Culturing microorganisms like bacteria - testing antibiotics

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 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 in a school/college laboratory. 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 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 Petri dishes and culture medium - agar gel, must be all sterilised before conducting the experiment by heating to a high temperature e.g. ~100oC ???

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!

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

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 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.

The antibiotics soaked into the circular paper discs will diffuse out into the agar jelly and may/may not kill the bacteria.

If the antibiotic works the bacteria are killed, inhibiting growth, a 'cleared' area will grow around the disc - an inhibition zone - see diagram above.

The bigger the inhibition zone, the more effective is the antibiotic against the particular strain of bacteria.

If you have an antibiotic resistant bacteria, then the bacteria will continue to grow around the paper disc.

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 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 A2 has weakly antibacterial action - small inhibition zone.

Antibiotic A3 is a 'moderately' effective in its antibacterial action.

Antibiotic A4 is very effective in killing this particular strain of bacteria - the largest inhibition zone.

You can quantitatively measure the effectiveness of the antibiotics by calculating the area of the dead bacteria - better and more accurate than just a superficial visual assessment.

You measure the diameter of the circular area with a ruler (e.g. in mm) where no bacteria are growing any longer.

relative effect of antibiotic = area of circle = π x r2 e.g. in mm2. (pi = 3.14, r = diameter/2)

 

Variations on the experiment

You can keep the antibiotic 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.

 


  Keywords for gcse biology revision notes on culturing microorganisms like bacteria - testing antibiotics: GCSE 9-1 biology biological science IGCSE revision notes culturing microorganisms like bacteria - testing antibiotics KS4 biology Science notes on culturing microorganisms like bacteria - testing antibiotics GCSE biology guide notes on culturing microorganisms like bacteria - testing antibiotics for schools colleges academies science course tutors images pictures diagrams for culturing microorganisms like bacteria - testing antibiotics science revision notes on culturing microorganisms like bacteria - testing antibiotics for revising biology modules biology topics notes to help on understanding of culturing microorganisms like bacteria - testing antibiotics university courses in biological science careers in science biology jobs in the pharmaceutical industry biological laboratory assistant apprenticeships technical internships in biology USA US grade 8 grade 9 grade10 AQA GCSE 9-1 biology science notes on culturing microorganisms like bacteria - testing antibiotics GCSE notes on culturing microorganisms like bacteria - testing antibiotics Edexcel GCSE 9-1 biology science notes on culturing microorganisms like bacteria - testing antibiotics for OCR GCSE 9-1 21st century biology science notes on culturing microorganisms like bacteria - testing antibiotics OCR GCSE 9-1 Gateway  biology science notes on culturing microorganisms like bacteria - testing antibiotics WJEC gcse science CCEA/CEA gcse science gcse biology revision notes on culturing microorganisms like bacteria - testing antibiotics

KS3 SCIENCE QUIZZES ALPHABETICAL INDEX
GCSE grade 9-1 & IGCSE CHEMISTRY Doc Brown's Travel Pictures & Notes
ADVANCED LEVEL CHEMISTRY [SEARCH BOX] - see below
GCSE 9-1 Physics Revision Notes GCSE 9-1 Biology Revision Notes
All website content Dr Phil Brown 2000 onwards. All copyrights reserved on revision notes, images, quizzes, worksheets etc. Copying of website material is NOT permitted. Exam revision summaries and references to science course specifications are unofficial. Email doc b: chem55555@hotmail.com

 Doc Brown's Biology

*

 For latest updates see https://twitter.com/docbrownchem

 Have your say about doc b's website

TOP OF PAGE