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 Plate Assay
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Materials
Culture of test
bacterium (This is the bacterium to be tested for bacteriocin production.)
Cultures of
indicator organism(s). (These are bacteria or fungi that will be inhibited
by the test bacterium.)
Sterile 0.45µm
membrane filter set-up
Nutrient agar
plate (One for each indicator organism)
Cork borer
(4-mm diameter)
Sterile Pasteur
pipette
Sterile cotton
swab
Procedure
- Grow a pure culture
of the test bacterium in an appropriate liquid culture medium.
- After good growth
is obtained, separate the cells from the growth medium by membrane
filtration. The medium will contain any bacteriocins secreted by the
test bacterium.
- Sterilize a cork
borer by autoclaving or disinfect it by rising in alcohol followed
by sterile water.
- Obtain a nutrient
agar plate and aspetically punch (4-mm) holes in the agar using a
cork borer. Obtain a nutrient agar plate and aseptically punch (4-mm)
holes in the agar using a cork borer.
- Swab the surface
of the agar with the indicator organism. Be sure to cover the entire
surface of the agar.
- Place 1, 2 or
3 drops of the filtered growth medium in the appropriate wells. Incubate
the plates and observe for zones of inhibition.
- Fill one well
with liquid culture medium without bacterial growth. This is a control.
- Incubate the
plate for 24 to 48 hr. and record the zones of inhibition about each
well.
- Discard all cultures
in the "To be autoclaved" area.
- You may have
to repeat steps 4 and 6 using different indicator organisms to find
the range of activity of your bacteriocin.
- After you have
found a bacteriocin and susceptible organism, you can determine the
minimum inhibitory concentration (MIC).
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