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Carbohydrates are organic molecules that comprise carbon, hydrogen, and oxygen in the ratio (CH2O)n. There are three types of carbohydrates based on their structure and number of carbon atoms present, they are monosaccharides (simple sugar containing 3-7 carbon atoms), disaccharides (comprising of two monosaccharides linked together by the glycosidic bond), polysaccharides (containing eight or more monosaccharide molecules). The energy is released by the process of catabolism referring to the breakdown of the complex organic molecules. The power of chemical breakdown of carbohydrates from the complex to simple forms is possessed by a large number of bacteria, fungi, and yeasts. However, the utilization of the carbohydrate and its breakdown is different depending upon the enzyme system of the organism. The pattern of the fermentation is the characteristics of certain species, genera, or groups of organisms. Due to this reason, the property of fermentative reaction has been extensively used as a method for biochemical differentiation of microorganisms.
Objectives of Carbohydrate fermentation test
- To demonstrate the ability of microorganisms to ferment the carbohydrate and production of organic acid end products.
- To determine the ability of the microorganism to produce gaseous end products in fermentation.
Principle of Carbohydrate fermentation test
Carbohydrate fermentation is the process by which the microorganism utilizes to produce energy in the form of ATP, the ultimate energy source of the organism. Glucose after entering a cell can be catabolized either aerobically (in the presence of O2), where molecular oxygen serves as the final electron acceptor (oxidative pathway), or anaerobically (in absence of O2) in which inorganic ions can serve as the final electron acceptor (fermentative pathway). The metabolic end products of a carbohydrate fermentation can either be organic acids (lactic, formic, acetic acid) or organic acid and gas (hydrogen or carbon dioxide). Fermentative degradation of the carbohydrates (monosaccharide, disaccharide, and polysaccharide) by microorganisms under the anaerobic condition is carried out in the fermentation tube, which comprises of Durham tube for the detection of the gas production. A fermentation medium is composed of a basal medium containing a specific carbohydrate (glucose, sucrose, or cellulose) along with a pH indicator (phenol red, Andrade’s indicator, or bromocresol). When the organism ferments carbohydrates, organic acid products (Lactic acid, formic acid, or acetic acid) are obtained which turns the medium into yellow color with a reduction in the pH (acidic-below pH of 6.8). The change in the pH indicator in the fermentation tube and the gas production in the Durham tube is indicative of the metabolic reaction with the production of acid end product and gas. Color change only occurs and is visible when a sufficient amount of acid is produced, as bacteria may utilize the peptone producing alkaline by-products. The degradation of peptones in the broth may result in the production of alkaline end products, which will change the broth color to pink often at the top of the tube.
The procedure of Carbohydrate fermentation test
Preparation of Carbohydrate Fermentation Broth
- Weigh and dissolve trypticase, Sodium chloride, and Phenol red in 100 ml distilled water and transfer into conical flasks.
- Add 0.5% to 1% of the desired carbohydrate into all flasks.
- Insert inverted Durham tubes into all tubes, the Durham tubes should be fully filled with broth.
- Sterilize in an autoclave at 115°C for 15 minutes. (Note: Do not overheat the Phenol red Carbohydrate fermentation broth. The overheating will result in breaking down the molecules and form compounds with a characteristic color and flavor. The process is known as the caramelization of sugar (the browning of sugar).
- Transfer the sugar into screw-capped tubes or fermentation tubes and label properly.
Inoculation of Bacterial Culture into fermentation medium tube
- Inoculate each tube with 1 drop of an 18 hour or 24-hour cultural broth in aseptic condition (keep uninoculated tubes as control tubes).
- Incubate the tubes at 18-24 hours at 37°C
- Examine the tube for acid and gas production.
Result and Interpretation of Carbohydrate fermentation test
Figure: Carbohydrate fermentation test results. Image Source: Quizlet Inc.
|The medium changes to yellow color||Acid production||Organism ferments the given carbohydrate and produces organic acids thereby reducing the pH of the medium into acidic condition.|
|The medium changes to yellow color and production of gas formation in the Durham tube||Acid and Gas production||Organism ferments the given Carbohydrate and produces organic acids and gas. Gas production is detected by the presence of small bubbles in the inverted Durham tubes.|
|No change in color (retains red color)||Absence of fermentation||The organism cannot utilize the carbohydrate but the organism continues to grow in the medium using other energy sources in the medium.|
- After inoculation into a particular sugar, sterilize the loop in order to avoid cross-contamination of the tube with other sugars.
- Do not use the tubes with Durham tubes that are partially filled or with bubbles.
- Over incubation will help the bacteria to degrade proteins and will result give false-positive results.
- Reading after 24 hours may not be reliable if no acid is produced.
- No color change or a result indicating alkalinity may occur if the organism deaminates the peptone, masking the carbohydrate fermentation evidence.
- 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.
- Hardy diagnostics
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