Introduction
You can grow bacteria, and other microorganisms
safely in a school or college
laboratory by using the correct procedures.
You can then test the cultures of the bacteria for the
effectiveness of various antibiotics, antiseptics and disinfectants in
inhibiting and killing a particular bacterial growth - this is described
in the 2nd section of this page on growing microorganisms.
The setup - equipment and materials (diagram ==>)
All equipment must be sterilised before use
to exclude the growth of any other bacteria except those you are
experimentally interested in, it also prevents contamination of the
laboratory environment.
The experiments are conducted in petri dishes - shallow round
plastic/glass containers over which a tight fitting lid can be fitted.
The bacteria are grown ('cultured') in a culture medium such as agar
jelly (gel) which contains the necessary food for the
microorganism-bacteria to grow.
The agar gel (nutrient broth solution) contains carbohydrates,
minerals, proteins and vitamins mixed with water - the bacteria cannot
grow without water containing nutrients, but it must be jelly like
nutritious broth.
In the pharmaceutical industry uses the
Mueller-Hinton agar for testing antibiotics.
One recipe uses a mix of beef, milk
protein and blood and is good for culturing human pathogens.
When inoculated with a pathogen, the agar
plates would be incubated at ~37oC (the human body's
temperature).
In schools the agar gels would never
be incubated above 25oC because of the risk of
growing colonies of pathogens.
Hot fluid agar jelly is poured into the Petri dish
and left to cool and set to a firm gel-like state.
The selected microorganism to be investigated can then be
transferred onto the surface of the culture medium using an inoculating
loop which is previously sterilised by heating it in a bunsen burner
flame.
Some liquid bacterial cultures will be in vials,
these should be opened for the minimum time as you spread the culture
over the gel to prevent any other microorganism from the air getting in.
The lid is placed over the petri dish and
secured with a bit of tape, but not completely sealed with adhesive tape.
The
culture (agar gel + microorganism in the petri dish) can be
incubated at an appropriate temperature to encourage the bacteria to
grow and multiply.
The plates are incubated upside down to stop drops of condensation
falling onto the agar surface.
When a particular bacteria is spread over the surface of the agar gel
(e.g. with an inoculating loop) you will see colonies growing
that will eventually spread over the whole surface, hopefully giving an
even coating of the selected bacteria as it multiplies.
With an ample supply of nutrients and an
appropriate temperature, the bacteria will multiply by binary
fission where one cell splits in two.
This continuous division in a live bacterium
results in colony that spreads across the agar gel.
A colony contains millions of bacteria.
A bacterium can have an average division time
of minutes e.g. 15 mins and the maths is scary!
I don't mean its difficult, but in 5 hours
(300 mins), there can be 600/15 = 20 divisions.
Therefore after 5 hours there will be 220
over 1 million bacteria!
(theoretically microorganism 1 048 576 cells,
but some may die in the process)
Note on bacterial growth curves
You can follow the growth of a bacterium
colony over many hours and end up with this kind of graph.
1. The lag phase:
For the initial lag phase there is no cell
division, but the bacteria are copying their DNA and synthesising
the necessary proteins.
2. The exponential growth phase:
Lots of food available, so cell division rapidly takes place
and the number of bacteria can double in a relatively short time
producing the 'acceleration' in the graph line.
3. The stationary phase
Growth of the colony cannot continue to
accelerate because the nutrient resources are becoming depleted.
Eventually, the rate of bacterial growth is equal to the rate of
bacterial death and the graph line becomes horizontal. If you
introduce more food, the colony can grow again, but, otherwise ...
4. The death phase:
Not only are resources being reduced, but
bacteria produce toxins as a waste product, poisoning the live
bacteria and the colony steadily declines in number.
For more on cell division see:
Cell division - including
binary fission, maths involved and bacterial growth curves
Safety notes and ensuring uncontaminated
cultures prior to testing 'antibacterial agents'
Cultures of microorganisms should not be kept
above 25oC because there is less chance of harmful pathogens
(microorganisms that cause disease) growing at the cooler temperatures.
In research laboratories in universities and
industry, cultures can be safely incubated at higher temperatures to
grow them faster - time is money!
If the culture is contaminated with unwanted
microorganisms, these will affect your results and some of them maybe
pathogens too!
Precautions to be taken - the
use of aseptic techniques
Aseptic techniques are designed to
avoid contamination by unwanted microorganisms which could
affect your results i.e. the growth of pathogens.
Disinfect all work surfaces in the
laboratory - alcohol is very effective, but very inflammable!
The agar gel, all glassware and other equipment must
be sterilised before use.
The Petri dishes and culture medium - agar
gel, must be all sterilised before conducting the experiment by
heating to a high temperature e.g. >100oC.
This can be done in an autoclave which
uses steam at high pressure to kill any
microorganisms/pathogens present.
The higher temperature should kill any
unwanted microorganisms.
The metal inoculating loop is sterilised
by placing it in a roaring blue bunsen flame until it glows red
- no microorganism will survive this heat treatment!
The liquid bacterial cultures should be
kept in special culture vials with lids.
The lids should only be removed
briefly, when transferring the bacteria to the petri dishes,
to stop other microorganisms getting in.
You can briefly flame the neck of a
glass container of bacteria just after its opened and just
before its closed - the hot convecting air moves air out of
the container preventing microbes in the air getting in.
When the Petri dish is ready with the agar
gel added and set, a lightly taped lid should be placed on it to
stop any microorganisms in air getting in.
The Petri dishes should be stored upside
down to prevent drops of condensation falling on the agar jelly.
The prepared agar gel petri dishes
must not be incubated in case other bacteria begin to
grow.
After use the agar plates should be
sterilised in an autoclave to complete the steps to avoid
contamination - to complete the aseptic procedure.
An autoclave is a device that
uses steam under pressure to kill harmful bacteria, viruses,
fungi, and spores on items that are placed inside the heated
pressurised vessel. The items to be sterilised are heated to
an appropriate sterilization temperature for a given amount
of time.
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.
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