Acetate Utilization Test Introduction

In the modern world, apart from life style changes, we come across many medical terms as a part of our daily activities, such as Lab test, diagnosis, vaccine, chemicals and so on, which became most common terms as a part of other things we hear.

Many tests are performed in a laboratory and in biochemical labs to detect the nature of the pathogen or a disease. Acetate utilization test is one such test which is used to determine whether the organism has the ability to use acetate as a sole source of carbon.

What is Acetate Utilization Test?

The acetate utilization test is generally used to test the ability of the organism to utilize the acetate as the single source of carbon.

This test is also performed as a qualitative test for the differentiation of the Gram-negative bacteria into fermentative and the oxidative group of bacteria.

This test is also being used to differentiate the species of Shigella from E. coli and the non-fermentative negative bacteria.

Acetate agar which contains sodium acetate as the sole source of nitrogen is used for inoculating the organism. Growth is indicated for the positive test of acetate utilization test.

When the bacteria metabolize acetate, the ammonium salts are broken down into ammonia, which increases the pH medium of the culture, this increase in pH turns the bromothymol blue indicator in the medium changes from green to blue.

Acetate Utilization Test Principle

In acetate utilization test, Acetate agar is employed as a test organism. This acetate agar has the ability to utilize acetate.

The culture medium is composed of Sodium acetate which acts as a sole carbon source and the inorganic ammonium salts as the source of the nitrogen. Where the growth of organisms suggests the positive results for the utilization of acetate.

During the metabolism process of acetate, by the bacteria, the ammonium salts are broken into ammonium, that elevates alkalinity.

The shift in the pH changes bromothymol blue indicator in the medium from green to blue. This medium is generally used for differentiating the Shigella spp from Escherichia coli.

As Shigella spp doesn’t have the capability to metabolize the acetate, However, approximately of about 94% of Escherichia coli plays a major role in utilizing the acetate.

Acetate Utilization Test Reagents

Media: The culture media is composed of Sodium acetate agar.

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
Agar	20.0
Bromothymol blue	0.08

 Sterilized sticks and inoculating loops

 Sterile pipette

 Incubator

 Sterile saline.

Acetate Utilization Test Procedure

Procedure for Acetate utilization test involves two steps;

1. Preparation of media

2. Utilization test

1. Preparation of Media

For the preparation of media, 69.1 grams of the dehydrated powder is added in beaker along with 1000 milliliters of the deionized or the distilled water.

Instead of dehydrated powder lab-prepared media can also be used.

The prepared suspension is then heated till boiling; so that the medium is dissolved completely.

Then the dissolved medium is dispensed into tubes and they are sterilized in an autoclave at 121ºC for about 15 minutes.

After completing the process of autoclaving the tubes are taken out and cooled at a slanted position to a temperature of about 40 to 45º.

The position is then maintained in order to obtain butts of depth 1.5 to 2.0 cm.

2. Utilization Test

The isolated colony is taken from an 18 to 24-hour culture with the help of a sterile inoculating needle.

A turbid suspension of saline is prepared by using 18-to-24-hour culture from a noninhibitor plate of culture.

The acetamide agar tube is inoculating by streaking the surface of a slant with the light inoculum which is picked from the culture.

The slant is then streaked back and froth with the loop or using an inoculating stick.

The cap or the test tubes are loosened to ensure whether the inoculum is getting sufficient aeration.

The tubes are then incubated aerobically at the temperature of about 35 to 37ºC for about seven days.

As incubation at 35 to 37ºC is not sufficient for thee Enterobacteriaceae, incubation at 30ºC is followed for seven days for the non-fermentation of Gram-negative rods.

The test tubes are examined regularly for at least 7 days before discarding the samples.

Acetate Utilization Test Result

For a positive result, the growth is represented as a change of color green to intense blue along the slant. Where as for negative result, the growth is absent and there will be no color change and the slant remain green as same.

Bacterial Control

Mostly two different organisms are taken for the positive and negative controls as the form of quality control for the acetate utilization test.

Control	Incubation	Results
Shigella flexneri	Aerobic incubation is followed for 24 to 48 hours at temperature of about 33 to 37ºC	Acetate negative, where there is no growth and no change in color and the medium remains green
Escherichia coli	Aerobic incubation is followed for 24 to 48 hours at temperature of about 33 to 37ºC	Acetate positive where the growth is shown and change of color to intense blue from green

Acetate Utilization Test Uses

• This test is usually used to test the ability of the organism to utilize acetate as the source of carbon.

• This test is also used in the form of qualitative test for differentiation of Gram-negative bacteria for fermentation and oxidative group of bacteria.

• Acetate agar can also be used as a selective media for the isolation of Escherichia coli.