Malonate Test Principle, Procedure, Results

Sourav Bio
  • The purpose of the malonate test is to see if the organism can get all of its energy from malonate.
  • To tell the difference between bacteria based on how well they use malonate.
  • It is a colourful test to see how well organisms can use malonate as their only source of carbon.
  • The result is the production of alkaline metabolites, which cause a change in colour.
  • This test is often used to tell the difference between different types of Enterobacteriaceae, especially Klebsiella and Salmonella species.
  • In 1933, Leifson came up with a way to tell Escherichia coli from Enterobacter spp. using a liquid medium.
  • Ammonium sulphate was the only source of nitrogen in this medium. Malonate was the only source of carbon, and bromothymol blue showed the pH.
  • Enterobacter grew and turned the green light into a blue one (alkalinization).
  • E. coli did not grow well, and the indicator did not change. Ewing et al. changed Leifson’s recipe by adding a small amount of dextrose and yeast extract to help some organisms grow that wouldn’t have grown otherwise and to see how they react to malonate.
  • Some subgroups of Salmonella can be told apart from other Enterobacteriaceae by using Malonate Broth.

Malonate Test Purpose

  • To tell the difference between different Enterobacteriaceae based on how they use sodium malonate.
  • To tell the difference between Enterobacter and Escherichia based on how they use malonate.
  • To see if the organism can live off of malonate as its only source of carbon.

Malonate Test Principle

  • The medium has sodium malonate in it. Malonate is an enzyme inhibitor that stops organisms from using succinic acid. This stops the Krebs and glyoxylic cycles from running.
  • When sodium malonate is used as a carbon source and ammonium sulphate is used as a nitrogen source, sodium hydroxide is made. This makes the environment more alkaline.
  • Inorganic ammonium salts are the only way nitrogen can get into a medium.
  • When the bacteria ferment the sodium malonate, they make sodium hydroxide and sodium bicarbonate. This makes the medium more alkaline.
  • The bromothymol blue indicator in the medium goes from green to blue when the pH changes.
  • Organisms that don’t have malonate and ferment glucose make the indicator turn yellow because they make acid.
  • When an organism that can’t use these substances is in the medium, the colour of the medium doesn’t change. Also, some malonate-positive organisms don’t make much alkalinity, which makes it hard to figure out what the results mean. So, these tubes should be compared to a malonate tube that hasn’t been seeded.

Requirements for Malonate Test

  • Sterile inoculating loops or sticks
  • Incubator
  • Malonate Broth

Malonate Broth Composition

The medium used is malonate broth. It is composed of mineral salts, sodium citrate as its carbon source, and ammonium phosphate as its nitrogen source. The indicator for pH is brom thymol blue, which is green at neutral pH, yellow at acidic pH 6.0, and blue at alkaline (basic) pH >7.4.

Sodium Malonate 3.0gm
Ammonium Sulfate 2.0gm
Sodium Chloride 2.0gm
Yeast Extract 1.0gm
Dipotassium Phosphate 0.6gm
Monopotassium Phosphate 0.4gm
Dextrose 0.25gm
Bromothymol Blue 0.025gm

Final pH 6.7 +/- 0.2 at 25ºC.

Malonate Broth Preparation

  1. Dissolve 8.02 grams in 1000 ml distilled water.
  2. Autoclave at 15 pounds of pressure (121°C) for 15 minutes to dispense and clean.
  3. Don’t put carbon and nitrogen from other places into the soil.

Malonate Test Procedure

Inoculation of Medium

  1. Go with the malonate broth setting.
  2. Get the Bunsen burner going.
  3. The inoculating loop should be chosen.
  4. In order to prevent contamination, you should flame your inoculating loop.
  5. Don’t forget to take the lids off your test tubes.
  6. Set your test tube openings on fire.
  7. A sample of the unknown bacterium should be taken from its culture tube using the sterile inoculating tool.
  8. The inoculum must be inoculated into the new, sterile medium immediately.
  9. Again, set fire to the openings of your pipes.
  10. Please re-cap the test tubes.
  11. Bring the inoculation back to a flame.

Incubation of the Inoculated Medium

  1. Place the inoculated tube into the 35-37 C incubator.  
  2. By pressing the “New Day” button, you can skip ahead 24 hours. 

Determination of Test Results

  1. Let it sit for the right amount of time. For this test, it only takes 24 hours to find out the result.
  2. Get the culture you want out of the incubator.
  3. Observe test result. If the test was done the way it was said above, the culture will have turned blue when alkali or bases were present (indicating a positive test). Almost never grows in negative tests.
  4. Record test result.
  5. Throw away the culture.

Result Interpretation of Malonate Test

  • Positive test: A blue colour appears in the medium.
  • Negative test: The medium stays green or turns yellow due to the fermentation of dextrose. 
Malonate Test
Malonate Test
Malonate Positive Malonate Negative
Klebsiella pneumoniae Klebsiella ozaenae
Citrobacter koseri Citrobacter amalonaticus
 Salmonella enterica subsp. arizonae  Shigella spp
 Hafnia alvei  Escherichia coli
 Bordetella trematum  Elizabethkingia anophelis

Quality control

The following quality control organisms were used to test all lot numbers of Malonate Broth (Ewing Modification) and found to be good. Control organisms should be tested using quality control methods that have already been set up in the lab. If there are problems with quality control, patient results shouldn’t be sent out.

  • Positive control (PC): Klebsiella pneumoniae ATCC 13883
  • Negative Control (NC): Escherichia coli ATCC 25922

Malonate Test Uses

  • Klebsiella ozaenae is malonate negative; Klebsiella pneumoniae is malonate positive.
  • Citrobacter amalonaticus doesn’t like malonate, but Citrobacter koseri does.
  • This test is used to find out what other Enterobacteriaceae are.

Malonate Test Limitation

  • A few malonate-positive organisms create only a trace amount of alkalinity. To confirm weakly positive results, compare the contents of the test isolate tube with an uninoculated tube of Malonate Broth. Any trace of blue hue after 48 hours of incubation indicates a positive result.
  • Do not discard the test prior to 48 hours of incubation.
  • Some malonate-negative bacteria create a yellow tint when dextrose is fermented.
  • Yeast extract and glucose are required to boost the growth of certain Salmonella organisms but are not often required for the growth of other species.


What is the purpose of the test? 

The objective is to determine whether or not the microorganism can use malonate as its sole source of carbon and energy for growth.

How is malonate use determined? 

If a bacterium can obtain carbon and energy from malonate, it will flourish in malonate broth. The addition of malonate raises the pH of the medium, causing the pH indicator to change colour.

What medium is used?  

The medium used is malonate broth. It is composed of mineral salts, sodium citrate as its carbon source, and ammonium phosphate as its nitrogen source. The indicator for pH is brom thymol blue, which is green at neutral pH, yellow at acidic pH 6.0, and blue at alkaline (basic) pH >7.4.

How is the test performed?  

An inoculum is transferred aseptically from a pure culture to a sterile tube of malonate broth. After incubating the infected tube at 35-37 degrees Celsius for 24 hours, the results are determined. A positive malonate growth test is indicated by abundant growth and a change from green to blue in the medium.


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