Standard Procedure SP 0004:2005

A method for evaluating the effectiveness of anti-microbial products

1 Scope

This Standard Procedure tests the anti-microbial effects of antiseptics and/or disinfectants. It can be used to compare different anti-microbial products, or the same product at different dilutions. Repeating the test with different cultures compares the effectiveness against different micro-organisms.
The plate pouring procedure is adapted from BS 4285-2.1 and similar standards.

2 Definitions

micro-organisms (microbes)
bacteria, yeasts and moulds

anti-microbial
a chemical product that kills microbes, or prevents their growth

antiseptic
an anti-microbial product designed to prevent sepsis (infection of a wound)

disinfectant
an anti-microbial product designed for use on objects (e.g. equipment) and surfaces (e.g. laboratory or kitchen worktops, toilets, floors and walls)

zone of inhibition
the clear area around a specimen disc, where no microbes have grown

3 Principle

Test solutions soaked into discs of filter paper are placed on the surface of an inoculated culture plate. After incubation, a clear zone around a disc shows that the chemical on that disc has anti-microbial properties. The chemical has diffused out of the disc and through the agar, preventing the growth of micro-organisms.

4 Apparatus

sterile apparatus:
- bottle containing 20 cm³ of molten nutrient agar, suitable for micro-organism being tested
- Petri dish
- dropper
bacterial culture (such as Bacillus subtilis, Escherichia coli, or Micrococcus luteus) Caution: biohazard
plain antibiotic assay discs (or 5 mm diameter discs of filter paper)
forceps
marker pen to write on Petri dish
adhesive tape
incubator set at 30 ±1^oC

5 Test Specimens

Depending on the purpose of the test, use either:

Three different concentrations of the same antiseptic or disinfectant, for example:
A - manufacturer’s recommended concentration
B - diluted to half strength with distilled water
C - diluted to quarter strength with distilled water
or:
Three different antiseptics or disinfectants at the manufacturer’s recommended concentration, labelled A to C.

6 Procedure

IMPORTANT: Before using this procedure you must be familiar with aseptic techniques, which must be used throughout.

Preparing the culture plate

Keep the molten agar at 45 to 47^oC until you are ready to use it.
Place the sterile Petri dish on the bench, lid upwards.
Using the sterile dropper, put 5 drops of bacterial culture into the dish. Add the 20 cm³ of molten agar. Replace the lid.
Immediately mix the contents by gently sliding the dish over the bench with the following movements:
- five times from side to side;
- five times in a clockwise circle;
- five times back and forth (at 90^o to the side to side movements);
- five times in an anticlockwise circle.
Leave the plate to set.

Testing the product(s)

When the agar plate is fully set, turn the dish over. Mark out the base with four quadrants (quarter circles) labelled A to D at the centre of the dish. Turn the dish lid upwards again.
Using forceps, dip a 5 mm assay disc into distilled water. Let the surplus liquid drain off. Place the disc onto the surface of the agar in the middle of quadrant D. Gently press it down with the forceps. This is the control specimen.
Repeat this with test specimen C instead of distilled water, then with B, then A.
NOTE: If using three concentrations of the same product, it is important to handle them in the correct sequence – from C (most dilute) to A (most concentrated).
Tape down the lid at four points at 90o to each other. (Do not seal it completely, since that would cause anaerobic conditions.)
Incubate the dish, with the lid upwards, at 30 ± 1^oC for 48 hours. (Do not turn it lid downwards, or the paper discs may fall off.)
After incubation, examine the culture plate without opening it. Measure the zone of inhibition, if any, around each disc. If the zones are all circular, measure their diameters. Otherwise, measure their areas, (for example, by standing the dish on graph paper and counting the squares beneath each zone).
When finished with, dispose of the culture plate as instructed by your teacher / supervisor.

7 Expression of Results

Record the name and concentration of specimens A to D, and the diameter (in millimetres) or area (in square millimetres) of the inhibitions zone, if any, for each.

8 Test Report

Your test report should include:

(a) reference to this Standard Procedure;

(b) the size of the inhibition zone produced by each specimen;