Cat. no. K36 Malonate Broth, 15x103mm Tube, 5ml 20 or 100 tubes/box


Hardy Diagnostics Malonate Broth is recommended for use in the differentiation of Enterobacteriaceae on the basis of malonate utilization.


Malonate Broth was developed by Leifson in 1933.(7) Leifson's medium allowed for the differentiation of Enterobacter from Escherichia spp. based on malonate utilization. The medium contained ammonium sulfate as the sole nitrogen source, malonate as the sole carbon source, phosphates as buffers, and bromothymol blue as the pH indicator.

Hardy Diagnostics Malonate Broth follows a modified formula developed by Edward and Ewing in 1962.(8,9) The medium contains the same basal medium as Leifson's Malonate Broth with the addition of dextrose and yeast extract. Yeast extract serves as a source of vitamins while dextrose provides carbohydrates and a minimal amount of carbon. Incorporation of both ingredients initiate growth of organisms incapable of utilizing malonate or ammonium salt.

Organisms which simultaneously utilize malonate and ammonium sulfate produce sodium hydroxide which thereby results in an alkaline reaction and changes the indicator from its original green color to light blue or Prussian blue. (4) Organisms which cannot utilize malonate and ammonium sulfate and do not ferment dextrose produce no color change. Organisms which are malonate-negative but do ferment dextrose result in the development of a yellow color due to increased acidity in the medium.


Ingredients per liter of deionized water.*

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.

*Adjusted and/or supplemented as required to meet performance criteria.


Storage: Upon receipt store at 2-30ºC. away from direct light. Media should not be used if there are any signs of deterioration, discoloration, contamination, or if the expiration date has passed. Product is light and temperature sensitive; protect from light, excessive heat, moisture, and freezing.



Specimen Collection: This medium is not intended for primary isolation of patient specimens. It should be used only with cultures of an isolated organism. This product is used in conjunction with other biochemical tests to identify cultures of an isolated organism.

Method of Use:

1. Using a light inoculum from an 18-24 hour pure culture, inoculate the tube.

2. Incubate the tube with loosened caps at 35ºC. in an aerobic atmosphere for 24-48 hours.

3. Observe for alkalization (blue color) at 24 and 48 hours.


A positive malonate test is indicated by the development of a blue color in the medium.

A negative malonate test is indicated by the media remaining green or turning yellow due to dextrose fermentation.


The test organisms must be in pure culture and 18-24 hours old.

Some malonate-positive organisms produce only a slight alkalinity which renders difficulty in interpretation. Questionable results should be compared with an uninoculated malonate tube. Any trace of blue color is indicative of a positive test after 48 hours of incubation. Negative results should not be reported until a full 48 hour incubation period.(4)

Some malonate-negative strains ferment glucose only and produce a yellow color in the medium.


Standard microbiological supplies and equipment such as loops, other culture media, swabs, applicator sticks, incinerators, and incubators, etc., as well as serological and biochemical reagents, are not provided.


Test Organisms
Inoculation Method*
Escherichia coli
ATCC ® 25922
A 24hr 35°C Aerobic Growth, broth remains green
Klebsiella pneumoniae
ATCC ® 13883
A 24hr 35°C Aerobic Growth, broth turns deep blue



Malonate Broth should appear clear, and bluish-green in color.

E. coli growing in Malonate Broth

Escherichia coli (ATCC® 25922) growing in Malonate Broth (Cat. no. K36). Incubated aerobically for 24 hours at 35ºC.

K. pneumoniae growing in Malonate Broth

Klebsiella pneumoniae (ATCC® 13883) growing in Malonate Broth (Cat. no. K36). Incubated aerobically for 24 hours at 35ºC.

Malonate Broth

Uninoculated tube of Malonate Broth (Cat. no. K36).


1. Anderson, N.L., et al. Cumitech 3B; Quality Systems in the Clinical Microbiology Laboratory, Coordinating ed., A.S. Weissfeld. American Society for Microbiology, Washington, D.C.

2. Tille, P., et al. Bailey and Scott's Diagnostic Microbiology, C.V. Mosby Company, St. Louis, MO.

3. Isenberg, H.D. Clinical Microbiology Procedures Handbook, Vol. I, II & III. American Society for Microbiology, Washington, D.C.

4. MacFaddin, J.F. 1985. Media for Isolation, Cultivation, Identification, Maintenance of Bacteria, Vol. I. Williams & Wilkins, Baltimore, MD.

5. Jorgensen., et al. Manual of Clinical Microbiology, American Society for Microbiology, Washington, D.C.

6. Quality Assurance for Commercially Prepared Microbiological Culture Media, M22. Clinical and Laboratory Standards Institute (CLSI - formerly NCCLS), Wayne, PA.

7. Leifson. 1933. J. Bacteriol. ; 26:329-330.

8. Ewing, et al. 1957. Public Health Lab; 15:153-167.

9. Davis, et al. 1957. Int. Bull. Bact. Nomencl. Taxon.; 7:151-157.

ATCC is a registered trademark of the American Type Culture Collection.