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PRACTICAL 2

Screening for Amylase Producing
Bacteria









INTRODUCTION

In recent years, the potential of microorganisms as biotechnological sources of industrially
relevant enzymes has stimulated renewed interest in the exploration of extracellular enzymatic
activity in several microorganisms. Amylases are enzymes that are used to break down starch
into glucose. Amylases represent a sector of industrial enzymes, which cover approximately 25%
of the enzyme market including many industrial processes such as sugar, textile, paper, brewing
and pharmaceuticals. Commercial producing amylases are: 1) Bacillus subtilis 2) Bacillus
licheniformis.

Classification of amylases:
-amylase - is an endo-acting enzyme, catalyzing the random hydrolysis of internal -1,4
glycosidic linkages present in the starch substrate.
-amylase - is an exoenzyme or exoglycosidase, because it attacks the ends and produces
maltose in its -form.
-Amylase - amyloglucosidase is an exoacting hydrolase which releases single glucose
molecules from the non-reducing end of -(1-4) oligo or polysaccharides.

Application of amylase in industry:
Amylase enzymes were used in bread and roll to give these products a higher volume,
better colour, improve flavour and a softer crumb.
The second type of enzymes used in the formulation of enzymatic detergent is amylases,
and 90% of all liquid detergents contain these enzymes.
The use of -amylases in the pulp and paper industry is for the modification of starch of
coated paper, for example the production of low-viscosity, high molecular weight starch
In the textile industry, sizing of paper with starch is carrying out to protect the paper
against mechanical damage during processing

MATERIAL

Sample of material that you believe will contain starch digesting organisms (i.e. soil)
OBJECTIVES

Student will learn to isolate starch-degrading bacteria from soil using
a starch agar medium.
Student will learn to how to screen starch-degrading bacteria using
starch agar plate method.

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Universal bottle 2 per group
Sterile 9 ml dilution blanks - distilled water (4 per group)
LB agar + 1% starch-agar plate 4 per group
Micropipettors and tips (sterile) (1000 l)
Ziplock bag 1 per group
37C and 50C incubators
Parafilm
Iodine stain

METHOD

A. Screening of Amylase Producing Bacteria Isolates

1. Weigh out 1 gram of your soil and add this to 9 ml of sterile distilled water (1/10
dilution). Mix well.
2. Further dilute the suspension by removing 1 ml with a micropipette and add it to a test
tube containing 9 ml of sterile distilled water(1/10 dilution).
3. Repeat steps 2 to make a third dilution.
4. Using sterile technique, use a loop to inoculate the bacteria on the LB+starch agar.
(Carefull: If your loop is not cool enough, it will kill the bacteria as you streak them
around!).
5. Repeat steps 4 to make a second plate (duplicate plate).
6. Incubate one plate in inverted position/ upside down in the 37C incubator.
7. Place your other plate in a ziplock bag with and incubate inverted position/ upside
down in the 50C incubator. (Why in a zip-lock bag?)
8. The next day, examine the plates. Look to see if clear areas are developing around
certain colonies. You may have to use an Iodine stain to visualize starch degradation on
you plates.
9. Iodine solution was added to the plates, iodine solution was poured on the plates. Clear
zones around the colonies indicated the presence of amylase activity.
10. Photograph the colonies on your plates in the results section in your lab report.
11. Dont forget to subculture the positive bacteria culture for your next lab (practical 3).

B. Gram Staining Analysis of Amylase Producing Bacteria Isolates

Emulsify your sample taken from one colony with a sterile loop or needle in a very small
drop of saline on a clean glass slide. Spread it out to leave a thin film:
1. Allow smear to air dry. (Heating destroys the morphology of the cells.)
2. Flame quickly to fix the smear to the slide.
3. Place slide on wire rack, over the staining tray.
4. Flood the smear with CRYSTAL VIOLET; let stand 15 seconds.
5. Wash stain off with a gentle flow of water; drain.
6. Flood smear with GRAM'S IODINE; let stand 15 seconds.
7. Wash stain off with a gentle flow of water; drain.
8. Decolorize with ACETONE-ALCOHOL until the solvent flows colourlessly from the slide.
Use less solvent and time for thin films or for smears taken from liquid cultures.
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9. Wash off quickly with a gentle flow of water; drain.
10. Flood smear with SAFRANIN; let stand 15 seconds.
11. Wash stain off with a gentle flow of water; drain.
12. Blot smear with bibulous paper (NOT LENS PAPER) and let dry before viewing.


RESULTS, OBSERVATIONS AND DISCUSSIONS

Inhibition zones:

Observation:
Illustration Justification
Plate @ 37C




Plate @ 50C


DISCUSSION:
Discuss on your results.

QUESTIONS AND LEARNING ACTIVITIES:

1. Given the industrial microbiologist scenario of this exercise, what might be an advantage
of having a starch degrading bacteria that grows at high temperatures (i.e. 50C)?

2. How might you isolate a new strain of bacteria to degrade, or decompose oil from spills
in marine environments?

CONCLUSION

REFERENCE