Acetate Utilization Test- Principle, Procedure, Results, Uses

Interesting Science Videos

What is Acetate Utilization Test?

The acetate utilization test is one of the biochemical tests performed for the identification of aerobic organisms by evaluating their ability to utilize acetate present in the growth media.

  • The principle of the acetate utilization test is based on the ability of an organism to utilize acetate as a sole source of carbon.
  • This test is similar to the acetamide utilization test and other such tests as it helps in the identification of a particular group of organisms on the basis of their metabolic activities.
  • Acetate utilization test is usually performed to differentiate Shigella from other members of the Enterobacteriaceae family.
  • This test is particularly effective in the differentiation of Shigella from coli as the majority of E. coli can utilize acetate while most of the species of Shigella cannot utilize acetate.
  • The acetate utilization test is also one of the methods for the differentiation of the fermentative and oxidative group of organisms.
  • The utilization of acetate causes a change in the pH of the medium, which then results in the change of the color of the pH indicator used in the medium.
  • The acetate utilization test is similar to citrate utilization test or citrate test, except that the medium has acetate instead of citrate as the sole source of carbon.

Objectives of Acetate Utilization Test

  • To differentiate between Gram-negative rods that are oxidase negative, nonmotile, and anaerogenic, which are likely to be either E. coli or Shigella.
  • To differentiate lactose-non-fermenting, Gram-negative microorganisms from fermentative bacteria.

Principle of Acetate Utilization Test

  • Acetate agar is a media used in biochemical tests to determine the ability of an organism to utilize acetate. Organic acids like citrate and acetate have been widely used as a method for the differentiation of the members of the Enterobacteriaceae family.
  • Most of these bacteria are capable of utilizing organic acids in the presence of organic nitrogen.
  • The acetate media contains inorganic ammonium salts as the source of nitrogen along with acetate as a source of carbon.
  • The growth of the organism on the media is indicative of a positive test for acetate utilization. The metabolism of acetate by the bacteria is followed by the breaking down of the ammonium salts into ammonia.
  • The release of ammonia then increases the pH of the medium. The shift in pH turns the bromthymol blue indicator in the medium from green to blue.
  • This medium is recommended in differentiating Shigella from Escherichia coli.
  • Approximately 84% of E. coli strains utilize acetate, whereas the majority of Shigella and Proteus species are incapable of acetate utilization.

Media and Reagent used for of Acetate Utilization Test

1. Media Used

  • The media used in the acetate utilization test is the growth medium containing sodium acetate as the sole source of carbon.
  • The composition of the acetate agar medium is given below:
S.N Ingredients Gram/liter
1. Sodium chloride 5.0
2. Magnesium sulfate 0.1
3. Ammonium phosphate, monobasic 1.0
4. Potassium phosphate, dibasic 1.0
5. Sodium acetate 2.0
6. Bacteriological gar 20.0
7. Bromothymol blue 0.08
Final pH at 25°C: 6.7 ±0.2
Store at 2°C to 8°C.

Acetate agar is also available commercially and might have a different composition.

S.N Ingredients Gram/liter
1. Peptic digest of animal tissue 5.0
2. Malt extract 5.0
3. Yeast extract 5.0
4. Glucose 5.0
5. Polysorbate 80 (Tween 80) 10.0
6. Bacteriological Agar 20.0
7. Sodium acetate 27.220
Final pH at 25°C: 5.4 ±0.2

2. Supplies Used

  • Sterile inoculating loops or sticks.
  • Sterile pipettes
  • Incubator at 35°C
  • Sterile saline

Procedure of Acetate Utilization Test

1. Preparation of media

  • In a beaker, 69.1 grams of the dehydrated powder or lab-prepared media is added to 1000 milliliters of distilled or deionized water.
  • The suspension is then heated to boiling to dissolve the medium completely.
  • The dissolved medium is then dispensed into tubes and sterilized in an autoclave at 15 lbs pressure (121°C) for 15 minutes.
  • Once the autoclaving process is complete, the tubes are taken out and cooled at a slanted position to a temperature of about 40-45°C. The position should be maintained in order to obtain butts of 1.5 – 2.0 cm depth.

2. Utilization test

  • A well-isolated colony is taken from an 18-24 hour culture with a sterile inoculating needle.
  • Instead, a turbid saline suspension can be prepared by using an 18- to 24-h culture from a noninhibitory culture plate.
  • The acetamide agar tubes are inoculated by streaking the surface of the slant with either the light inoculum picked from the culture plate or with a drop of the saline suspension. The slant should be streaked back and forth with the loop or the inoculating stick.
  • The cap of the test tubes should be left loosened to ensure adequate aeration.
  • The tubes are then incubated aerobically at 35-37°C for up to 7 days. Incubation at 35-37 °C for up to 5 days insufficient for Enterobacteriaceae but incubation at 30°C for 7 days is recommended for nonfermenting, Gram-negative rods.
  • The test tubes should be examined daily for 4 days and again at 7 days before discarding the result as a negative.

Result Interpretation of Acetate Utilization Test

Acetate Utilization Test- Principle, Procedure and Result Interpretation

Figure: Acetate Utilization Test Results. Image Source: Bailey and Scott’s Diagnostic Microbiology. Elsevier.

  • A positive test is represented by growth and change from green to intense blue color along the slant.
  • A negative test is represented by no growth, no color change, with the slant remaining green.

Control bacteria

  • As a form of quality control for acetate utilization test, two different organisms can be taken as a positive and negative control.
Control Incubation Results
Escherichia coli Aerobic incubation for 24-48 hours at 33-37°C. Acetate positive (growth; intense blue color)
Shigella flexneri Aerobic incubation for 24-48 hours at 33-37°C. Acetate negative (no growth; green color)

Uses of Acetate Utilization Test

  • The acetate utilization test is used to test the ability of an organism to utilize acetate as a sole source of carbon.
  • It is also used as a qualitative test for the differentiation of Gram-negative bacteria into the fermentative and oxidative group of bacteria.
  • Acetate agar is also used as a selective medium for the isolation of E. coli.

Limitations of Acetate Utilization Test

  • Growth on the slant without an accompanying color change may indicate a positive test. However, if the agar does not turn blue on further incubation, the test should be repeated with less inoculum.
  • Tests with equivocal results should be repeated.
  • The slant should not be stabbed as the test requires an aerobic environment.
  • The inoculums should not be taken from broth cultures as there is a chance of carryover of media with broth cultures.
  • A light inoculum should be taken to prevent the carryover of substances from previous media.

References and Sources

  • Acetate agar. M1225. HiMedia Laboratories.
  • Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook,–
  • Bergman JM, Wrande M, Hughes D (2014) Acetate Availability and Utilization Supports the Growth of Mutant Sub-Populations on Aging Bacterial Colonies. PLOS ONE 9(10): e109255.
  • Trabulsi LR, Ewing WH. 1962. Sodium acetate medium for differentiation of Shigella and Escherichia cultures. Public Health Lab 20:137–140.
  • Bailey and Scott’s Diagnostic Microbiology. Elsevier.

About Author

Photo of author

Anupama Sapkota

Anupama Sapkota has a bachelor’s degree (B.Sc.) in Microbiology from St. Xavier's College, Kathmandu, Nepal. She is particularly interested in studies regarding antibiotic resistance with a focus on drug discovery.

Leave a Comment

This site uses Akismet to reduce spam. Learn how your comment data is processed.