisms that can ferment sodium malonate and utilize it as a carbon source grow in this medium. The medium also contains inorganic ammonium salts as the sole source of nitrogen.

The fermentation of sodium malonate results in sodium hydroxide and sodium bicarbonate, increasing the medium’s alkalinity. The increase in pH turns the bromthymol blue indicator from green to blue. Malonate-negative organisms that ferment glucose cause the indicator to turn slightly yellow or no color change at all.

Materials Required

  1. Sterile inoculating loops or sticks
  2. Incubator
  3. Test Organism: Enterobacteriaceae as part of the identification to the species level.
  4. Medium: Malonate test medium
IngredientsAmount (Gram/Liter)
Yeast extract*1.0 g
Ammonium sulfate2.0 g
Dipotassium phosphate0.6 g
Monopotassium phosphate0.4 g
Sodium chloride2.0 g
Sodium malonate3.0 g
Glucose*0.25 g
Bromothymol blue0.025 g
Final pH: 6.7 
* Yeast extract and glucose are needed to stimulate the growth of some Salmonella species but are generally not necessary for others.

Quality Control

Perform quality control on each new lot or media shipment before using it. Inspect broth for contamination before storage and use. Discard any blue tubes.

  1. Klebsiella pneumoniae ATCC 13883: Malonate positive (good growth, blue color)
  2. Escherichia coli ATCC 25992: Malonate negative (no growth, green color)


  1. Using a loop or stick, pick a light inoculum from the center of a well-isolated colony.

  2. Inoculate the malonate test tube.

    The turbidity should be less than a no. 0.5 Mc-Farland standard, i.e., no visible turbidity

  3. Incubate aerobically at 35 to 37°C for up to 48 hours.

    Do not read reactions before 48 hours.

  4. Observe a color change from green to blue along the slant.


Malonate test
Malonate broth inoculated with Klebsiella aerogenes (+ve), Escherichia coli (-ve), and an uninoculated control (Left to Right)
  1. A positive test is a growth and change to light blue or deep Prussian blue color throughout the medium.
  2. A negative test is no color change or change from green to yellow due to the fermentation of glucose.

Test Results

Malonate Utilization PositiveMalonate Negative
Klebsiella pneumoniaeKlebsiella ozaenae
Citrobacter koseriCitrobacter amalonaticus
 Salmonella enterica subsp. arizonae Shigella spp
 Hafnia alvei Escherichia coli
 Bordetella trematum Elizabethkingia anophelis

References and further readings

  1. Andrea J. Linscott, 2016.  Clinical Microbiology Procedures Handbook 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.
  2. Leboffe MJ and Pierce BE. A Photographic Atlas for the Microbiology Laboratory, 5th Edition (2021). Morton Publishing Company.

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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