Carbohydrate fermentation test 1

The carbohydrate fermentation test is used to determine whether a bacterium can utilize a certain carbohydrate or not.
It tests for the presence of acid and/gas produced from the fermentation of a single carbohydrate.

1. CARBOHYDRATE FERMENTATION TEST. Yousef Elshrek

2. Objective The carbohydrate fermentation test is used to determine whether a bacterium can utilize a certain carbohydrate or not. It tests for the presence of acid and/gas produced from fermentation of a single carbohydrate. Principle Fermentation is the metabolic process to breaking down or catabolism of the sugars under anaerobic conditions. The result of this process is the production of energy source of ATP by substrate level phosphorylation, in the absent of aerobic respiration electron transport chain. Microorganisms utilize the carbohydrates by their enzymatic reactions and produce organic acids , acids, and gas. The types and proportion of the products depend on species and carbohydrates. Formation of acids in sugar broths can be detected by using the pH indicator phenol red which is red at neutral pH and changes to yellow at acidic conditions, slight amount of acid can cause color change. Gas formation can be detected by using an inverted Durham tube

3. Materials Phenol red carbohydrate fermentation broth. Bacterial culture. Inoculation loop. Incubator (37 o C). Procedure How to prepare Carbohydrate Fermentation Broth Weight and dissolve trypticase, Sodium chloride, and phenol red in 100 ml distilled water and transfer into conical flasks. Add 0.5% to 1% 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 o C for 15 mins. Do not overheat the phenol red carbohydrate fermentation broth. The overheating will result in breaking down the molecules and form compounds with characteristic color. The process is known as caramelization of sugar the browning of sugar). Transfer the sugars into screw-capped tubes or fermentation tubes and label properly.

4. Ingredients of The Fermentation Broth Trypticase: 1gm Carbohydrate: 0.5gm Sodium Chloride: 0.5 gm Phenol red: 0.0189 gm Autoclave at 115 o C for 15 mins. Inoculation of Bacterial Culture into the Phenol Red Carbohydrate Broth Aseptically inoculate each labeled carbohydrate broth with bacterial culture ( keep uninoculated tubes as control tubes). Incubate the tubes for 18-24 hrs. at 37 o C. Observe the reaction.

5. Precautions After inoculation into sugar, sterilize the loop in order to avoid cross contamination of the tube with other sugars. Keep uninoculated sugars tubes as control tubes. Do not use the tubes with Durham tubes that partially filled or with bubbles. Over incubation will help the bacteria to degrade proteins and will result give false positive results.

6. Result Acid production: Changes the medium into yellow color, organism ferments the given carbohydrate and produce organic acid there by reducing the pH of the medium into acidic.

7. Acid and Gas production: Changes the medium into yellow color-organism ferments the given carbohydrate and produce organic acids and gas. Gas production can be detected by the presence of a small bubbles in the inverted Durham tubes.

8. Absence of fermentation: The broth retains the red color. The organism cannot utilize the carbohydrate, but the organism continues to grow in the medium using other energy source in the medium.

9. E. coli and Klebsiella sp., shows both yellow color changes in the medium and bubble formation inside Durham’s tube in all the sugars. Type of organism Glucose Lactose Sucrose E. coli Acid /Gas Acid /Gas Acid /Gas Klebsiella sp. Acid /Gas Acid /Gas Acid /Gas

10. Salmonella species shows yellow color changes in the medium and gas bubbles formation in glucose, only yellow color change in sucrose and no reaction in lactose sugar.

11. 6. Pseudomonas sp. Shows yellow color change in glucose, no reaction in sucrose and lactose. 7. E. coli , Klebsiella sp. Ferment the sugars glucose, lactose and sucrose and giving the following results. Fig. (1) Fermentations of carbohydrate by certain type of bacteria

12. Table (7) Sugar fermentation by different bacterial species   https://1.bp.blogspot.com/-PIqg1UxoDyo/WdySP8pd-hI/AAAAAAAAAzw/egZCitygxsYny2Ev0Or-VUQqvShKxRoUwCLcBGAs/s1600/tab1.png Source: - senthilprabhu.blogspot.com/2017/10/carbohydrate...

13. Interpretation: Organism ferments the given carbohydrate and produces organic acids thereby reducing the pH of the medium into acidic condition. 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. The organism cannot utilize the carbohydrate, but the organism continues to grow in the medium using other energy sources in the medium.

14. References Cappuccino G .James, Sherman Natalie, Microbiology A laboratory manual, seventh edition, Pearson Education Morello A. Josephine, Mizer E. Helen, Granato A. Paul, Laboratory manual and work book in Microbiology Applications to patient care, seventh edition, McGraw-Hill Higher Education Prescott M. Lansing, Harley P. John, Klein A. Donald, Laboratory Exercises in Microbiology, fifth edition, McGraw-Hill college division senthilprabhu.blogspot.com/2017/10/carbohydrate... Prescott M. Lansing, Harley P. John, Klein A. Donald, Microbiology, sixth edition, McGraw-Hill Higher Education http://famsbc.wordpress.com/2009/07/28/carbohydrate-oxidation-fermentation  http://www.mesacc.edu/~johnson/labtools/Dbiochem/cho.html http://openwetware.org/wiki/BISC209:_Carbohydrate_Fermentation_Medium http://spot.pcc.edu/~jvolpe/b/bi234/lab/differentialMedia/CarboFermentationTest.html ] ttps://1.bp.blogspot.com/-PIqg1UxoDyo/WdySP8pd-hI/AAAAAAAAAzw/egZCitygxsYny2Ev0Or-VUQqvShKxRoUwCLcBGAs/s1600/tab1.pn   https://micro-biotech.blogspot.com/2014/10/carbohydrate-fermentation.html https://microbenotes.com/oxidation-fermentation-of-test/ https://www.researchgate.net/publication/330449115_Carbohydrate_ fermentation_test_starch_hydrolysis_test