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School biology: How to culture microorganisms - testing anti-bacterial agents

method of growing bacteria petri dish of agar gel/jelly for culturing microorganisms testing effectiveness of antibiotics antiseptics petri dish agar gel/jelly innoculated with microorganims

Culturing microorganisms like bacteria

How to grow microorganisms and

testing antibiotics and antiseptics-disinfectants

 Doc Brown's school biology revision notes: GCSE biology, IGCSE  biology, O level biology,  ~US grades 8, 9 and 10 school science courses or equivalent for ~14-16 year old students of biology 

 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 - 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

culturing bacteria microorganisms bacterial growth curve lag phase exponential growth phase stationary phase death phase

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.


To test the comparative effectiveness of antibiotics or antiseptics

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.

 


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.


Some associated pages

Keeping healthy - communicable diseases - pathogen infections   gcse biology revision notes

Keeping healthy - defence against pathogens, infections, treating diseases, vaccination, new drugs and testing, monoclonal antibodies, detecting diseases  gcse biology revision notes

Doc Brown's School Biology Revision Notes

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