Acetamide Utilization Test Introduction

Many of the biochemical tests are used to determine the ability of the micro-organism to utilize the chemicals and their reaction to certain enzymes. One such biochemical test is Acetamide utilization test.

This test is completely based on the acetamide agar, which is used to detect the ability of the organism to use acetamide by performing deamidation in the culture medium. This medium also contains carbon as its sole source along with inorganic ammonium salts.

What is Acetamide Utilization Test?

Acetamide utilization test is one of the biochemical tests which helps us to identify the aerobic organisms.

This test is based on the utility of acetamide by the organism during the process of deamidation.

This process occurs only during the presence of an enzyme acalamidase. This acetamide utilization test is performed to differentiate the group of fermentative organisms from the oxidative group of species.

This test is most commonly used for differentiating the Gram-negative, non-fermentative group of bacteria on the basis of their capability to utilize acetamide.

A medium containing acetamide as the sole source of carbon is used for utilizing the acetamide by the organism present in the growth medium.

Thus, the change in color in the growth medium of the medium, detects the growth of the organism present in the medium.

Acetamide Utilization Test Objective

 The main aim of the test is to differentiate the bacteria based on their ability to use the acetamide as the sole source of carbon.

 To identify the species of Aeruginosa from the other non-glucose-fermenting, Gram negative rods.

Acetamide Utilization Test Principle

As mentioned above, Acetamide utilization test is usually used to determine the ability of the specific organism to utilize acetamide as a source of carbon. This process usually takes place by deamidation.

The medium consists of acetamide. As a sole source of carbon along with the inorganic ammonium salts which serves as a sole of nitrogen.

The growth of organisms in the medium of acetamide agar indicates the positive Test for utilization of utilization.

During the metabolism of acetamide by the organism, the enzymatic action of acetamidase breaks the ammonium salts into ammonia. The ammonia thus released increases the alkalinity in the medium and results in change in the pH.

The change in the pH in the medium causes bromothymol blue indicator present in the medium to turn the color from green to blue, and it indicates the positive test.

When there is no release of ammonia in the medium, the color remains same and it indicates the negative result. In some cases, assimilation of the acetamide results in a formation of yellow color and it is mistaken for a positive result.

But the digestion of the acetamide by a deamination is limited to only some of the organisms. Hence this test is only performed for differentiating Pseudomonas aeruginosa from the other non-glucose fermenting, Gram-negative rods.

Acetamide Utilization Test Reagents

Media: Acetamide agar, it can be purchased commercially or it can also be prepared in laboratory in the following composition.

Ingredients	Gram/liter
Acetamide	3.0
Dextrose	0.2
Yeast extract	0.5
Potassium dihydrogen phosphate	1.0
Phenol red	0.03
Bacteriological agar	15.0
Sodium agar	5.0

Lab Supplies:

• Sterile inoculating loops or sticks

• Incubator

Acetamide Utilization Test Procedure

About 24.7 grams of the dehydrated powder or a lab prepared medium is added in a beaker containing about 1000 milli liters of distilled water.

The suspension is heated until it boils; so that the solution dissolves completely.

The dissolved medium is then dispersed into the tubes and it is sterilized in an autoclave at a temperature of about 121ºC for about 15 minutes.

After completing the autoclave process the tubes are taken out and they are cooled at a slanted position until it forms a butt at a depth of about 1.5 to 2.0 cm.

Acetamide Utilization Test

Here, a well isolated colony is taken from an 18 to 24-hour culture using a sterile inoculating loop or a needle.

Then the acetamide agar inoculated tubes are stroked in the slant in the direction of back and froth using the sterile inoculating loops or sticks.

After streaking the caps of the tubes are closed loosely and adequate aeration is ensured.

Then the tubes are incubated at a temperature of 35 to 37ºC for 7 days.

After incubating they are examined daily for 4 days and at the 7th day before discarding in order to ensure the negative result.

Acetamide Utilization Test Result

Positive Result: In case of positive result, there will be change in change and a growth is detected in the medium

Negative Result: Here, negative result is indicated by no change in color and the slant remains green and there will be no change in growth

Controlled Organism

In case of controlling the quality for the acetamide utilization test, two different organisms are considered here as negative and positive controls.

Control	Incubation	Results
Pseudomonas aeruginosa	It is incubated in an aerobic incubation of about 24 to 48 hours in a temperature of 33 to 37ºC	It results in Acetamide positive with a growth and change in color to blue
Escherichia coli	In an aerobic incubation for about 24 to 48 hours in a temperature of about 33 to 37ºC	It results in Acetamide negative, hence there will be no growth and there will also be no change in color

Acetamide Utilization Test Uses

• Acetamide utilization test is usually used to detect the ability of the particular organism to utilize the acetamide as the only source of carbon.

• It is also used as qualitative test for differentiating Gram-negative bacteria into the fermentative and the oxidative group bacteria into fermentative and the oxidative group bacteria. Acetamide agar is used in the selective medium for isolating P. aeruginosa

• This medium has can also be used as a modification of Simmon Citrate agar to determine the ability of acetamide in acting a carbon source without the presence of peptone and other protein sources.