Carbohydrate Fermentation Test Method

In this carbohydrate fermentation test method post we have briefly explained carbohydrate fermentation test principle, objectives, requirements, carbohydrate fermentation test microbiology procedure, uses and limitations of carbohydrate fermentation test microbiology.

Carbohydrate fermentation test method

Carbohydrate fermentation test microbiology is performed to see if bacteria can ferment a certain carbohydrate. Carbohydrate fermentation patterns can help distinguish between various bacterial groups or species.

It looks for acid and/or gas that is created during carbohydrate fermentation. Carbohydrate fermentation test microbiology done with a basal medium containing a single carbohydrate source such as glucose, lactose, sucrose, or any other carbohydrate.

In addition, a pH indicator (such as Andrade’s solution, bromcresol purple (BCP), bromothymol blue (BTB), or phenol red) is included in the medium to detect the medium’s pH dropping due to acid formation. Durham tubes are small inverted tubes that are immersed in the medium to test for gas production (hydrogen or carbon dioxide).

Objectives

To demonstrate microorganisms’ ability to ferment carbohydrates and produce organic acid end products. Determine the microorganism’s ability to produce gaseous end products during fermentation.

Principle

A wide range of carbohydrates or similar substances can be fermented by several bacteria. They are decomposed into acids such as lactic acid and pyruvic acid, as well as gases like as CO2 and hydrogen.

A particular bacterial species fermentation range is usually stable. We can identify a species by doing a battery of sugar fermentation tests. Various carbohydrates such as glucose, lactose, maltose, inulin, salicin, esculin, mannitol, sucrose, and others are used in a typical laboratory.

When an organism ferments a certain carbohydrate, the pH of the medium changes to acidic, which can be detected with a pH indicator. An inverted Durham’s tube in the medium can be used to monitor gas generation.

Requirements

Materials

1. Conical flasks

2. Durham tubes

3. Screw-capped tubes

4. Autoclave

Reagents

1. Peptone water

2. Glucose

3. Fructose

4. Mannose

5. Galactose

6. Phenol red

Test Procedure

Carbohydrate Fermentation Test

Carbohydrate fermentation test microbiology

1. The carbohydrate broth medium ingredients, or their ready-made powder, are weighed and dissolved in 100 ml of distilled water in a 250 ml conical flask by shaking and swirling.

2. The pH is measured with a pH metre and then corrected to 6.8 with 0.1N HC1. If necessary, the flask is heated to completely dissolve the materials. Five test tubes are filled with the broth (approximately 10 ml each).

3. In each test tube, one Durham tube is inverted and placed in the soup. The Durham tubes float because they are filled with air. Hot steam displaces this air during sterilisation, causing them to submerge in the broth.

4. As a result, there is no need to eliminate air from the Durham tubes by filling them with broth before sterilisation in order to maintain them submerged in broth. Cotton is inserted into the test tubes, which are then wrapped with craft paper and secured with thread or rubber bands.

5. In an autoclave, the broth tubes are sterilised at 121°C (15 psi pressure) for 15 minutes. Alternatively, sterilise 90 mL of the basal medium (medium without carbohydrate) in the autoclave, then add 10 mL of the carbohydrate solution (10%) sterilised by membrane filtration after cooling. This is especially important for heat-sensitive carbohydrates, which degrade during the heat sterilisation process.

6. Allow the broth tubes to cool to room temperature. With the use of an inoculating loop sterilised over bunsen flame, the test bacteria are injected aseptically into the broth, preferably in a laminar flow chamber. In an incubator, the inoculated broth tubes are incubated for 24 hours at 37°C.

Observation

Carbohydrate Fermentation Test

Carbohydrate fermentation test microbiology

Pink colour with gas bubble in Carbohydrate fermented with acid the Durham’s tube and gas (+) (e.g. E.coli, Klebsiella).

Pink colour with no gas Carbohydrate is fermented without bubbles in the Durham’s tube gas production (+) (e.g. Salmonella Shigella, Vibro).

 No color change or yellow colour Carbohydrate not fermented (-) and no gas in Durham’s tube e.g. Pseudomonas, Alkaligenes.

Advantages

1. Carbohydrate fermentation test microbiology is performed to see if bacteria can digest a particular carbohydrate. Carbohydrate fermentation test microbiology examines the presence of acid and/or gas produced during the fermentation of a single carbohydrate.

2. Carbohydrate fermentation test microbiology evaluates an organism’s ability to ferment glucose as well as convert pyruvic acid, the end product of glycolysis, into gaseous by-products.

3. Carbohydrate fermentation test microbiology is a typical test for identifying Gram-negative enteric bacteria that are all glucose fermenters but only some of which generate gas.

Limitations

1. For complete identification, biochemical, immunological, molecular, or mass spectrometry testing on colonies from pure culture is indicated.

2. If bubbles are trapped in the durham tube, it may be required to invert the tube before inoculation. False-positive results may come from trapped bubbles that are not released.

3. If no acid is formed after 24 hours, the reading may not be accurate. If the organism deaminates the peptone, no colour change or result indicating alkalinity may occur, hiding the carbohydrate fermentation evidence.

Further Readings

Reference