Exercise 9

Tests for Carbohydrate Fermentation

Fermentation is an anaerobic degradation of carbohydrates in which the final electron acceptor is an organic
molecule. Fermentation test is a common laboratory procedure in microbiology to determine which specific carbohydrate
can a certain microorganism ferment or degrade.Conventionally, there are three basic requirements needed to do the said
test and these are the specific carbohydrates of interest, pH indicator and an inverted tube (usually a Durham tube) to trap
gas production as a result of such process. During the process of in vitro fermentation, microorganisms would degrade the
carbohydrates that would turn the medium acidic. Hence, the presence of appropriate pH indicator is necessary to
determine if fermentation has indeed taken place.Carbohydrate fermentation is also invaluable for determining sugar
utilization of some bacteria. This may aid microbiologists in the probable identification of their suspected organisms.

Leaming Outcomes:

At the end of the exercise, it is expected that the student must be able to:
1. Understand and apply the principles of various methods of carbohydrate fermentation tests.
2. Perform various methods carbohydrate fermentation tests.
3. Interpret correctly the results of various methods of carbohydrate fermentation tests.

A. Sugar Fermentation

The action of many species of microorganisms on a carbohydrate substrate result in the acidification of the
medium. A basal fermentation medium typically includes tryptic hydrolysate of casein (source of carbon and nitrogen),
sodium chloride (osmotic stabilizer), phenol red (indicator)and carbohydrate to be tested (0.5% - 1% concentration).

Materials:
1. Triple sugar iron tubes
2. Alcohol lamp
3. Inoculating needle
4. Test organisms (Escherichia coli, Pseudomonas aeruginosa and Proteus vulgaris)

Procedure:
1. Inoculate TSI by stabbing the medium through the butt or deep and the slant is streaked in a zigzag manner.
2. Incubate overnight at 35-37℃.
3. Observe for color reactions (acid = yellow, alkaline = red or orange):
a. Alkaline slant, alkaline butt-no sugars fermented
b. Alkaline slant, acid butt-Only glucose is fermented, lactose and sucrose not fermented
c. Acid slant, acid butt - glucose and lactose (or sucrose in TSI) fermented.