Malonate broth foundation, preparation and uses

4477
Egbert Haynes

The malonate broth It is the liquid culture medium used for the diagnostic test (malonate test), used to differentiate some genera of the Enterobacteriaceae family. It was created by Leifson in 1933 and later modified by Ewing, who added a small amount of dextrose and yeast extract to the original formula..

The medium is currently composed of yeast extract, ammonium sulfate, dipotassium phosphate, monopotassium phosphate, sodium chloride, sodium malonate, dextrose, and bromothymol blue. This test is generally included in the biochemical identification battery for Enterobacteriaceae, helping to differentiate certain genera and species..

Graphic representation of the malonate test. A. Negative test, B. (Acidification) Negative test, C. (Medium alkalization) Positive test. Source: Color tubes publicdomainpictures.net

The malonate test is mainly based on the ability of some microorganisms to use sodium malonate as their sole carbon source and ammonium sulfate as their nitrogen source..

The malonate test is usually positive in some species of the Enterobacter, Klebsiella, and Citrobacter genera. Whereas, most of the species of the genera Escherichia, Salmonella, Shigella, Edwardsiella, Yersinia, Serratia, Morganella, Proteus and Providencia, give negative reaction.

Article index

  • 1 Rationale
    • 1.1 Interpretation
  • 2 Preparation
    • 2.1 Malonate broth
    • 2.2 Phenylalanine Malonate Broth
  • 3 Use
    • 3.1 Procedure
  • 4 Quality control
  • 5 Limitations
  • 6 References

Basis

The malonate test consists of showing those bacteria that are capable of using sodium malonate as the sole carbon source and ammonium sulfate as a nitrogen source..

Most of the Enterobacteriaceae that do not use malonate are able to grow in this medium, taking dextrose and yeast extract as nutrients..

In this case, any attempt to alkalinize by the use of peptones will be counteracted by the production of acids generated by the fermentation of dextrose. Likewise, dipotassium and monopotassium phosphates act as a buffer, maintaining the pH at 6.7.

That is why, when the test is negative, the broth remains the same original color (green). On rare occasions the medium may become acidic due to dextrose fermentation; without the use of the peptones and the pH indicator it would turn the color of the medium towards yellow. For this to happen, the pH must drop to 6.

Now, when this test is positive, it is said that the microorganism used malonate and ammonium sulfate as carbon and nitrogen sources respectively, without making use of the other components..

In this case, the medium becomes alkaline due to the release of sodium and the consequent formation of NaOH. In this sense, the pH indicator (bromothymol blue) turns the color of the medium from green to blue when the pH is equal to or greater than 7.6. Blue can be light or intense (Prussian blue).

Finally, sodium chloride maintains the osmolarity of the medium and water is the diluent of all the components..

Interpretation

Same Color Broth (Green) - Negative Test

Yellow broth: negative test

Light or deep blue broth: test positive

There is a variant called phenylalanine malonate broth, also called Shaw and Clarke's medium. In this case, two tests can be analyzed, the use of malonate as a carbon source and the production of pyruvic acid from phenylalanine..

Preparation

Malonate broth

The number of grams specified by the insert of the chosen commercial company is weighed (it may vary from one to another). The weighed grams are suspended in one liter of distilled water. Heat slightly until completely dissolved. Distribute 3 ml of the medium into 13/100 test tubes with cotton caps.

Sterilize in autoclave at 121 ° C for 15 to 20 minutes.

Cool before using. If they are not going to be used immediately, store in the refrigerator until use. Bring broths to room temperature before inoculating.

The pH of the medium should be 6.7 ± 0.2. The color of the prepared medium is bottle green.

Phenylalanine Malonate Broth

Weigh out 11 g of the dehydrated medium and dissolve in 1 liter of distilled water. The rest of the preparation equal to that described above.

It can also be prepared by adding 2 g / L of phenylalanine to the malonate broth medium before it is sterilized..

Use

It is used as part of the battery of biochemical tests that are assembled for the identification of bacteria of the Enterobacteriaceae family.

Helps distinguish between:

-The genus Klebsiella and Enterobacter (+) of the genus Escherichia and Serratia (-).

-The species of Salmonella enterica ssp arizonae, Salmonella enterica ssp salami and Salmonella enterica ssp diarizonae (+), of the species Salmonella enterica ssp enterica (-).

-Of the genus Klebsiella usually (+) of the genus Actinobacillus (-).

-It can sometimes help to differentiate genera and species of bacteria not belonging to the Enterobacteriaceae family, such as among non-fermenting Gram negative rods. Alcaligenes faecalis (+) and Acinetobacter sp (-).

Source: Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana S.A. Argentina.

Process

Under a lighter, a portion of a pure cologne is taken, using a properly sterilized and cooled platinum handle. The sample taken (light inoculum) is dissolved in the malonate broth. Incubate with the lid loose in aerobiosis at 35 ° C ± 0.2 for 24 to 48 hours.

Malonate broth can also be inoculated from an 18-24 hour culture in trypticase soy broth. In this case, 0.01 ml is taken with a sterile pipette and the malonate broth is inoculated. Incubate with the lid loose in aerobiosis at 35 ° C ± 0.2 for 24 to 48 hours.

At the end of the time, the results are interpreted. Any trace of blue color after 48 hours of incubation should be considered positive. The test should not be interpreted as negative until the 48-hour incubation time has elapsed..

In the case of using the phenylalanine malonate broth variant, the malonate is first interpreted and then 5 drops of 1N HCl and 3-5 drops of 8% ferric chloride are added. A dark green color is interpreted as a positive test for phenylalanine. If, on the contrary, the medium turns pale blue, the test is negative for phenylalanine..

QA

To perform the sterility control of the medium, one or two broths should be incubated at 35 ° C ± 0.2 for 24 hours of incubation. After this time there should be no cloudiness or color change.

Known or certified strains can be used for quality control, such as: Enterobacter aerogenes ATCC 13048, Klebsiella pneumoniae ATCC 33945, Salmonella enterica ssp arizonae ATCC 13314 and Escherichia coli ATCC 25922.

The expected results are:

  • Enterobacter aerogenes, Klebsiella pneumoniae Y Salmonella enterica ssp arizonae give positive reaction (half blue color).
  • In order to Escherichia coli the result should be negative, that is, it is expected that there will be no color change (green) or that it will turn yellow due to the fermentation of glucose.

Limitations

Do not use broth that show turbidity, precipitates, color change or any sign of deterioration.

References

  1. Pedraza J, Sanandres N, Varela Z, Aguirre E, Camacho J. Microbiological isolation of Salmonella spp. and molecular tools for its detection. Uninorte Health. Barranquilla (Col.) 2014; 30 (1): 73-94. Available at: scielo.org.co
  2. BBL. Malonate Broth, Ewing modified. 2007. Available at: bd.com
  3. Senna Laboratories. Malonato broth. Available at: cientificasenna.com
  4. RenyLab. Malonato broth. 2013.Available at: es.renylab.ind.br
  5. Mbiolog Diagnostics. Malonato broth. Available at: mbiolog.com
  6. Koneman E, Allen S, Janda W, Schreckenberger P, Winn W. (2004). Microbiological Diagnosis. 5th ed. Editorial Panamericana S.A. Argentina.
  7. Conda Pronadisa Laboratories. Phenylalanine malonate broth. Available at: condalab.com

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