Test Name	Acetamide utilization Test
Purpose	Differentiate microorganisms according to their capacity to utilise acetamide as their only carbon source.
Microorganisms Tested	Isolated colonies of Gram-negative, non-glucose-fermenting rods that emit a luminous pigment and are suggestive of P. aeruginosa.
Result	Positive: The slant shows growth, and its colour changes from green to intense blue. Negative: There is no growth, no change in colour, and the slope stays green.
Require Media	Acetamide Agar
Quality Control	Acetamide Positive: Pseudomonas aeruginosa (ATCC 27853), growth; blue color Acetamide Negative: Escherichia coli (ATCC 25922), no growth; green color

What do you mean by Acetamide Utilization Test?

• The acetamide utilisation test is a good way to tell Pseudomonas aeruginosa apart from other gram-negative rods that don’t ferment glucose. It tells the difference between microorganisms based on whether they use acetamide as their only source of carbon.
• The easiest amide to make from acetic acid is acetamide, also called ethanamide. This organic compound is written as CH3CONH2.
• Bacteria that can grow on this medium make the enzyme acylamidase, which breaks down acetamide into ammonia and acetamide back into acetamide.
• When ammonia is made, the pH becomes more alkaline. This makes the medium change colour from green to royal blue.
• In 1961, Bühlmann et al. wrote that acetamide deamination and growth at 42°C can help find P. aeruginosa strains that don’t make pyocyanin.
• A modified Christensen medium was used that didn’t have any agar, urea, or glucose and had 1% acetamide added to it.
• Hedberg used acetamide in a modified Simmons Citrate Agar without peptone to separate P. aeruginosa from strains of swarming Proteus.
• The formula showed that acetamide could be the only source of carbon when peptone was not present.
• Oberhofer and Rowen also used a modified version of Simmons Citrate Agar to separate non-fermenting bacteria based on their ability to deaminate acetamide.
• This mixture is suggested by the Association of Official Analytical Chemists (AOAC) and the American Public Health Association (APHA). It can be used to grow P. aeruginosa using the multiple tube technique.

Acetamide utilization Test Principle

• With acetamide agar, you can see if an organism can use acetamide by deamidation.
• The only source of carbon is acetamide, and the only source of nitrogen is morankc ammonium salts.
• Growth is a sign that a test for acetamide uliation is positive.
• Bacterium breaks down acetamide using an enzyme called acylamidase. This breaks down ammonium salts into ammonia, which makes the water more alkaline.
• The bromothymol blue indicator in the medium changes from green to blue when the pH changes. This means that the test is positive.
• When acetamide is broken down, it turns yellow, which is not the same as a positive result.
• In general, only a small number of organisms can deaminate.
• This medium is recommended for telling Pseudomonas aeruginous apart from other gram-negative rods that don’t ferment glucose.

Purpose of Acetamide utilization Test 

• Differentiate microorganisms according to their capacity to utilise acetamide as their only carbon source.

Microorganisms Tested

• Isolated colonies of Gram-negative, non-glucose-fermenting rods that emit a luminous pigment and are suggestive of P. aeruginosa.
• Gram-negative, non-glucose fermenting rods, as part of the identification.

Media, Reagents, and Supplies

• Sterile inoculating loops or sticks
• Incubator
• Acetamide Agar

Acetamide Agar composition

Ingredients	Gram/liter
Sodium chloride	5.0
Magnesium sulfate	0.2
Ammonium phosphate, monobasic	1.0
Potassium phosphate, dibasic	1.0
Acetamide	10.0
Agar	15.0
Bromothymol blue	0.08

Final pH at 25°C: 6.8 ±0.2

Store at 2°C to 8°C.

Acetamide Agar Preparation

1. In a beaker, 1000 millilitres of distilled or deionized water is mixed with 24.7 grammes of the dehydrated powder or lab-made media.
2. The suspension is then brought to a boil to completely dissolve the medium.
3. The dissolved medium is then put into tubes and sterilised in an autoclave for 15 minutes at 15 pounds of pressure (121°C).
4. Once the autoclaving is done, the tubes are taken out and cooled at an angle until the temperature is between 40 and 45°C. Keep the position the same if you want to get butts that are 1.5 to 2 cm deep.

Quality Control

Using the following quality control organisms, all lot numbers of Acetamide Agar have been tested and found to be good. Control organisms should be tested using quality control methods that have already been set up in the lab. If there are problems with quality control, patient results shouldn’t be sent out.

• Acetamide Positive: Pseudomonas aeruginosa (ATCC 27853), growth; blue color
• Acetamide Negative: Escherichia coli (ATCC 25922), no growth; green color

Procedure of of Acetamide utilization Test 

1. Pick the inoculum from the centre of a colony that has been well separated for 18 to 24 hours. Do not inoculate from a broth culture because the growth will be too strong and the carryover of media may cause false positive reactions.
2. Use a needle to inoculate acetamide slant by running it back and forth across the slant. Do not stab the slant because the test needs a place with oxygen.
3. Put the cap on the tube in a loose way. At 35°C to 37°C and with oxygen for up to 4 days.
4. Along the slant, you can see that the colour changes from green to blue.
5. If you’re not sure, put the slant back in the incubator for two more days and watch the colour change.
6. Repeat the test, but the results aren’t clear.

Expected Results of Acetamide utilization Test

• Positive: The slant shows growth, and its colour changes from green to intense blue.
• Negative: There is no growth, no change in colour, and the slope stays green.

Delftia (Comamonas) acidovorans, P. aeruginosa, and Alcaligenes faecalis can perform acetamide deamination. Pseudomonas aeruginosa can be found with the help of fluorescent pigment, acetamide deamination, and a positive oxidase test.

Limitations

• Growth without a change in colour could be a sign of a good test result. If more time in the incubator doesn’t cause a change in colour, repeat the test with less of the inoculum.
• A negative test does not rule out the possibility of P. aeruginosa, and 6% of Pseudomonas fluorescens organisms have been said to give a false positive result. Other fluorescent species of Pseudomonas do not respond in a good way.
• Acetamide hydrolysis is not a good way to find out which strains of Pseudomonas make pyocyanin.
• Only 38% of P. aeruginosa strains that don’t make pyocyanin will show a positive result in Acetamide Agar. For a sure identification, it may be necessary to do more biochemical tests.

Uses

• This medium was made by changing Simmon Citrate agar to see if acetamide could act as a carbon source when peptone or other protein sources were not present.
• The Acetamide Utilization Test is used to see if a living thing can get all of its carbon from acetamide.
• Acetamide agar is also used to separate P. aeruginosa from other bacteria.
• It is also used as a quality test to tell the difference between the fermentative and oxidative groups of Gram-negative bacteria.

References

• Bailey & Scott’s Diagnostic Microbiology, Forbes, 11th edition
• https://www.himedialabs.com/TD/M148.pdf
• Andrea J. Linscott, 2016.  Clinical Microbiology Procedures Handbook, 4th Edition. ASM Press, Washington, DC. doi: 10.1128/9781683670438.
• https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/390/787/00185dat-twinpack.pdf
• http://microbesinfo.com/2016/04/acetamide-utilization-test-principle-procedure-expected-results-quality-control-and-limitation/
• https://www.studocu.com/in/document/rajiv-gandhi-university-of-health-sciences/general-microbiology-and-immunity/acetamide-utilization-test/32709511
• https://www.coursehero.com/file/95586733/Acetamide-utilization-Test-Principle-Procedure-Uses-and-Interpretationdocx/
• https://microbenotes.com/acetamide-utilization-test-principle-procedure-and-result-interpretation/
• https://tools.thermofisher.com/content/sfs/manuals/IFU60012.pdf
• https://www.phdnest.com/acetamide-utilization-test-objective-principle-procedure-results/
• https://microbeonline.com/acetamide-utilization-test/
• https://clinmicronow.org/doi/10.1128/9781683670438.CMPH.ch3.17-1
• https://microbiologyinfo.com/acetamide-utilization-test/