Unit 8: Carbohydrate catabolism ability of microorganism

You will determine:

- how a microorganism uses glucose for energy supply
- if a microorganism has extracellular enzymatic activities on starch

Microorganisms will be used for test:

- Bacteria: Bacillus subtilis, Lactobacillus acidophilus
- Yeast: Saccharomyces cerevisiae
- Mold: Aspergillus oryzae

Culture media:
- OF-glucose (M9) for carbohydrate catabolism test: glucose (10 g/l), peptone (2 g/l), NaCl
(5 g/l), KH2PO4 (0.3 g/l), bromothyol blue solution 1 % (3 ml/l), agar (3 g/l). pH adjusted
to 7.1 ± 0.2
- Starch medium (M8) for exoenzyme activity test: peptone (3 g/l), solubilized starch (10
g/l), agar (20 g/l)
Other reagent:
- Lugol solution: for detection of zone of hydrolyzed starch

Each group of 2-3 students will prepare:

- 2-3 plates containing M8 medium
- 6 essay tubes containing 10 ml M9 medium
- 1 box of 1000 µl tips
- 1 erlenmeyer of 10 ml of sterilized liquid paraffin

Procedure to prepare culture media (for all class):

- Prepare 500 ml of M9 medium:
o Dissolve all ingredients completely
o Distribute into essay tubes
o Fill the Durham tubes with the medium and dispose into essay tubes
o Cover the tubes by cotton bud
o Take to autoclave
- Prepare 500 ml of M8 medium:
o Dissolve all ingredients (except agar) completely in Erlenmeyer
o Add agar
 o Cover the erlenmeyer by cotton bud or aluminum foil
o Take to autoclave
- Prepare the plates:
o Pour the autoclaved M8 medium into sterilized Petri disks (see Unit 6 for pouring
plate)
- Prepare sterilized liquid paraffin:
o Prepare 6 tubes of 15 ml liquid paraffin
o Cover with aluminum foil
o Take to autoclave

Procedure to test glucose catabolism ability of bacteria:

Principle: Organisms use carbohydrates differently depending on their enzyme complement.
Some organisms are capable of fermenting sugars such as glucose anaerobically, while others use
the aerobic pathway. Still others, facultative anaerobes, are enzymatically competent to use both
aerobic and anaerobic pathways, and some organisms lack the ability to oxidize glucose by either
in fermentation, substrates such as carbohydrates and alcohols undergo anaerobic dissimilation
and produce an organic acid (for example, lactic, formic, or acetic acid) that may be accompanied
by gases such as hydrogen or carbon dioxide. Facultative anaerobes are usually the so-called
fermenters of carbohydrates.
In M9 medium, after adjusted pH to 7.1, bromothyol blue gives the medium blue color. If the
bacteria catabolize the glucose and produce acid, so the culture will turn to yellow. Durham tube
can be used to test the ability of gas formation during fermentation.
At the bench:
- Inoculate 1 ml of bacteria culture into tubes. Prepare 2 tubes per strain: tube A and tube B
- Prepare the anaerobic condition for tube B: Add 1 ml of sterilized liquid paraffin into
tube B; eliminate the air in tube by heating the upper of tube on the flame; close with
cotton bud and cover by candle wax
- Incubate the tubes at 37oC for 48 h
- Read the result

Procedure to test extracellular enzymatic activities on starch of mold:

Principle: The medium is composed of nutrient agar supplemented with starch, which serves as
the polysaccharide substrate. The detection of the hydrolytic activity following the growth period
is made by performing the starch test to determine the presence or absence of starch in the medium.
Starch in the presence of iodine will impart a blue-black color to the medium, indicating the
absence of starch-splitting enzymes and representing a negative result. If the starch has been
hydrolyzed, a clear zone of hydrolysis will surround the growth of the organism.
At the bench:
- Inoculate the mold in the middle of the plate
- Incubate at 32oC for 48 h
- Dispose the Lugol solution over the colony and abounding zone
- Read the result

Glassware and materials need to be autoclaved and dried before use.

Please review:
- Unit 6 for preparation of glassware
- Unit 10 for preparation of solid medium and pouring plate.

Please read the document and solve the following questions before class:
- Distinguish between respiration and fermentation.
- Name and state the purpose of each component that comprises a carbohydrate
fermentation medium.
- State the different result can be obtained (color, gas) while the bacteria catabolize glucose
in M9 medium.
- Give a reason for what polysaccharide need to be hydrolyzed outside the cell?