Malonate Test: Introduction, Composition of Medium, Principle, Procedure, Uses, Result-Interpretation, and Limitations

Introduction of Malonate Test

Malonate test uses to check the ability of the organism to utilize malonate as the sole source of carbon for energy. To differentiate bacteria on the basis of a malonate utilization test. It is a colorful test to check the ability of organisms for using as the sole source of carbon as malonate. The endpoint of  which is  the  production of alkaline metabolites that create a color change. This test is recommended as part of differentiating among the Enterobacteriaceae, especially species of Klebsiella and Salmonella.

Composition of Medium

IngredientsGm per litre
Yeast extract        1.0
ammonium sulfate2.0
Dipotassium phosphate0.6
Monopotassium phosphate0.4
Sodium chloride2.0
Sodium malonate   3.0
Glucose   0.25
Bromthymol blue   0.025
Deionized or distilled water1000 ml
Final pH6.7
Table: Malonate Test Medium
  • Store the medium at 2 to 8°C.

Principle of Malonate Test

The sodium malonate present in the medium. Malonate is an enzyme inhibitor and inhibits the utilization of succinic acid by organisms, shutting down the Krebs and glyoxylic cycles. Growth is indicative of malonate utilization as a carbon source; a small amount of glucose is also present to enhance the growth of some organisms. The presence of Inorganic ammonium salts in a medium acts as the sole source of nitrogen. When the bacterium ferments sodium malonate, sodium hydroxide and sodium bicarbonate are formed, which increases the alkalinity of the medium. The shift in pH turns the bromthymol blue indicator in the medium from green to blue. Malonate-negative organisms that ferment glucose cause the indicator to turn yellow due to acid.

Requirements for Test

  1. Malonate Test Medium
  2. Sterile inoculating loops or sticks
  3. Test organism
  4. Incubator
  5. Control Organisms
  • Positive control (PC): Klebsiella pneumoniae ATCC 13883
  • Negative Control (NC): Escherichia coli ATCC 25922

Procedure of Malonate Test

  1. Using a loop inoculate tube with a light inoculum picked from the center of a well-isolated colony (turbidity <0.5 McFarland standard, i.e. no visible turbidity).
  2. Incubate aerobically at 35 to 37°C for up to 2 days.
  3. Observe a color change from green to blue.


  1. Klebsiella ozaenae is malonate negative; Klebsiella  pneumoniae is malonate positive.
  2. Citrobacter amalonaticus is malonate negative; Citrobacter koseri is malonate positive.
  3. This test uses as part of the identification of other Enterobacteriaceae.

Result and Interpretation

Malonate test positive: Development of a blue color

Malonate Test Positive and Negative bacteria
Fig. Malonate Test Positive and Negative bacteria

Malonate test negative:  green or turning yellow due to dextrose fermentation

Klebsiella pneumoniae ATCC 13883: malonate positive (blue color)

Escherichia coli ATCC 25922: malonate negative (green color)

List of Malonate Test Positive and Negative Bacteria

Positive BacteriaNegative Bacteria
EnterobacterEscherichia coli
Klebsiella pneumoniaeKlebsiella ozaenae
Salmonella arizonaeSalmonella species
Citrobacter koseriCitrobacter amalonaticus
Table: Malonate Test Positive and Negative Bacteria

Limitations of Test

  1. Some reactions are slight and therefore compare to an uninoculated tube when reading.
  2. Do not read reactions before 48 hours.
  3. Yeast extract and glucose are needed to stimulate the growth of some Salmonella, organisms but are not generally necessary for other species.

Further Readings

  1. Cowan and Steel’s, manual for the identification of medical bacteria
  2. Lynne S. Garcia, Clinical Microbiology Procedures Handbook

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