This activity involves skills for safe handling of microbial material.

Students compare the activity

of an enzyme extract with that of two enzyme-producing bacteria and can consider the industrial
importance of enzymes and microbes.

This practical is based on an investigation called Breakdown of starch by

microbes published in Practical Microbiology for Secondary Schools © Society for General
Microbiology.

Give each working group (1-2 students) a starch-nutrient agar plate. They will have to use sterile
paper discs to collect samples of two bacteria and one enzyme extract, all using sterile technique.
Organise the work so that not all the groups want bottle A at the same time!

Written information about industrial processes using microbes and enzymes, particularly amylases
for starch processing, would be a useful resource for this lesson. See Web links at end.

There is scope here for developing skills of aseptic technique to become the focus as well as
exploring the enzyme activity.

Forceps (1)

Sterile paper discs (4)

Ruler (1)

Marker pens to label the plates

Tape to close the plates (Note 3)

Bacillus subtilis nutrient broth culture, 2 cm3 in a bottle/ test tube

Escherichia coli nutrient broth culture, 2 cm3 in a bottle/ test tube

Starch-nutrient agar plates, 2 per group (Note 1)

VirKon solution 1% w/v (see manufacturer’s instructions) (Note 2)

Bottles of sterile distilled water, 1 per 4 groups

Bottles of 0.1% amylase solution, 1 per 4 groups

Carry out a full risk assessment before planning any work in microbiology (Note 1 for more details).
Check the standard techniques for more details of Aseptic techniques, Making up nutrient
agars, Pouring an agar plate, Maintaining and preparing cultures and Incubating and viewing plates
safely.
Wear eye protection when handling iodine solution to test for starch.

Read our standard health & safety guidance

1 Before embarking on any practical microbiological investigation carry out a full risk assessment.
For detailed safety information on the use of micro-organisms in schools and colleges, refer to Basic
Practical Microbiology – A Manual (BPM) which is available, free, from the Society for General
Microbiology (email education@sgm.ac.uk) or go to the safety area of the SGM website
(http://www.microbiologyonline.org.uk/teachers/safety-information) or refer to the CLEAPSS
Laboratory Handbook, section 15

2 Make nutrient agar into starch-nutrient agar following the recipe in Standard techniques: Making up
nutrient agars.

3 Suitable disinfectants include sodium chlorate(I) (hypochlorite) at concentrations providing 1000

ppm available chlorine for general surface cleaning, or 2 500 ppm chlorine for discard pots, or
VirKon (follow manufacturer’s instructions).

4 Make up 0.1% amylase solution or use purchased stock. See CLEAPSS Hazcard and Recipe
card. Powdered enzyme is harmful, but solutions less than 1% are considered low hazard. Make up
fresh for each day of use as it degrades over time.

5 Make your own assay discs by punching discs around 6mm diameter from filter paper, or
chromatography paper using a cork borer or a hole punch. Wrap in aluminium foil, in sets of four,
and sterilise by autoclaving.

6 Make up iodine solution. See CLEAPSS Hazcard and Recipe card. A 0.01M solution is suitable for
starch testing. Make this by 10-fold dilution of 0.1M solution. Once made, the solution is a low
hazard but may stain skin or clothing if spilled and may irritate the eyes.

SAFETY:
Handle all cultures of microbes according to good laboratory practice.

Wear eye protection when handling iodine solution.

Preparation

a Inoculate nutrient broth with fresh cultures of Bacillus subtilis and Escherichia coli at least 48 hours
before use. (See Standard technique: Maintaining and preparing cultures)
b Calculate the quantity required and prepare just enough starch-agar for the investigation (12-15 ml
for normal depth in a 90 mm Petri dish). Any surplus will keep for 6-12 months in tightly-sealed
screw-top bottles if sterile.

c Prepare a suitable solution to disinfect the work area for the investigation and afterwards (Note 3).

d Prepare 0.1% amylase solution (Note 4).

e Sterilise distilled water in enough McCartney/ Universal bottles.

f Paper discs can be purchased (Whatman Antibiotic Assay discs) or you can make your own (Note
5).

g Stop the growth of the culture before the students inspect the plates. Refer to standard
procedure Incubating and viewing plates.

h Make up iodine solution (See CLEAPSS Hazcard and Recipe card).

Investigation

a Keep one starch-nutrient agar plate unopened as a control. Flood with iodine at the same time as
the other plates.

b Provide each working group with one starch-agar plate.

c Students use a marker pen to mark four sections on the base. Label the sections A, B, C, and D.
Write a key to record the treatment of each disc, for example

Area Treatment

A Bacillus subtilis

B Escherichia coli

C 0.1% amylase solution

D Distilled water
d Pass the forceps through a Bunsen burner flame. Allow them to cool and use them to pick up one
of the paper discs.

e Open a bottle containing bacterial culture (or remove the cotton wool stopper from the test tube).
Flame the neck and dip the disc into the broth. Allow excess to drip off. Flame the neck again and
replace the top or stopper. Transfer the disc to the middle of the correct section on the agar plate.

f Repeat d and e using another disc and the other bacterial culture.

g Repeat d and e using another disc and the amylase solution.

h Repeat d and e using another disc and the distilled water.

i Place the forceps in a discard beaker.

j Tape up the dish and incubate the plates (see Standard techniques).

k After 2-3 days, technically-qualified staff will kill the culture.

l Open the Petri dish and use a dropper to put in just enough iodine solution to cover the surface of
the agar. Replace the lid.

m Put the dish on squared paper. Measure and record the diameter of any clear zones around the
discs. A wider diameter suggests a greater concentration of amylase.

This protocol introduces a quantitative assay technique for enzyme activity. It is worth discussing the
value and limits of this as a technique.

The fact that enzymes (of microbial origin) will digest starch to produce sugars is important to the
starch-processing industry – manufacturing sugars and syrups for foods, beverages and possibly
biofuels. A text-based exercise, such as gathering useful information and making notes from a
complex source, would be a useful companion to this practical work.

Amylase breaks down starch molecules to sugars. We produce amylase (in our saliva and
pancreatic secretions) to break down the starch we eat. Some microbes also produce amylase.
These enzymes are secreted by microbes to digest large external starch molecules. The microbes
then absorb the sugars produced, because they are small enough to pass into their cells.

Health & Safety checked, May 2009