Malonate Test – Principle, Procedure, Results, Uses

The Malonate Test is a biochemical test which is used for the identification and differentiation of bacteria belonging to Enterobacteriaceae. It is mainly employed to distinguish genera such as Enterobacter and Klebsiella from Escherichia coli. It is the process by which the ability of a microorganism to utilize sodium malonate as the sole source of carbon and energy is determined. In this test ammonium sulfate acts as the only source of nitrogen for the organism.

The principle of Malonate Test is based on the structural similarity between malonate and succinate. Malonate acts as a competitive inhibitor of succinate dehydrogenase enzyme of Krebs cycle. Due to this inhibition the normal energy production is blocked. Malonate-negative organisms are not able to overcome this block and hence shows poor or no growth. Malonate-positive organisms possess specific enzyme system such as malonate decarboxylase which helps in converting malonate into acetate and carbon dioxide.

The test is carried out using Malonate broth medium which contains bromothymol blue as pH indicator. The medium is green in colour initially. A small amount of dextrose is present to support initial growth. If the organism is unable to utilize malonate it ferments dextrose producing acid which turns the medium yellow. If the organism utilizes malonate alkaline products are formed which increase the pH of medium and change the colour from green to deep blue. The appearance of blue colour indicates a positive Malonate Test while yellow colour indicates a negative result.

Objectives of Malonate Test

• To determine whether an organism is able to utilize sodium malonate as the sole source of carbon and energy.
• To test the ability of bacteria to use ammonium sulfate as the only source of nitrogen during growth.
• To help in differentiation of members of family Enterobacteriaceae based on malonate utilization.
• To differentiate Escherichia coli which is malonate negative from Enterobacter species which is malonate positive.
• To identify certain Salmonella subspecies such as Salmonella enterica subsp. arizonae which shows positive malonate reaction.
• To differentiate between different species of Klebsiella on the basis of malonate metabolism.
• To assist in identification of Enterobacteriaceae isolated from food and dairy products.

Principle of Malonate Test

The principle of Malonate Test is based on the ability of certain bacteria to utilize sodium malonate as the sole source of carbon and energy and ammonium sulfate as the only source of nitrogen. Malonate is structurally similar to succinate and acts as a competitive inhibitor of succinate dehydrogenase enzyme of Krebs (TCA) cycle. Due to this inhibition the normal respiratory pathway is blocked and energy production is affected. Malonate-negative organisms are not able to overcome this metabolic block and hence they show no significant growth in the medium.

Malonate-positive organisms possess specific enzyme system such as malonate decarboxylase which helps in bypassing this inhibition. In this process malonate is converted into acetate and carbon dioxide and alkaline products such as sodium hydroxide is formed. The accumulation of alkaline products increases the pH of the medium. Bromothymol blue which is used as pH indicator changes its colour from green to deep blue indicating a positive test. In malonate-negative organisms the trace amount of glucose present in the medium may be fermented producing acidic products which turns the medium yellow or remains green due to lack of growth.

Requirements for Malonate Test

• Malonate broth (Ewing modified) containing sodium malonate as carbon source and ammonium sulfate as nitrogen source.
• Bromothymol blue is used as pH indicator in the medium.
• Trace amount of dextrose is present to support initial growth of organism.
• Fresh pure culture of test organism preferably 18–24 hours old.
• Light inoculum should be used to avoid carryover of nutrients.
• Sterile inoculating loop or needle for transfer of culture.
• Clean test tubes containing malonate broth with loosely fitted caps.
• Incubator maintained at temperature of 35–37°C.
• Aerobic condition is required during incubation.
• Incubation period of 24–48 hours for observation of results.
• Known malonate positive organism as positive control (e.g. Klebsiella).
• Known malonate negative organism as negative control (e.g. Escherichia coli).

Malonate Broth Composition and Preparation

Composition (per litre)

• Sodium malonate – 3.0 g
• Ammonium sulfate – 2.0 g
• Sodium chloride – 2.0 g
• Yeast extract – 1.0 g
• Dipotassium phosphate – 0.6 g
• Monopotassium phosphate – 0.4 g
• Dextrose (glucose) – 0.25 g
• Bromothymol blue – 0.025 g
• Final pH – 6.7 ± 0.2 at 25°C

Preparation

1. About 9.3 g of dehydrated malonate medium is weighed and suspended in 1000 ml of distilled water.
2. The medium is heated with gentle agitation to dissolve completely.
3. The prepared medium is dispensed into clean test tubes or suitable containers.
4. Sterilization is done by autoclaving at 121°C (15 lbs pressure) for 15 minutes.
5. After autoclaving the medium is allowed to cool to room temperature.
6. Care should be taken to avoid contamination or addition of any extra carbon or nitrogen source during preparation.

Storage

• The dehydrated medium should be stored in a tightly closed container below 30°C.
• The prepared malonate broth should be stored at 2–8°C and protected from light until use.

Malonate Test Procedure

1. A fresh pure culture of the test organism (18–24 hours old) is selected.
2. A light inoculum is taken from a well isolated colony using sterile inoculating loop or stick.
3. The organism is aseptically inoculated into tube containing sterile Malonate broth.
4. The cap of the tube is kept loosened to maintain aerobic condition.
5. The inoculated tubes are incubated at 35–37°C.
6. The tubes are observed after 18–24 hours for any colour change.
7. If no definite colour change is observed the tubes are further incubated up to 48 hours.
8. The final result is noted where change of green colour to deep blue indicates positive test and green or yellow colour indicates negative test.

Result Interpretation of Malonate Test

Positive result– In a positive Malonate Test the colour of medium changes from green to light blue or deep Prussian blue. This indicates that the organism is able to utilize sodium malonate as the sole source of carbon and ammonium sulfate as the source of nitrogen. During utilization of malonate alkaline end products such as sodium hydroxide and sodium bicarbonate is formed which increases the pH of medium above 7.6. Due to rise in pH the bromothymol blue indicator turns blue. Organisms such as Klebsiella pneumoniae Enterobacter aerogenes and Salmonella enterica subsp. arizonae shows positive reaction.

Negative result– In a negative Malonate Test the medium remains green or turns yellow. This indicates that the organism is unable to utilize malonate for growth. Yellow colour is due to fermentation of trace amount of dextrose present in medium resulting in acid production which lowers the pH below 6.0. If no growth or metabolic activity occurs the medium remains green. Organisms such as Escherichia coli Salmonella typhi and Shigella species gives negative reaction.

Doubtful or weak reaction– In some cases the medium may appear blue green in colour. Such tubes should be compared with an uninoculated control. Any trace of blue colour observed after 48 hours of incubation is considered as positive Malonate Test.

List of organisms showing Malonate Test reaction

Malonate positive organisms

• Buttiauxella agrestis
• Cedecea davisae
• Citrobacter koseri (C. diversus)
• Enterobacter aerogenes
• Enterobacter cloacae
• Enterobacter gergoviae
• Hafnia alvei
• Klebsiella oxytoca
• Klebsiella pneumoniae subsp. pneumoniae
• Klebsiella pneumoniae subsp. rhinoscleromatis
• Pantoea agglomerans
• Providencia rettgeri
• Salmonella enterica subsp. arizonae
• Salmonella enterica subsp. diarizonae

Malonate negative organisms

• Bordetella trematum
• Cedecea lapagei
• Citrobacter amalonaticus
• Citrobacter freundii
• Elizabethkingia anophelis
• Escherichia coli
• Klebsiella pneumoniae subsp. ozaenae
• Morganella morganii
• Proteus mirabilis
• Proteus vulgaris
• Providencia alcalifaciens
• Salmonella enterica subsp. enterica
• Salmonella Typhi
• Salmonella Typhimurium
• Serratia marcescens
• Shigella species
• Yersinia enterocolitica

Quality Control of Malonate Test

• The prepared malonate broth should be clear bluish green in colour and free from any precipitate.
• Any tube showing blue colour before inoculation should be discarded as it indicates contamination or instability of medium.
• The dehydrated malonate medium should be free flowing and light yellow to light green in colour.
• The final pH of the prepared medium should be around 6.7 ± 0.2 at 25°C.
• Quality control should be performed for each new batch or lot of malonate medium.
• A known malonate positive organism such as Klebsiella pneumoniae or Enterobacter aerogenes should be used as positive control.
• A known malonate negative organism such as Escherichia coli or Salmonella Typhimurium should be used as negative control.
• An uninoculated malonate broth tube should be incubated along with test tubes for colour comparison.

Precautions of Malonate Test

• A light inoculum should be used to avoid carryover of nutrients from the primary medium.
• Heavy inoculum should be avoided as it may give false positive result.
• The test tubes should be incubated with loosened caps to maintain aerobic condition.
• The result should not be interpreted before 18–24 hours and negative result should be confirmed after 48 hours.
• Weak or doubtful colour change should be compared with an uninoculated control tube.
• Any malonate broth showing blue colour before inoculation should be discarded.
• Fresh and pure culture of 18–24 hours should be used for the test.
• Care should be taken to avoid contamination or introduction of other carbon or nitrogen sources during inoculation.

Uses of Malonate Test

• It is used to differentiate Escherichia coli from Enterobacter species on the basis of malonate utilization.
• It helps in differentiation of Salmonella enterica subspecies arizonae and diarizonae from other Salmonella subspecies.
• It is useful in distinguishing Klebsiella pneumoniae from Klebsiella ozaenae.
• It helps in differentiation of Citrobacter koseri from Citrobacter freundii and Citrobacter amalonaticus.
• It is used as a part of biochemical tests for identification of Enterobacteriaceae in clinical and non clinical samples.
• It is used in analysis and differentiation of Enterobacteriaceae isolated from food and dairy products.
• It is used to determine the ability of organism to utilize sodium malonate as sole source of carbon and energy.

Advantages of Malonate Test

• It is useful for clear differentiation between Enterobacter species and Escherichia coli.
• It helps in identification of certain Salmonella subspecies such as Salmonella enterica subsp. arizonae and diarizonae.
• It aids in differentiation of Klebsiella pneumoniae from Klebsiella ozaenae.
• It is helpful in distinguishing Citrobacter koseri from Citrobacter freundii and Citrobacter amalonaticus.
• It provides specific information about the metabolic ability of organism to utilize malonate.
• It gives distinct and easily interpretable colour change from green to deep blue in positive reaction.

Limitations of Malonate Test

• Heavy inoculum may give false positive result due to carryover of nutrients from primary medium.
• Early reading of test may give false negative result as initial acid production occurs due to dextrose fermentation.
• The test requires aerobic condition and tight caps may interfere with proper utilisation of malonate.
• Trapping of carbon dioxide may neutralize alkaline products and prevent colour change.
• Some organisms show weak blue green colour which makes interpretation difficult.
• Bromothymol blue indicator may become unstable on prolonged storage or contamination.
• Some fastidious strains may require longer incubation period for clear result.
• The test cannot be used alone and should be performed along with other biochemical tests.
• A pure and fresh culture is required and direct clinical specimens cannot be used.

FAQ

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.