Carbohydrate Fermentation Test

• The carbohydrate fermentation test can be used to determine if bacteria are able to ferment a certain carbohydrate.
• It is a test to determine any presence of acids or gas resulting from carbohydrate fermentation.
• Carbohydrates are organic molecules which consist of carbon, hydrogen as well as oxygen, in the proportion (CH2O)n.
• There are three kinds of carbohydrates, based upon their structure and the amount of carbon atoms These include monosaccharides (simple sugar that has 3- 7 carbon atoms) and disaccharides (comprising of two monosaccharides bonded to each other by the glycosidic link) and polysaccharides (containing eight or more monosaccharide molecules).
• It releases energy through catabolism which refers to the breaking down of complicated organic compounds. The capability of chemical breakdown of carbohydrates from their complex to the simpler forms is used by many bacteria, fungi and yeasts. But, the process of using carbohydrate and its breakdown differs depending on how the body’s enzyme system functions.
• The process of fermentation has specific to certain genera, species or species of living organisms. This is why the characteristic of fermentation is widely used to aid in biological differentiation among microorganisms.

Purpose of Carbohydrate fermentation test

• To demonstrate the capacity of microorganisms in fermenting carbohydrates and to produce organic acid-end products.
• To test the capability of the microorganisms to produce gases as end products from fermentation.

Principle of Carbohydrate fermentation test

Carbohydrate fermentation is the method in which microorganisms utilize carbohydrates to create energy through the production of ATP which is the primary energy source for the organism. After entering a cell, glucose can be catabolized in an aerobic manner (in with the help of O2) and in which molecular oxygen is an electron acceptor (oxidative pathway) as well as the other way (in the absence of O2) where organic ions may function as an electron acceptor (fermentative pathway). The metabolic end-products of the carbohydrate fermentation could include organic acids (lactic acid, formic, and acetic acids) or organic acids and gases (hydrogen as well as carbon dioxide). 

The degradation of carbohydrates (monosaccharide disaccharide as well as polysaccharide) by microorganisms during the anaerobic environment is performed in the fermentation tube made up of Durham tube to detect of gas production. A fermentation medium is made up of a base medium that contains one specific carbohydrate (glucose sucrose, sucrose, or cellulose) and an indicator for pH (phenol red, the indicator of Andrade bromocresol, or Andrade’s indicator). 

As the organism ferments sugars, acid organic compounds (Lactic acid or formic acid as well as acetic acids) are produced, which change the medium yellow and a decrease in acidity (acidic-below the pH value of 6.8). The alteration in the pH indicator of the fermentation tube and in the gas production within the Durham tube are a sign of the metabolic reaction that results in the production of acid end products and gas.

The change in color only happens and is evident when an adequate amount of acid is produced, since bacteria can utilize peptone for creating alkaline products. The breakdown of peptones in the broth can lead to the production of alkaline ending products, which change the color of the broth, which is pink frequently on the highest point in the tube.

Media for Carbohydrate fermentation test

Phenol red carbohydrate broth can be typically used in tests for carbohydrate fermentation. The carbohydrate sources may differ depending on the test specifications. The most common broth media include:

• Phenol red glucose broth
• Phenol red lactose broth
• Phenol red maltose broth
• Phenol red mannitol broth
• Phenol red sucrose broth

Preparation and Composition of the media 

Make sure you purchase phenol red test media from commercial vendors or the base of phenol red broth and include specific carbohydrate sources in accordance with your test specifications You can also make your own media using with the ingredients listed below.

Composition of Phenol Red Carbohydrate Broth

Ingredients	Amount
Trypticase or protease peptone No. 3	10 g
Sodium chloride (NaCl)	5 g
Beef extract (optional)	1 g
Phenol red (7.2 ml of 0.25% phenol red solution)	0.018 g
Carbohydrate source	10g

Procedure of Carbohydrate fermentation test

Preparation of the media

1. Make broth media by mixing all the ingredients in 1000 mL of deionized or distilled water, and then heating it gently to dissolve it. (Note that you should use a single carbohydrate source according to your needs).
2. Fill 13x100mm test tubes with about 4-5 ml in phenol-red carbohydrate soup.
3. Inject the Durham tube to measure the production of gas.
4. Make sure to autoclave the test media (at 120°C for 15 minutes) for sterilization. The sterilization process will force the broth into the reverse Durham tube. (Note that when using lactose, arabinose, salicin, maltose, sucrose and xylose autoclave at 121°C just 3 minutes since these carbohydrates are susceptible to decomposition through autoclaving.)

Note: The broth will have light red in color, and the pH of the final product will be 7.4 + 0.2.

If you prefer, make the base of phenol red broth that you heat sterilize and cool to 45 degrees Celsius. Separately prepare a specific carbohydrate mixture then remove the solution using membrane filters (pore dimension: 0.45 mm). Add the solution of carbohydrate in the broth base, and then mix. The ideal carbohydrate content is 1 %.

Inoculation  and Incubation into the fermentation medium

• Inoculate every test tube using the microorganism of the test using an inoculating needle, loop. Or, you can inject each test tube with 2 to 3 drops of an 18-to 24-hour brain-heart infusion broth of the organism you wish to test.
• Incubate the tubes at 35 to 37 degrees Celsius over 18-24 hours. More time incubation could be needed to verify a negative test.
• Examine the tube to determine if it contains gas and acid production.

Result and Interpretation of Carbohydrate fermentation test

Acid production

• Positive: After the incubation process, the tube’s liquid changes color to the color of yellow (indicated via the alteration in color of the indicator for phenol red). This indicates a reduction in pH as a result of the production of acid from the process of fermentation of carbohydrates (sugar) that is present in the medium.

NOTE: If there is a reason you’re using different pH indicator, please look up Table 1 for their colors with respect to pH.

• Negative: Tubes that contains the medium will remain in red meaning that the bacteria can’t ferment the specific carbohydrate source within the media.

Gas Production

• Positive:  A bubble (small or large, based on the quantity of gas produced) will be visible inside the reversed Durham tube.
• Negative: There will not be any bubbles inside this reversed Durham tube i.e. bacteria do not generate gas as a result of the process of fermentation of the specific carbohydrate within the medium i.e. anaerogenic organisms.

Uses of Carbohydrate Fermentation Test 

The patterns of carbohydrate fermentation are used as a way to identify between species of bacterial or bacterial groups.

• All members of the Enterobacteriaceae family are glucose-fermenters (they can also metabolize glucose anaerobically).
• Maltose fermentation is a way to distinguish Proteus vulgaris (positive) from Proteus mirabilis (negative).
• The two Neisseria on gonorrhoeae (gonococci) as well as Neisseria meningitides (meningococci) ferment glucose, however only meningococci ferments maltose.
• Tests for the utilization of carbohydrate in rapid fashion (RCUTs) are conducted to identify Corynebacterium diphtheriae as well as various Corynebacterium species.

Precautions of Carbohydrate Fermentation Test 

• After the inoculation of a specific sugar, clean the loop to prevent cross-contamination tube by other sugars.
• Don’t use tubes that have Durham tubes which are only partially filled or have bubbles.
• The prolonged incubation process will allow bacteria to break down proteins, and can in false-positive results.

Limitations of Carbohydrate Fermentation Test 

• After 24 hours, readings may not be accurate if there is no acid is being produced.
• A lack of color or result indicating alkalinity can occur when the organism disintegrates the peptone, obscuring the evidence of carbohydrate fermentation.

References

• Tille P.M (2014)Bailey and Scott’s diagnostic microbiology, Thirteen editions, Mosby, Inc., an affiliate of Elsevier Inc., 3251 Riverport Lane, St. Louis, Missouri 63043
• Aneja K.R. 2003. Experiments in Microbiology, Plant Pathology, and Biotechnology, fourth revised edition, New Age International (P) limited, Ansari road, Daryaganj, New Delhi-110002.