Culturing microorganisms like bacteria

and testing antibiotics and antiseptics

Doc Brown's Biology Revision Notes

Suitable for GCSE/IGCSE/O level Biology/Science courses or equivalent

 

 This page will help you answer questions such as ...

 How do you grow microorganisms safely in a laboratory?

 How do you fairly test the effectiveness of antibiotics?

 How do you analyze the results of testing antibiotics?



How to grow bacteria in the laboratory

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.

The setup - equipment and materials (diagram ==>)

The experiments are conducted in glass 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 jelly (nutrient broth solution) contains carbohydrates, minerals, proteins and vitamins.

The culture medium can be a nutrient broth solution or the semi-solid agar gel.

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 must then be transferred onto the surface of the culture medium.

You can use a dropping pipette and spreader to get an even coating of bacteria across the surface of the agar jelly.

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.

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!

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.


To test the comparative effectiveness of antibiotics or antiseptics

NOTE: An antibiotic kills bacteria in the body.

Antiseptics kill bacteria outside the body e.g. on your skin or disinfecting a worktop in the kitchen.

Preparation of the test samples and conducting the experimental investigation

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.

You soak small circular paper discs (all the same size) with different types of antibiotics/antiseptics and place them on the surface so they are spread out across a evenly bacteria coated surface of the agar gel.

The bacteria must be evenly spread out to make it a fair test, and the antibiotic test discs spread out to allow for the formation of inhibition zones - 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 left 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.

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/antiseptics/ 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 the concentration.

You can keep the antibiotic constant and coat the agar surface with 'strips' of different strains of bacteria.

Alternatively, you can mix an 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.

 


Practical work in schools and colleges - health and safety considerations

  • Uncontaminated cultures of microorganisms are required for investigating the action of disinfectants and antibiotics.

    • For this:

      • Petri dishes and culture media must be sterilised before use to kill unwanted microorganisms.

      • Inoculating loops used to transfer microorganisms to the media must be sterilised by passing them through a flame.

      • The lid of the Petri dish should be secured with adhesive tape to prevent microorganisms from the air contaminating the culture.

  • In school and college laboratories, cultures should be incubated at a maximum temperature of 25 C, which greatly reduces the likelihood of growth of pathogens that might be harmful to humans.

  • In industrial conditions higher temperatures can produce more rapid growth of unwanted, potentially harmful microorganisms.

  • Any practical work and investigations you did should also be revised - good context material for exam questions! See below!

Hopefully your school practical work will include the following (which should also be revised, helps in understanding 'how science works' and context examination questions):

  • Investigate the effectiveness of various antibiotic discs in killing bacteria.

  • Growing microorganisms in Petri dishes to demonstrate sterile technique and growing pure cultures.

    • Microorganisms are cultured in a culture medium which usually consists of agar jelly containing carbohydrates, minerals, proteins and vitamins that supply all the nutrients needed for cell growth.

  • Using pre-inoculated agar in Petri dishes to evaluate the effect of disinfectants and antibiotics.

    • The hot liquid agar jelly is poured into shallow Petri dishes to cool and set - just like a jelly!

    • The Petri dishes should be covered with an air-tight lid to stop microorganisms from the surrounding air contaminating the experiments.

    • Wire loops, sterilised in a hot flame, are used to transfer microorganisms onto the agar jelly, where they multiply producing many colonies quite rapidly - lots to feed on!

      • If the wire loop is not sterilised, other bacteria may contaminate the experiment and these other microorganisms will confuse the results.

    • Little bits of porous paper (filter paper?) soaked in different antibiotics are placed on the bacterial colonies on the jelly.

    • You can then see which antibiotics kill what bacteria, but the antibiotic-resistant bacteria will continue to grow.

    • In the pharmaceutical and medical industries where extremely dangerous pathogens are being investigated, extremely strict health and safety regulation is essential for both the safety of workers and members of the general public.

  • Seeing computer simulations to model

    • the effect of ...

      • (i) the growth of bacterial colonies in varying conditions,

      • (ii) the action of the immune system and the effect of antibiotics and vaccines.


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