8.2: Starch Hydrolysis w. Procedures and Results

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Starch is a polysaccharide which appears as a branched polymer of the simple sugar glucose. This means that starch is really a series of glucose molecules hooked together to form a long chain. Additional glucose molecules then branch off of this chain as shown below.

GLU
|
( ---GLU-GLU-GLU-GLU-GLU-GLU-GLU--- )n

Some bacteria are capable of using starch as a source of carbohydrate but in order to do this, they must first hydrolyze or break down the starch so it may enter the cell. The bacterium secretes an exoenzyme which hydrolyzes the starch by breaking the bonds between the glucose molecules. This enzyme is called a diastase.

( ---GLU / GLU / GLU / GLU / GLU / GLU / GLU--- )n
action of diastase

The glucose can then enter the bacterium and be used for metabolism.

Starch agar (one plate) See Fig. \(\PageIndex{1}\) .

Trypticase Soy broth cultures of Bacillus subtilis and Escherichia coli.

PROCEDURE (to be done in pairs)

1. Using a wax marker, draw a line on the bottom of a Starch agar plate so as to divide the plate in half. Label one half B. subtilis and the other half E. coli.

2. Make a single streak line with the appropriate organism on the corresponding half of the plate as shown in Fig. \(\PageIndex{2}\).

3. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section until the next lab period.

4. Next period, iodine will be added to see if the starch remains in the agar or has been hydrolyzed by the exoenzyme diastase. Iodine reacts with starch to produce a dark brown or blue/black color. If starch has been hydrolyzed there will be a clear zone around the bacterial growth (see Fig. 3A) because the starch is no longer in the agar to react with the iodine. If starch has not been hydrolyzed, the agar will remain a dark brown or blue/black color (see Fig. 3B).

Fig. \(\PageIndex{1}\) : Starch Agar	Fig. \(\PageIndex{2}\) : Inoculation of Starch Agar	Fig. \(\PageIndex{3A}\) : Starch Hydrolysis by Bacillus subtilis on Starch Aga	Fig. \(\PageIndex{3B}\) : No Starch Hydrolysis by Escherichia coli on Starch Agar

Starch agar before inoculation.		The clear zone around the growth of Bacillus subtilis no longer contains starch to react with the iodine. The starch was hydrolized by a diastase produced by the bacterium.	The starch was not hydrolized by the Escherichia coli on the left. The starch reacts with the iodine producing the dark color.
(Copyright; Gary E. Kaiser, Ph.D. The Community College of Baltimore County, Catonsville Campus CC-BY-3.0)

Results

When iodine is added to starch, the iodine-starch complex that forms gives a characteristic dark brown or deep purple color reaction. If the starch has been hydrolyzed into glucose molecules by the diastase exoenzyme, it no longer gives this reaction.

Flood the surface of the Starch agar plate with gram's iodine.

If the bacterium produced an exoenzyme that hydrolized the starch in the agar, a clear zone will surrround the bacterial growth because the starch is no longer there to react with the iodine (see Fig. \(\PageIndex{3A}\)).
If the bacterium lacks the exoenzyme to break down the starch, the agar around the growth should turn dark brown or blue/black color (see Fig. \(\PageIndex{3B}\)) due to the iodine-starch complex.

Record your results and indicate which organism was capable of hydrolyzing the starch (+ = hydrolysis; - = no hydrolysis).

Escherichia coli	Bacillus subtilis
Starch hydrolysis =	Starch hydrolysis =

Contributors and Attributions

Dr. Gary Kaiser (COMMUNITY COLLEGE OF BALTIMORE COUNTY, CATONSVILLE CAMPUS)