The carbohydrate fermentation test is utilized to assess a bacterium's ability to ferment specific carbohydrates, producing acids and gases. It involves anaerobic fermentation where changes in pH, detected by phenol red, indicate acid production, while gas formation is observed using inverted Durham tubes. Results reveal whether an organism ferments the carbohydrate and can suggest alternative energy sources when fermentation is absent.

1. CARBOHYDRATE
FERMENTATION TEST.
Yousef Elshrek

2. • Objective
1. The carbohydrate fermentation test is used to determine whether a bacterium can utilize
a certain carbohydrate or not.
2. It tests for the presence of acid and/gas produced from fermentation of a single
carbohydrate.
• Principle
1. Fermentation is the metabolic process to breaking down or catabolism of the sugars
under anaerobic conditions.
2. 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.
3. Microorganisms utilize the carbohydrates by their enzymatic reactions and produce
organic acids , acids, and gas.
4. The types and proportion of the products depend on species and carbohydrates.
5. 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.
6. Gas formation can be detected by using an inverted Durham tube

3. • Materials
1. Phenol red carbohydrate fermentation broth.
2. Bacterial culture.
3. Inoculation loop.
4. Incubator (37oC).
Procedure
• How to prepare Carbohydrate Fermentation Broth
1. Weight and dissolve trypticase, Sodium chloride, and phenol red in 100 ml distilled
water and transfer into conical flasks.
2. Add 0.5% to 1% the desired carbohydrate into all flasks.
3. Insert inverted Durham tubes into all tubes, the Durham tubes should be fully filled with
broth.
4. Sterilize in an autoclave at 115oC 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).
5. Transfer the sugars into screw-capped tubes or fermentation tubes and label properly.

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

5. • Precautions
1. After inoculation into sugar, sterilize the loop in order to avoid cross
contamination of the tube with other sugars.
2. Keep uninoculated sugars tubes as control tubes.
3. Do not use the tubes with Durham tubes that partially filled or with
bubbles.
4. 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. 1. 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. 1. 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. 1. 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:
1. Organism ferments the given carbohydrate and produces organic
acids thereby reducing the pH of the medium into acidic condition.
2. 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.
3. 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