Acetate utilization test is a biochemical test used to determine the ability of an aerobic organism to use acetate as the sole source of carbon and energy. It is mainly employed in diagnostic microbiology for differentiation of Gram-negative bacteria especially members of Enterobacteriaceae family. This test is commonly used to differentiate Escherichia coli from Shigella species and other closely related organisms.
The test is carried out on a chemically defined medium which contains sodium acetate as the only carbon source and inorganic ammonium salts as the only nitrogen source. Bromothymol blue is added in the medium as a pH indicator. The initial colour of the medium is green at neutral pH. Growth of the organism on this medium indicates that acetate is being utilized.
The principle of this test is based on the ability of certain organisms to metabolize acetate under aerobic conditions. When sodium acetate is utilized as a carbon source, ammonium salts are also broken down to obtain nitrogen. During this process ammonia is released into the medium which increases the alkalinity. This rise in pH changes the colour of bromothymol blue from green to blue. This is referred to as positive result.
If the organism is unable to utilize acetate there will be no growth and no breakdown of ammonium salts. Ammonia will not be produced and the pH of the medium remains unchanged. The medium remains green which indicates a negative result. Most strains of Escherichia coli are able to utilize acetate and produce blue colour whereas Shigella species generally do not utilize acetate and the medium remains green. Thus this test is useful in differentiation of these closely related bacteria.
Purpose of Acetate Utilization Test
The following are the objectives of Acetate Utilization Test–
- To determine whether an organism is capable of utilizing acetate as the sole source of carbon for its growth and metabolism.
- To differentiate Escherichia coli from Shigella species which are otherwise very similar Gram-negative, oxidase-negative and non-motile rods.
- To help in differentiation of lactose non-fermenting Gram-negative bacteria from fermentative organisms.
- To categorize enteric bacteria based on their ability to grow on acetate containing medium.
Principle of Acetate Utilization Test
The principle of Acetate Utilization Test is based on the ability of a microorganism to use acetate as the sole source of carbon for its growth. The test is carried out on a chemically defined medium which contains sodium acetate as the only carbon source and inorganic ammonium salts as the only nitrogen source. Bromothymol blue is incorporated in the medium as a pH indicator.
When an organism capable of utilizing acetate is inoculated on this medium, it metabolizes the sodium acetate under aerobic conditions. At the same time ammonium salts are broken down to obtain nitrogen for cellular activities. During this process ammonia is released into the surrounding medium.
The liberation of ammonia results in an increase in alkalinity of the medium. Due to this rise in pH the bromothymol blue indicator changes its colour from green to blue. This change in colour indicates a positive acetate utilization reaction.
If the organism is unable to utilize acetate there will be no growth and ammonium salts will not be decomposed. Ammonia will not be produced and the pH of the medium remains neutral. The medium remains green which indicates a negative result. Thus the test helps in differentiating organisms based on their ability to utilize acetate.
Media and Reagents, Materials used for Acetate Utilization Test
The media, reagents and materials used are as follows–
Media and Reagents
- Acetate Differential Agar (Sodium Acetate Agar)
It is a chemically defined medium.
Sodium acetate is the sole source of carbon in this medium.
The medium does not contain organic nitrogen. - Sterile physiological saline (0.85%)
It is used for emulsifying the colonies.
A light suspension is prepared before inoculation.
It prevents the carryover of nutrients from broth culture.
Composition of Acetate Differential Agar (per liter of water)
The composition is as follows–
- Sodium Acetate – 2.0 g
It is the sole source of carbon. - Monoammonium Phosphate – 1.0 g
It is the sole source of nitrogen. - Sodium Chloride – 5.0 g
It maintains osmotic equilibrium of the medium. - Dipotassium Phosphate – 1.0 g
It acts as a buffer. - Magnesium Sulfate – 0.1 g
It provides essential ions for enzymatic activity. - Bromothymol Blue – 0.08 g
It is the pH indicator.
It turns blue in alkaline condition due to utilization of acetate. - Agar – 15–20 g
It is the solidifying agent. - Distilled / Deionized water – 1 liter
It is used as solvent for preparation of the medium.
Supplies and Equipment
- Sterile inoculating loop / straight needle / stick
It is used for picking the colony and streaking the agar slant. - Sterile pipette
It is used for transferring saline suspension. - Test tubes
The medium is dispensed into test tubes.
Agar slants with deep butt are prepared. - Incubator (33–37°C, usually 35°C)
It is used to maintain required aerobic condition for growth. - Autoclave (121°C, 15 lbs pressure, 15 minutes)
It is used for sterilization of the prepared medium before use.
Procedure of Acetate Utilization Test
The procedure of Acetate Utilization Test is carried out in the following steps–
1. Preparation of light inoculum
A well–isolated colony from a pure 18–24 hour culture is selected. The colony is emulsified in 1.0 ml of sterile physiological saline (0.85%) to prepare a light suspension.
Broth culture is not used because carryover of peptones and organic nutrients may give false positive reaction.
2. Inoculation of the medium
Using a sterile inoculating loop or straight needle, a loopful of saline suspension is transferred to the Acetate Differential Agar slant. The surface of the slant is streaked back and forth in a serpentine pattern.
The butt of the agar is not stabbed because acetate utilization is an oxidative process and it requires strictly aerobic condition.
3. Ensuring adequate aeration
The caps of the inoculated test tubes are kept loosened. It allows proper exchange of oxygen. Oxygen is required for growth of organism and for alkaline shift of the medium.
4. Incubation
The inoculated tubes are incubated aerobically at 33–37°C (usually 35°C).
The tubes are incubated for up to 7 days.
5. Observation of results
The tubes are examined daily for 7 days before declaring the test negative. Positive test is indicated by visible growth and change in colour of medium from green to intense blue along the slant.
If there is no growth and the medium remains green after 7 days, the result is considered negative.
Result Interpretation of Acetate Utilization Test
The result of Acetate Utilization Test is interpreted on the basis of growth of organism and colour change of the medium. It is observed along the slant. The interpretation is as follows–
Positive Result
- It is indicated by visible bacterial growth on the slant.
- The colour of the medium changes from original green to intense blue.
- The blue colour appears along the slant surface due to alkaline reaction.
- In some cases growth is present without immediate colour change.
- This may still indicate positive reaction.
- The test should be repeated with lighter inoculum to rule out false positive reaction due to carryover of nutrients.
Negative Result
- It is indicated by complete absence of bacterial growth.
- There is no change in colour of the medium.
- The medium remains green after the incubation period.
Quality Control Organisms
Quality control organisms are used to ensure that the medium and procedure is working properly. These are as follows–
- Positive Control – Escherichia coli (ATCC 25922)
- It shows good growth on the medium. The medium turns blue indicating utilization of acetate.
- Negative Control – Shigella species (Shigella flexneri ATCC 12022 / Shigella sonnei ATCC 25931)
- There is no growth on the medium. The medium remains green indicating negative reaction.
Acetate Utilization Test positive and negative organism
| Organisms that yield a positive result | Organisms that yield a negative result |
|---|---|
| Most Escherichia coli strains, including some Enteroinvasive E. coli (EIEC), grow well on the medium and produce a positive reaction | Most Shigella species, including S. sonnei, S. dysenteriae, and S. boydii, are unable to utilize acetate and fail to grow |
| Klebsiella species, such as Klebsiella pneumoniae, yield a positive reaction | Proteus species, such as Proteus vulgaris, fail to grow and yield a negative result |
| Enterobacter species, including Enterobacter cloacae and Enterobacter aerogenes, give a positive result | Providencia species are also inhibited and incapable of acetate utilization |
| Citrobacter species, such as Citrobacter freundii, test positive | Salmonella Typhi uniquely produces a poor or negative reaction, distinguishing it from most other fermentative Salmonella species |
| Most Salmonella species, including Salmonella Arizonae, Salmonella Typhimurium, and Salmonella Enteritidis, successfully utilize acetate | — |
| Serratia species typically exhibit growth and test positive | — |
| Certain Shigella flexneri 4a biotypes, specifically 86% of mannitol-negative and 7.7% of mannitol-positive strains, are notable exceptions within their genus that give a positive result | — |
Limitations of Acetate Utilization Test
The limitations of Acetate Utilization Test are as follows–
- Some strains of Escherichia coli utilize acetate very slowly or may fail to utilize it. This can give false–negative reaction.
- Most Shigella species do not utilize acetate but certain atypical strains are exceptions. Shigella flexneri 4a may show acetate positive reaction and it can lead to misidentification as Escherichia coli.
- If a heavy inoculum is applied, internal nutrient stores and enzymes may be carried over. It can cause transient pH increase and false–positive blue colour without actual acetate utilization.
- If inoculum is taken directly from broth culture or enriched media, exogenous nutrients such as peptones and amino acids may be introduced into the medium. This allows acetate–negative organisms to grow and turn the medium blue falsely.
- Acetate utilization depends on oxidative pathway (glyoxylate cycle). The test requires strictly aerobic condition. If agar butt is stabbed or caps are tightly closed oxygen is restricted and false–negative result may occur.
- Sometimes visible growth is observed without expected colour change. Such results are considered equivocal and the test must be repeated with lighter inoculum.
- If the initial pH of the medium is not properly adjusted (around 6.7), minor metabolic activity may change the indicator colour and it leads to incorrect interpretation.
- Acetate utilization test alone cannot give definitive identification at species level. It is used along with other biochemical and molecular tests for confirmation.
Uses of Acetate Utilization Test
The uses of Acetate Utilization Test are as follows–
- It is used to determine whether the microorganism can utilize acetate as the sole source of carbon and energy.
- It is used to differentiate Escherichia coli from Shigella species.
- It helps in distinguishing non–motile, anaerogenic E. coli biotypes which closely resemble Shigella.
- It serves as a qualitative test to differentiate Gram–negative bacteria into fermentative and oxidative groups.
- It helps in characterization of non–fermentative Gram–negative bacilli.
- When it is combined with other biochemical tests, it is used for taxonomic classification of Enterobacteriaceae family.
- Acetate agar can be employed as a selective medium for isolation of Escherichia coli.
References
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- 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
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