Introduction to testing and the
reasons for doing tests
Many harmless bacteria live on
our skin and in our digestive system.
In our digestive system they
inhibit harmful bacteria from accumulating in our body and they help
produce useful nutrients for your body to absorb.
Gut bacteria have
a range of enzymes that can break down complex sugars and facilitate
lots other metabolic chemical reactions.
Unfortunately, we can be invaded
by harmful organisms e.g. pathogens like some bacteria that cause
disease.
Various laboratory methods have
been developed to culture (grow) potentially harmful bacteria and
test various chemicals to kill or counteract their harmful effects
i.e. how to test antibiotics and antiseptics-disinfectants.
You need to know how effective
an antibiotic or antiseptic chemical is.
Unfortunately, bacteria can
mutate and different strains emerge which are resistant to currently
used antibiotics, therefore there is a constant demand for the
pharmaceutical industry to develop new effective antibiotics.
The preparation agar plates has
already been described above in the first section, so here we pick
up the story with pre-prepared agar gel plates ready to test
antibiotics or antiseptics etc.
For more details on pathogens see
Keeping healthy - defence against
pathogens, infections, treating diseases, vaccination, new drugs and testing, monoclonal antibodies,
detecting diseases
gcse biology revision notes
NOTE: Agents for fighting against
pathogens
An
antibiotic drug kills bacteria
in the body.
Antiseptics kill bacteria
outside the body e.g. applied to your skin..
Disinfectants are
used to clean surfaces other than your body e.g. kitchen
worktops and toilets.
Preparation of the test samples and conducting
the experimental investigation.
The preparation of inoculated petri dishes
has already been described above and NOT incubated.
You can use petri dishes of agar jelly plus a
single selected bacteria to test the effectiveness of various
antibiotics, antiseptics and disinfectants in inhibiting and killing a
particular the selected bacterial growth.
It is very important that the strain of
bacteria chosen for the tests is representative of the
population of bacteria.
The disc-diffusion technique
You soak small circular paper discs (all the same
size) impregnated with different types of antibiotics/antiseptics, allow
them to drain, and place them on the surface
so they are spread out across an evenly bacteria coated
surface of the agar gel.
The bacteria must be very evenly spread out to make
it a fair test, and the antibiotic test discs spread out to allow
for the formation of inhibition zones
An inhibition zone is where the antibiotic is
effective in killing the bacteria (see diagram below, with a
fictitious bacteria strain and four fictitious antibiotics).
The petri dish and contents are for e.g.
48 hours at ~25oC after which it is ready to be examined
and the results analysed.
Using this set-up you can test antibiotics,
antiseptics and plant extracts (*) to investigate their
effectiveness in killing or inhibiting the growth of cultured
bacteria.
(* Some plants produce their own antiseptics
as part of their defence systems against pathogens))
The antibiotics/antiseptics (samples A1 to A4
on the diagram) soaked into the circular paper
discs will diffuse out into the agar jelly and may/may not kill the
bacteria.
If the antibiotic/antiseptic works the bacteria are killed,
inhibiting growth, a 'cleared' area will grow around the disc - called an
inhibition zone - see diagram above.
If the bacteria are resistant to the
antibiotic/antiseptic, the colony will continue to grow on the agar
gel around the paper discs.
It is important that the bacteria were
originally spread very evenly across the agar gel. If so, the
inhibition zones should be uniformly circular and there
should be uniform growth of the bacteria across the rest of the agar
plate.
Any agar plates should be discarded where
the zones are not circular or there is poor inconsistent growth
of the bacterial colony.
You can then make measurements if
everything seems ok.
You measure the diameter (radius = diameter /
2) of the inhibition zone with a mm ruler.
You than calculate the area of the inhibition
zone for a specific antibiotic from π x r2.
The bigger the inhibition zone, the more effective
is the antibiotic/antiseptic against the particular strain of bacteria
growing on the agar gel.
If you have an antibiotic/antiseptic resistant bacteria, then
the bacteria will continue to grow around the paper disc.
You can use this experimental procedure to test both
antibiotics and antiseptics.
Analysing the results
On the diagram
C is just a paper disc soaked in
sterile water to act as a control ...
... it should have no effect on bacterial
growth
... neither should it introduce any other
contaminating microorganism
... this is all about a fair test to show that
any inhibition is due to the antiseptic
... and any lack of inhibition is due the
antibacterial properties of the bacteria being investigated.
Antibiotic/antiseptic A1 is an ineffective
antibiotic with respect to the particular bacteria under
investigation - this bacterial strain is antibiotic-resistant with
respect to A1 only.
Antibiotic/antiseptic A2 has weakly antibacterial
action - small inhibition zone.
Antibiotic/antiseptic A3 is a 'moderately'
effective in its antibacterial action.
Antibiotic/antiseptic A4 is very effective in
killing this particular strain of bacteria - the largest inhibition
zone.
You can quantitatively measure the
effectiveness of the antibiotics/antiseptics by calculating the area of the dead
bacteria - better and more accurate than just a superficial
visual assessment.
You accurately measure, as best you can, the diameter of the circular
area with a ruler (e.g. in mm) where no bacteria are growing any
longer - see on the right of the experiment diagram above.
relative effect of antibiotic/antiseptic
= area
of circle = π x r2 e.g. in mm2.
(pi = 3.14, r = diameter/2)
Sample calculation of relative
effectiveness:
Suppose in the experiment the diameter of
the inhibition zones was 10 mm for test sample A3 and 20 mm for
sample A4.
Relative effect of A3 = 3.14 x (10/2)2
= 78.5
Relative effect of A4 = 3.14 x (20/2)2
= 314
314/78.5 = 4.0: therefore
antibiotic/antiseptic A4 is four times more effective than A3.
You can use the same diameter measurement
and calculation technique to calculate the area of a colony.
of a strain of bacteria.
Variations on the experiment
You can keep the antibiotic/antiseptic
constant and vary its concentration on the filter paper discs.
You can keep the antibiotic constant and coat
the agar surface with 'strips' of different strains of bacteria.
Alternatively, you can mix a specific antibiotic with the agar gel and then
treat the surface with various strains of bacteria.
You can then
measure the area of growth to test the effectiveness of the
antibiotic in killing that particular bacterium.
