Core practical 13 biology - investigating bacteria for investigation

What are microorganisms?

What are microorganisms?

Organisms visible under a microscope e.g. bacteria, fungi and parasites

How can we grow microbes?

Either in agar jelly (solid) or broth (liquid)

What must our growth media contain?

• Source of energy

• Carbon source

• Nitrogen course

• Mineral salts

• Growth factors

• Water

What is sterilisation

1. Ensure medium is sterilised before we grow cultures, otherwise organisms will grow and compete with the culture

2. Medium is sterilised by heating in autoclave oven at 121 degrees for 15 mins

3. Autoclave oven - uses boiling H2O under high press to kill bacteria and fungi

4. When cool enough to use, pour in into Petri dishes and allow to set

5. Keep lid firmly on dish until usage to prevent contamination - open lid minimally and flame bottle neck

6. Flame any metal or glassware before + after use to kill unwanted microbes

7. Open any vessels or containers to a minimal degree less exposure to air

What is inoculation?

Introduce culture to a medium

Use streaking - use an inoculation loop to transfer a drop of liquid medium onto agar surface

Make sure not to damage the agar surface as may affect population growth

Remember to zig-zag wire across surface in same motion

What is incubation

Producing conditions to encourage growth

• Make sure Petri dish is labelled and placed taped down - do not cover gap completely as anaerobic bacteria to dom + harm culture bacteria - may require O2

• Place dish in warm environment to incubate, encourage culture growth

• Place it upside down to prevent condensation droplets disrupting agar and to stop it drying out + could affect their growth

• Do not open dish when examining colonies as it will intro microorganisms

• Sterilise dishes before + after use

• Throughly wash hand after examining colonies as some could be infectious and harmful to humans

• Temp will depend on culture

• Examine after 24 to 36 hours

Planning for this practical

1. Agar powder with 1% or 2% conc solution

2. Boil in thermostatically controlled water bath

3. Cool this to a temp of about 50 degrees

4. Add nutrients, if not already applied with powder

5. When molten, pour into Petri dishes and keep preserved in fridge

6. If using liquid broth, contains all nutrients necessary to agar

How to test for antimicrobial agents?

Antimicrobial agents las IV e.g. penicillin

• In order to test use spreader to place even coat of bacteria culture on agar plate

• When incubation has finished this will leave and even and continuous layer of bacteria called a bacterial lawn

• If we use multiple sample, we use multi disk - contains ring for adding diff microbial agents

• Add microbial agents use paper discs, soaked in antimicrobial adding it with sterile forceps

How to investigate population growth?

• Liquid broths we take samples from turn cloudy once bacteria have begun to flourish - must wait for this

• Samples must have same vol

• Shake broth each time a sample is taken - cultures may settle in broth and be missed

• Incubate all agar plates at same temp (25 degrees)

• Units: density of cells per Cm3

• Produce table to record data as follows

• Always stir broth before taking sample and always pipette from halfway down

Analysis of results

• Effective antimicrobials = stop microorganism growth = zone of inhibition surrounding the effective antimicrobials

• More effective antimicrobials will inhibit a wider area so zone of inhibition can be larger

• We can measure zone of inhibition to assess the effectiveness of antimicrobial

• Measure area by measuring radius of zone + use the equation for the area of circle

• Area = Pi x Radius squared

Presenting the results

• Photo or draw diagram of Petri dish

• Display our results graphically, plotting area of zone of inhibition on y-axis and our IV on x -axis

• Testing affects of diff antimicrobials we could draw a bar chart

• Testing effects of different concs of an antimicrobial - we could draw a line graph

Evaluation

Anomalous results - zone of inhibition may be larger or smaller than expected - same conc + type of antimicrobial or it doesn’t fit the trend

What does the zone of inhibition indicate

• If a zone is unusually large = measuring error + antimicrobial disc prepared incorrectly

• If a zone is unusually small = antimicrobial resistance could have developed, agar plate may have not been prepared correctly - lacking nutrients or powder and improperly mixed.

• Contamination with other organisms, in prep failure to sterilise appropriately

What are Limitations

• Takes a lot of time - 24 to 36 hrs - observe growth of single culture - rate of growth after time but initially v slow

• Some contamination may be hard to identify - colonies of different of bacteria organisms could be different colour/size/shape may be misinterpreted

• Can never be completely aseptic process - airborne organisms are always present

• Manually counting colonies - opens for opportunities for errors

• Every time a culture is transferred/opened, there is potential for damage or contamination

• Only grow and study aerobic bacteria, since O2 is present within Petri dish system

• Although colony indicates origins of a single cell, some colonies merge and combine which makes it harder to determine how many cells produced this