Acetate Utilization Test: Introduction, Composition, Principle, Test Requirements, Test Procedure, Result and Interpretation, and Keynotes

Introduction of Acetate Utilization Test

Acetate Utilization Test ueses to determine the ability of an organism to use acetate as the sole source of carbon same as in the citrate utilization test for Enterobacteriaceae. It is the recommended assay for the differentiation of Shigella species from Escherichia coli.

Composition of the Medium

IngredientsGms / Litre
Sodium acetate2.0
Magnesium sulfate:0.1
Sodium chloride5.0
Monoammonium phosphate:1.0
Dipotassium hydrogen phosphate1.0
Bromothymol blue0.08
Final pH 6.7±0.2
Table: Composition of Acetate Agar

 Principle of Acetate Utilization Test

Breakdown of the sodium acetate causes the pH of the medium to shift toward the alkaline range, turning the indicator, bromothymol blue from green to blue.

Test Requirements Acetate Utilization Test

  1. Acetate agar slant
  2. Cotton plug
  3. Sterile Inoculating wire/ sticks
  4. Test organism
  5. Bunsen burner
  6. Incubator
  7. Test tube rack
  8. Control strains

Test Procedure of Acetate Utilization Test

  1. Streak the slant back and forth with a light inoculum picked from the Center of a well-isolated colony.
  2. Place cap loosely on the tube.
  3. The tube is then incubated aerobically at 35-37°C for up to 7 days. Incubation at 35-37 °C for up to 5 days is insufficient for Enterobacteriaceae but incubation at 30°C for 7 days is recommended for non-fermenting, Gram-negative bacilli/ rods.
  4. The test tube should be examined daily for 4 days and again at 7 days before discarding the result as a negative.
  5. Observe a color change from green to blue along the slant.

Quality Control(QC)

Quality Control strains used in citrate utilization test

Positive Control (PC)-Escherichia coli

Negative Control (NC)-Shigella flexneri

Result and Interpretation of Acetate Utilization Test

Positive – Medium becomes alkalinized (blue) because of the growth of the organism

Negative – no growth or no indicator change to blue

Acetate Utilization Test-Negative (left) and Positive (right side)
Fig. Acetate Utilization Test-Negative (left) and Positive (right side)

Limitations of Acetate Utilization Test

  • Slant growth without an accompanying color change may indicate a positive test. However, if the test medium does not turn blue on further incubation, the test should be repeated with a light inoculum.
  • The equivocal test results should be repeated.
  • The slant stabbing should be omitted since the test requires an aerobic environment.
  • Broth culture inoculum should be avoided since there is a chance of the carryover of media with broth culture.
  • A light inoculum is preferred to prevent the carryover of substances from previous media.

Keynotes on Acetate Utilization Test

  1. It uses to identify the ability of an organism to utilize acetate as a sole source of carbon.
  2. It is also used as a qualitative assay for the differentiation of Gram-negative bacteria into the fermentative and oxidative group of bacteria.
  3. Acetate agar is also applied as a selective medium for the isolation of Escherichia coli.
  4. Approximately 84% of Escherichia coli strains utilize acetate, whereas the majority of Shigella and Proteus species can not utilize acetate.
  5. Composition of Acetate differential agar

Further Readings

  2. Biochemical Tests for the Identification of Aerobic Bacteria. (2016). Clinical Microbiology Procedures Handbook,– 10.1128/9781555818814.ch3.17.1
  3. 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.
  4. Trabulsi LR, Ewing WH. 1962. Sodium acetate medium for differentiation of Shigella and Escherichia cultures. Public Health Lab 20:137–140.
  5. Bailey and Scott’s Diagnostic Microbiology. Elsevier.
  6. Jean F. Mac Faddin Biochemical tests for Identification of Medical bacteria

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