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.
Sugar fermentation tests, Cetrimide agar medium, Hugh Leifson medium Shivam kumar Sriwas
Combined presentation on:-
1. Sugar fermentation tests
2. Cetrimide agar
3. Hugh Leifson medium
Covering principle, preparation of the medium, test protocol, result interpretations, test conclusions.
Capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Staining such an layer is difficult with the normal stains so it is necessary to stain the background and the cell itself which makes the capsule appear colourless.
Sugar fermentation tests, Cetrimide agar medium, Hugh Leifson medium Shivam kumar Sriwas
Combined presentation on:-
1. Sugar fermentation tests
2. Cetrimide agar
3. Hugh Leifson medium
Covering principle, preparation of the medium, test protocol, result interpretations, test conclusions.
Capsule is an layer around the bacteria cell which gives bacteria the protection and pathogenicity. Staining such an layer is difficult with the normal stains so it is necessary to stain the background and the cell itself which makes the capsule appear colourless.
Biochemical analysis of unknown bacteriaAerotolerance TeChantellPantoja184
Biochemical analysis of unknown bacteria
Aerotolerance Test
Fluid Thioglycollate broth (FTB) is a medium designed to test the aerotolerance of bacteria.
Along with nutrients to support bacterial growth, it contains sodium thioglycollate, thioglycollic acid, L-cystine, methylene blue, and 0.05% agar.
The sodium thioglycollate, thioglycollic acid, and L-cystine reduce the oxygen to water.
Methylene blue is an indicator that is colorless in an anaerobic environment and greenish-blue in the presence of oxygen.
The agar helps retard oxygen diffusion and helps maintain the stratification of organisms growing in different layers of the broth.
Oxygen is driven out of the broth by autoclaving, but as the broths sit at room temperature, oxygen begins to diffuse back into the tube.
Obligate aerobes will only grow in this oxygen-rich top layer. On another hand, obligate anaerobes will only grow in the lower areas of the tube. Microaerophiles will grow in a thin layer below the richly-oxygenated layer. Facultative or aerotolerant anaerobes can grow throughout the medium but will primarily grow in the middle of the tube, between the oxygen-rich and oxygen-free zones
Reactions typically take up to 1-2 days to develop at 37⁰C
Media is inoculated using an inoculating loop
(A) Escherichia coli and (C) Staphylococcus aureus: both are Facultative Anaerobe, grows both aerobically and anaerobically and growth is seen throughout the tube. Some are capable of growth respiring with oxygen and anaerobically by fermentation.
(B) Clostridium botulinum: Obligate Anaerobe: can not grow in the presence of oxygen, growth is seen approximately 1/4 to 1/2 of the way from the top of the tube.
(D) Neisseria sicca: Microaerophile, requires oxygen but at concentrations below atmosphere, grows just below the surface of the media but not at the top.
(E) Pseudomonas aeruginosa: Obligate Aerobe: oxygen is required for growth and grows at the top of the tube only. The Organism will “settle” and sink into the media if grown longer than 24 hrs.
Aerotolerance Test
Phenol red test
Phenol red broth is a differential test medium prepared as a base to which a carbohydrate such as sucrose, lactose, dextrose or glucose is added.
Included in the base medium are peptone and the pH indicator is phenol red. Phenol red is yellow below pH 6.8, pink to magenta above pH 7.4, and red in between. During preparation, the pH is adjusted to approximately 7.3 so it appears red.
Deamination of peptone amino acids produces ammonia which rises the pH and turns the broth pink.
An inverted Durham tube is added to each tube as an indicator of gas production.
Gas production, also from fermentation, is indicated by a bubble or pocket in the Durham tube where the broth has been displaced.
Acid production from fermentation of the carbohydrate lowers the pH below the neutral range of the indicator and turns the medium yellow. Deamination of peptone amino acids produces ammonia which rises the pH and ...
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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