Acetate utilization test: Principle, media composition, Procedure and Results

Acetate utilization test measures an organism’s capacity to use acetate as its only source of carbon. This is especially important for distinguishing members of the Enterobacteriaceae family. Organisms that can grow on acetate create an alkaline reaction, which changes the medium’s pH indicator from green to blue.

Acetate utilization test

Acetate utilization test is a biochemical experiment used to assess an organism’s capacity to use acetate as its only source of carbon. This test is very useful for distinguishing members of the Enterobacteriaceae family from other Gram-negative rods. The test’s idea is based on bacteria’s metabolic pathways. Some bacteria have the enzymatic machinery needed to use acetate via the tricarboxylic acid (TCA) cycle. When an organism uses acetate, it produces alkaline byproducts, increasing the pH of the medium. This pH change is measured by a pH indicator in the media, which causes a noticeable color shift.

Media Content

The acetate utilization test is typically conducted using acetate agar, which is a modified form of Simmons Citrate Agar. The sole carbon source that the organism has access to in this minimal environment is acetate thanks to the chemistry of this medium. The following are the medium’s constituent parts:

• 2.0 g of sodium acetate (CH3COONa)
• One gram of ammonium dihydrogen phosphate (NH4H2PO4)
• 7.0 g of dipotassium phosphate (K2HPO4)
• 0.2 g of magnesium sulfate (MgSO4•7H2O)
• 5.0 g of sodium chloride (NaCl)
• The indicator, bromothymol blue, is 0.08 g.
• 15.0 g of Agar
• 1.0 L of distilled water
• pH: Raised to 6.9

Part Functions:

• Sodium acetate is the only carbon source present.
• Ammonium dihydrogen phosphate and dipotassium phosphate: These nutrients assist buffer the medium and supply the nitrogen and phosphate required for growth.
• Magnesium sulfate: Provides the necessary magnesium ions needed for enzyme functions.
• Osmotic equilibrium is preserved by sodium chloride.
• As a pH indicator, bromothymol blue changes from green to blue in an alkaline environment.
• Agar: This solidifies the medium and creates a slanted surface that makes it easier to observe color changes and growth.
• Procedure: There are multiple procedures involved in the acetate utilization test, which include preparing the medium, inoculating the test organism, and incubating. Carefully following each step is necessary to guarantee accurate and trustworthy outcomes.

Setting Up the Media:

• In one liter of distilled water, dissolve the prescribed amounts of sodium acetate, magnesium sulfate, sodium chloride, ammonium dihydrogen phosphate, dipotassium phosphate, and bromothymol blue.
• As needed, add sodium hydroxide (NaOH) or hydrochloric acid (HCl) to the solution to bring its pH down to 6.9.
• To fully dissolve the agar, add it to the mixture and heat it.
• To disinfect the medium, autoclave it for 15 minutes at 121°C.
• Allow the medium to cool to between 45 and 50°C after autoclaving, and then transfer it into sterile test tubes.
• As the material solidifies, arrange the test tubes such that the slanted surface is on the inside.

vaccination

Acquire a pristine culture of the examined organism cultivated on a nutrient agar medium.
Select one test organism colony at a time using a sterile inoculating loop or needle.
Apply the inoculum evenly across the slant’s surface by splattering it on.
Using a mild inoculum is essential to prevent false-positive results caused by rapid development.

Embryology

For 24 to 48 hours, incubate the inoculation tubes at 35 to 37°C.
To keep the medium from drying out, make sure the tubes are properly sealed.
Check the tubes every day for any changes in color or growth.

Interpretation of the Results
The presence or lack of growth and the medium’s color change determine the outcome of the acetate consumption test. The medium’s bromothymol blue indicator serves as a visual cue for pH variations brought on by metabolic processes.

Good Outcome:

The creature is shown to be growing down the slant surface.
The medium turns blue in color instead of green.
Because acetate is being used and alkaline byproducts are being produced, the blue color denotes an alkaline reaction.
This finding suggests that the creature can use acetate as its only source of carbon.

Adverse Outcome:

Along the slant surface, there is no development or very little growth visible.
The medium’s green color doesn’t alter much, suggesting that the pH hasn’t changed.
This finding indicates that acetate cannot be used by the organism as its only source of carbon.

Question for Acetate Utilization Test Goal:

Finding out if the test organism can use acetate as its only carbon source is the aim of the acetate utilization test. In particular, the Enterobacteriaceae family benefits greatly from this knowledge for the purpose of distinguishing and recognizing various bacterial species.

Supplies Required:

• Acetate agar medium (made in the manner specified)
• sterile inoculation needle or loop
• the test organism’s pure culture
• sterile test tubes
• 35–37°C is the incubator’s temperature setting.

Method:

Getting Ready for the Media:

• One liter of distilled water should be used to dissolve the acetate agar medium’s ingredients.
• Use HCl or NaOH to bring the pH down to 6.9.
• When the agar is fully dissolved, add heat.
• For fifteen minutes, autoclave the medium at 121°C.
• Transfer the mixture into sterile test tubes and permit it to harden at an inclined angle.

vaccination

Select a single colony of the test organism from a nutrient agar plate using a sterile inoculating loop or needle.
Apply the inoculum to the acetate agar medium’s slant surface.

Embryology

For 24 to 48 hours, incubate the inoculation tubes at 35 to 37°C.
Check the tubes every day for any changes in color or growth.

Analysis of the Findings:

Positive Outcome: Green to blue color shift and growth along the slant.
shows that acetate can be used as the only carbon source by the organism.
Negative Outcome: Medium stays green with little to no growth. There is also no color change.
shows that acetate cannot be used by the organism as its exclusive supply of carbon.

Detailed Justification

In order to classify and identify microorganisms, the acetate consumption test is an essential technique. An organism’s capacity to use acetate as its only carbon source depends on the existence of particular enzymes and metabolic pathways. Organisms harboring the enzymes required for the breakdown of acetate will flourish in the absence of any other carbon source, while those without these enzymes would not develop.

Carefully designed to eliminate other carbon sources, the test medium guarantees that any development seen is exclusively the result of using acetate. As a visual cue of metabolic activity, bromothymol blue’s existence as a pH indicator is essential. Bromothymol blue is green at neutral pH values; at alkaline pH values, it becomes blue. The medium’s pH rises and turns blue when organisms metabolize acetate, producing alkaline byproducts in the process.

Adjusting the pH and maintaining sterility are two important aspects of the medium preparation that need for accuracy. Better monitoring of development and color change is made possible by the medium’s slanted surface in test tubes. To avoid overwhelming the medium and producing false-positive results, it is imperative to utilize a mild inoculum during inoculation. During the incubation stage, metabolic activities have enough time to materialize as observable growth and colour changes.

The findings are simple to interpret. Growth and a shift in hue to blue verify that the organism can use acetate, which is a desirable outcome. This finding can help identify particular bacterial species and broaden our knowledge of their metabolic potential. If, on the other hand, the medium stays green, the result is negative and suggests that the organism has to grow from other sources of carbon because it is unable to use acetate.

To sum up, evaluating the metabolic capacities of bacteria can be achieved by the useful and dependable acetate utilization test. The complex metabolic processes underlying microbial growth and survival are brought to light by its use in microbial identification and differentiation. We can better comprehend microbial ecology and taxonomy thanks to the test’s ease of use and clarity, which makes it a vital tool in microbiology labs.

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