Carbohydrate Fermentation Test

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Carbohydrate
Carbohydrates are biological molecules which are made up of carbon, hydrogen and oxygen
molecules.

● Monosaccharides
●Monosaccharides are simple molecule of sugar. It is water-soluble and crystalline in nature.
Examples are glucose, fructose, glyceraldehyde and galactose.
● Diasaccharides
●Diasaccharides is a simple carbohydrate formed when two monosaccharide molecules are
joined together and a water molecule is removed. Examples are lactose,sucrose and maltose.
● Oligossacharides
●Oligosaccharide is a polymer containing small number of monosaccharide.monosaccharide.
Examples are fructo-oligosaccharide, galacto-oligosaccharide and mannan.
● Polysaccharide
●Polysaccharide are complex carbohydrate containing long chain of monosaccharides.Examples
are starch,glycogen,cellulose and chitin.
 Fermentation
●is a metabolic process in which bacteria or yeast
convert substrate into a product like acid, gas or
alcohol.
●Carbohydrate fermentation tests demonstrate
fermentation of sugars like glucose, lactose or sucrose.
The fermentation is noted by acid and gas production
by bacterial cells.
 Objective
●To find the ability of microorganisms to ferment the
given Carbohydrate.
●To determine the ability of microorganism to produce
gaseous end products in fermentation.
Contains: single carbohydrate peptone broth with durham tube for gas
collection, Phenol red pH indicator: alkaline pH = red, acidic pH =
yellow
Discriminates the ability to ferment a single carbohydrate (glucose,
lactose, or mannitol) into acid products (e.g. pyruvic acid) or acid
plus gas
Results: Red = inert, negative for fermentation of specified
carbohydrate
Yellow = positive for fermentation of carbohydrate to
acid products
Yellow with bubble = positive for fermentation of
carbohydrate to acid + gas
 Materials
1.Nutrient broth medium
●

a) Glucose broth medium

●

b) Sucrose broth medium

●

c) Lactose broth medium

●

2. Indicator – Phenol red or Andrade’s indicator.

●

3. Durham’s tube
●

4. Bacterial cell culture

●
 Composition of Sugar Broth Media

Peptone 1 gm
Meat extract 0.3 g
NaCl 0.5 gm
Distill water 100 ml
Indicator 0.008 gm
Sugar(glucose/lactose/sucrose) 0.5 gm
 Procedure

1. Take three different tubes containing three different types of sugar broth and invert a
Durham’s tube in it and screw cap the tubes.
2. Further the three sugar broth tubes are sterilized by autoclaving.
3. After sterilization, the tubes are cooled down to room temperature and inoculated
with a cell suspension in aseptic condition.
4. The tubes are incubated at 37°C for 24 hours.
5. After incubation, the tubes are examined for acid and gas production and results are
noted down.
 Mechanism

1. The three sterile sugar broth tubes are inoculated with bacterial suspension and
incubated for 24 hours. During incubation the bacterial cells may or may not utilise
sugar.
2. If the cells utilize sugar, there is acid and gas production and this change is observed
by change in colour of broth.
3. First of all, in sugar broth phenol, red indicator is added this pH sensitive indicator
is added for determination of acid production. Initially the broth tubes are red in colour
and after incubation, if there is acid production, the colour of broth changes from red to
yellow. If there is no change in colour of broth, then there is no acid production.
4. Secondly in sugar broth, a Durham’s tube is inverted to determine gas production.
After incubation, if a gas bubble is observed then there is gas production.
Note
●

– Phenol red is a pH sensitive indicator its pH range is at acidic pH it is yellow in

colour and at alkaline pH it is red in colour.

Observation
●

– There are three possibilities and they are after incubation

● If red colour of broth is observed then there is no fermentation.

● If yellow colour of broth is observed then there is acid formation.
● If yellow colour of a broth plus a gas accumulation in Durham’s tube is
observed then there is acid plus gas formation.
Result

● If there is no change in colour of a broth then the test is negative.

● If yellow colour of a broth is observed, fermentation is carried out resulting in acid
production and it is indicated by + sign
● If yellow colour of a broth along with gas bubble accumulation is observed then
fermentation is carried out resulting in acid and gas production.
1. Acid production: Changes the medium into yellow color- organism ferments the given
carbohydrate and produce organic acids there by reducing the ph of the medium into acidic.
2. 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 etected by the presence of
small bubbles in the inverted durham tubes.
3. 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 sources in the
medium.
 Glucose Lactose Sucrose

E. coli AG AG V

P. vulgaris AG - AG

P. aeruginosa - - -

S. aureus A A A