Principle of Acetate utilization test:
- Acetate agar is employed to test an organism’s ability to utilize acetate. The medium consists of sodium acetate as the sole carbon source and inorganic ammonium salts as the sole source of nitrogen. Growth of organisms suggests the positive test for acetate utilization. During the metabolism of acetate by the bacteria , the ammonium salts are broken down to ammonia, which elevates alkalinity. The shift in pH turns the bromothymol blue indicator in the medium from green to blue. This medium is usually used for differentiating Shigella spp from Escherichia coli. Shigella spp are not able to metabolize actetate whereas approximately 94% Escherichia coli utilize acetate.
Requirements:
- Media: Composition of Sodium acetate agar
- Ingredients gm/l
- sodium chloride …………………………………….5.0 g
- magnesium sulfate ………………………………..0.1 g
- ammonium phosphate, monobasic …….1.0 g
- potassium phosphate, dibasic …………….1.0 g
- sodium acetate …………………………………….2.0 g
- agar ………………………..………………………….20.0 g
- bromothymol blue ……………………………. 0.08g
- Sterile inoculating loops or sticks
- Sterile pipette
- Incubator at 35°C
- Sterile saline
Procedure of Acetate utilization test:
- Using an 18- to 24-h culture from a non-inhibitory culture plate, prepare a turbid saline suspension.
- Inoculate the slant with 1 drop of the suspension.
- Alternatively, streak the slant back and forth with a light inoculum picked from the centre of a well-isolated colony.
- Place cap loosely on tube.
- Incubate aerobically at 35 to 37°C for up to 5 days for Enterobacteriaceae; incubate at 30C for nonfermenting, gram-negative rods for up to 7 days.
- Observe a colour change from green to blue along the slant.
Results interpretations:
- Positive test:
- It is indicated by the growth of organisms and conversion of colour from green to intense blue along the slant.
- Negative test:
- No colour change indicates no growth and is suggestive of negative test.
Limitations:
- Stab on slant should be avoided as the test requires aerobic environment.
- Inoculation from broth cultures is avoided due to carry over of media.
- Light inoculum is preferred to avoid false positive results.