Acetate Utilization Test – Principle, Procedure, Results

Test Name	Acetate Utilization Test
Detection	Differentiate species based on ability to utilise acetate as the sole source of carbon. Typically used to distinguish Shigella spp. from Escherichia coli.
Test organism	It is best to use an acetate utilisation test to tell Shigella spp. from Escherichia coli. Test isolates are Gram-negative rods that don’t ferment lactose, aren’t motile and don’t grow in air. They are likely either E. coli or Shigella.
Result	Positive – As a result of the organism’s development, the medium turns alkaline (blue). Negative – no growth or no indicator change to blue
Required Media	Sodium acetate agar slants
Quality Control	E. coli ATCC 25922—acetate positive (growth; blue color) Shigella flexneri ATCC 12022—acetate negative (no growth or trace of growth)

• Similar to the citrate utilization test for Enterobacteriaceae, the Acetate Utilization Test is used to examine an organism’s capacity to utilize acetate as its only source of carbon and is recommended for distinguishing Shigella species from Escherichia coli.
• The organism is inoculated with agar containing sodium acetate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source. The presence of growth indicates a positive acetate consumption test.
• When bacteria metabolize acetate, ammonium salts are converted to ammonia, which raises the pH of the medium and causes the bromothymol blue indicator to change from green to blue.
• The ability of an organism to use acetate as a sole supply of carbon is the basis of the acetate utilization test.
• This test is equivalent to the acetamide utilization test and other tests of a similar nature.
• Since the majority of E. coli are E. coli, this test is particularly beneficial for separating Shigella from E. coli. coli strains are able to utilize acetate, but the majority of Shigella species cannot.
• The acetate utilization test is one method for distinguishing between the fermentative and oxidative groups of organisms.
• The use of acetate alters the pH of the medium, resulting in a change in the colour of the pH indicator used in the media.

Purpose of Acetate Utilization Test

• Differentiate species based on ability to utilise acetate as the sole source of carbon. Typically used to distinguish Shigella spp. from Escherichia coli.

Principle of Acetate Utilization Test

Acetate agar is a biochemical test medium used to determine an organism’s acetate utilisation capability. Long ago, organic acids such as citrate and acetate were utilised to differentiate between members of the Enterobacteriaceae family. The majority of these bacteria can utilise organic acids when organic nitrogen is present.

The acetate medium comprises ammonium ions as a source of nitrogen and acetate as a source of carbon. A positive acetate utilisation test is shown by the growth of the organism on the media. The breakdown of ammonium ions into ammonia follows the breakdown of acetate by bacteria.

The pH of the medium rises as a result of the emission of ammonia. As the pH changes, the bromothymol blue indicator in the medium changes colour from green to blue. This medium is suggested for discriminating Shigella from E. coli. E. uses acetate in around 84% of its products. coli strains, but not by the majority of Shigella and Proteus species.

Test organism

It is best to use an acetate utilisation test to tell Shigella spp. from Escherichia coli. Test isolates are Gram-negative rods that don’t ferment lactose, aren’t motile and don’t grow in air. They are likely either E. coli or Shigella.

Materials required

• Sodium acetate agar slants
• Sterile inoculating loops, Sterile pipette, Sterile saline

Sodium acetate agar Composition

Ingredients	Gram/liter
Sodium chloride	5.0
Magnesium sulfate	0.1
Ammonium phosphate, monobasic	1.0
Potassium phosphate, dibasic	1.0
Sodium acetate	2.0
Bacteriological gar	20.0
Bromothymol blue	0.08

Final pH at 25°C: 6.7 ±0.2

Sodium acetate agar Preparation

1. Pour 1000 ml of purified or distilled water over 61.9 grammes of dehydrated medium. This is the weight of dehydrated medium per litre.
2. Bring to a boil to completely dissolve the medium.
3. Use an autoclave for 15 minutes at 15 pounds of pressure (121°C) and 121°F.
4. Bring the temperature down to 45-50°C.
5. Mix well and pour into a clean test tube or Petri plate.

Quality Control

Before you use a new batch of media, you should check its quality. Before putting media away or using it, check for signs of contamination, cracks, dehydration, and bubbles. Discard any blue tubes. Do the testing of how well the test organisms work:

• E. coli ATCC 25922—acetate positive (growth; blue color)
• Shigella flexneri ATCC 12022—acetate negative (no growth or trace of growth)

Procedure of Acetate Utilization Test

1. Move a light inoculum from the centre of a well-separated colony back and forth along the slope.
2. Put the cap on the tube in a loose way.
3. The tube is then incubated aerobically at temperatures between 35 and 37 degrees Celsius for up to seven days. For Enterobacteriaceae, incubation at 35–37°C for up to 5 days is not enough, but non-fermenting, Gram-negative bacilli/rods should be kept at 30°C for 7 days.
4. The test tube should be looked at every day for 4 days and then again after 7 days. If the result is still negative, the result can be thrown out.
5. Along the slant, you can see that the colour changes from green to blue.

Result

• Positive – As a result of the organism’s development, the medium turns alkaline (blue).
• Negative – no growth or no indicator change to blue

Keynote 

• Without an accompanying colour change, growth on the slant may indicate a positive test. If the agar does not turn blue after additional incubation, the test must be redone with less inoculum.
• Use a light inoculum to avoid false-positive reactions by preventing the carryover of chemicals from prior media.
• Do not pierce the slant, as the test demands an oxygenated atmosphere.

Limitations of Acetate Utilization Test

• A positive test may be indicated by a growth pattern that is not accompanied by a change in colour. However, if additional incubation does not cause the test medium to become blue, the test should be redone with a mild inoculum.
• The ambiguous test findings need to be redone.
• Since the test requires an aerobic atmosphere, the slant stabbing should be removed.
• Broth culture inoculum should be avoided due to the possibility of media carryover with broth culture.
• To prevent the transfer of chemicals from prior media, it is preferable to use a mild inoculum.
• Certain strains of Escherichia coli are resistant to antibiotics. coli may utilise acetate very slowly or not at all, resulting in a false-negative identification reaction.

Uses

• It is used to determine whether an organism can use acetate as its only carbon source.
• In addition, it is employed as a qualitative test to differentiate Gram-negative bacteria into fermentative and oxidative groups.
• Also used as a selective medium for the isolation of Escherichia coli is agar acetate.

References

• Duncan SH, Barcenilla A, Stewart CS, Pryde SE, Flint HJ. Acetate utilization and butyryl coenzyme A (CoA):acetate-CoA transferase in butyrate-producing bacteria from the human large intestine. Appl Environ Microbiol. 2002 Oct;68(10):5186-90. doi: 10.1128/AEM.68.10.5186-5190.2002. PMID: 12324374; PMCID: PMC126392.
• Xu, Q., Bai, C., Liu, Y. et al. Modulation of acetate utilization in Komagataella phaffii by metabolic engineering of tolerance and metabolism. Biotechnol Biofuels 12, 61 (2019). https://doi.org/10.1186/s13068-019-1404-0
• Mepham, T., Davis, S., & Humphreys, J. (1976). Acetate utilization by the isolated perfused guinea-pig mammary gland. Journal of Dairy Research, 43(2), 197-203. doi:10.1017/S0022029900015740
• Bellion, E., Kim, Y.S. Acetate utilization by a methylotrophic bacterium. Current Microbiology 2, 31–34 (1979). https://doi.org/10.1007/BF02601730
• Ampe F, Lindley ND. Acetate utilization is inhibited by benzoate in Alcaligenes eutrophus: evidence for transcriptional control of the expression of acoE coding for acetyl coenzyme A synthetase. J Bacteriol. 1995 Oct;177(20):5826-33. doi: 10.1128/jb.177.20.5826-5833.1995. PMID: 7592330; PMCID: PMC177405.
• https://himedialabs.com/TD/M1225.pdf
• https://universe84a.com/acetate-utilization-test/
• https://histogene.co/acetate-utilization-test/
• https://clinmicronow.org/doi/10.1128/9781683670438.CMPH.ch3.17-2
• https://www.tgw1916.net/Tests/acetate.html
• https://www.phdnest.com/acetate-utilization-test-principle-procedure-result/
• https://www.slideshare.net/HusseinAltameemi2/medical-microbiology-laboratory-biochemical-tests-ii
• https://himedialabs.com/TD/M339.pdf
• https://journals.asm.org/doi/full/10.1128/JB.181.17.5521-5526.1999
• https://microbenotes.com/acetate-utilization-test-principle-procedure-and-result-interpretation/
• https://microbeonline.com/acetate-utilization-test-principle-procedure-results-uses.
• https://microbiologyinfo.com/acetate-utilization-test/