Acetate differential Agar principle and interpretation of test

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Acetate utilization test MLTGEEKS

Trabulsi and Ewing first formulated acetate Differential Agar. Tatum, Ewing and Weaver later on modified the medium by replacing sodium citrate by sodium acetate, which enables the differentiation of Shigella species from Escherichia coli. Organic acids have been used widely as an aid in the differentiation of Enterobacteriaceae, usually in formulae that contained organic nitrogen sources.

The basal mineral medium is alkalinized by bacteria, which utilize acetate as sole carbon source shifting the pH of the medium toward the alkaline range resulting in a change in color of the indicator from green to blue.

This test is use to differentiate Shigella species from Escherichia coli and non-fermentative gram-negative bacilli.

Principle of Acetate Differential Agar

  • The differentiation of groups is base on the ability or failure of inoculated microorganism to utilize acetate in a medium devoid of trace organic nitrogen.
  • This medium contains sodium acetate as the sole source of carbon. Trabulsi and Ewing demonstrated that Shigella and Proteus species are unable to utilize acetate and therefore fails to grow.
  • Majority of Escherichia coli and closely related organisms grow well within 24-48 hours but some strains grow very slowly and a few strains are unable to utilize acetate as a sole carbon source.
  • Acetate utilization is indicated by formation of blue color, which is due to the utilization of sodium acetate and subsequent formation of an alkaline reaction detected by the presence of bromothymol blue indicator.
  • Some strains of Escherichia coli utilize acetate slowly or not at all and therefore may produce a false negative reaction. Sodium acetate is utilized as a sole source of carbon by some serobiotypes of flexneri such as Shigella flexneri 4a
  • Magnesium sulphate is an essential ion. Sodium chloride maintains osmotic equilibrium and phosphates act as buffers.
Also read  Brain infusion Broth and Agar Principle and composition and growth response

Composition of acetate differential agar

Sodium acetate2.000
Magnesium sulphate0.100
Sodium chloride5.000
Monoammonium phosphate1.000
Dipotassium phosphate1.000
Bromothymol blue0.080
Final pH ( at 25°C)6.7+/-0.2

Preparation of acetate differential agar

  • Suspend 29.18 grams in 1000 ml distilled water.
  • Heat to boiling to dissolve the medium completely.
  • Distribute in tubes in sufficient amounts to give butt and slant.
  • Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.
  • Allow the tubes to cool in a slanted position.

Appearance of Medium after preparation

Emerald green colored clear to slightly opalescent gel forms in tubes as slants

acetate differential agar slant
acetate differential agar slant

Inoculation of Inoculum on Acetate Differential agar slant

  • Emulsify a portion of a colony from a pure, 18-24 hour culture in 1.0 ml of sterile physiological saline (0.85%).
  • A broth suspension should not be used due to carryover of peptones (a carbon source).
  • Using a sterile inoculating loop, transfer a loopful of the suspension to the surface of the Acetate Differential Agar slant.
  • Incubate aerobically at 33-37°C for at least 7 days with intermittent observation.
  • Observe for a color change from green to blue along the slant.

Interpretation of test after inoculation and Incubation

Cultural Response characteristics after inoculation and Incubation at 25-30°C for upto 1-7 days.

A (Positive) - B(negative)
A (Positive) – B(negative)
MicroorganismAcetate utilization
Citrobacter freundiipositive reaction, blue color
Enterobacter cloacaepositive reaction, blue color
Escherichia colipositive reaction, blue color
Klebsiella pneumoniaepositive reaction, blue color
Proteus vulgarisInhibited (No growth)
Salmonella Arizonaepositive reaction, blue color
Salmonella Typhinegative reaction green color
Shigella sonneinegative reaction, no change, medium remains green
Also read  MacConkey Agar Principles, Composition, Preparation, colony characteristics

Limitation of Acetate Differential agar

  • This test is usually performed when other biochemical reactions fail to differentiate coli from Shigella.
  • Some strains of E. coli do not use acetate as a carbon source.
  • A heavy inoculum may cause false-positive results.


  • Trabulsi and Ewing, 1962, Public Health Lab., 20:137.
  • Tatum H. W., Ewing W. H., and Weaver R. E., 1974, Manual of Clinical Microbiology, , 2nd Ed., American Society for Microbiology, Washington D.C. Pg.-270
  • Ewing, 1986, Edwards and Ewings Identification of Enterobacteriaceae , 4th Ed. Elsevier Science Publishing Co., Inc., New York
  • Talukder K. A, Islam M. A., Dutta D.K., Hasan F., Sada A., Nair G. B . and Sack D. A., 2002, J. Clin. Microbiol., 40:2490

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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