culturing (growing) microorganisms Flashcards
What does culture medium contain?
An energy source e.g. sugars -> needed for cellular respiration, to make ATP.
A source of carbon -> needed to make all organic molecules.
A source of nitrogen (ammonia, amino acids) -> needed for protein synthesis.
Mineral ions/vitamins -> used as co-enzymes and co-factors.
Growth factors -> hormone -like substances that stimulate growth.
Water -> all metabolic reactions occur in solution.
If microbes are grown in a broth, the “TURBIDITY” also has to be regulated - what does this mean?
How cloudy/thick/viscous the mixture is.
The more growth of the microbe, the more turbid the mixture becomes - difficult to stir and to evenly distribute nutrients and oxygen.
Why is the contamination of cultures bad?
- Contamination of cultures by unwanted microorganisms, may affect the growth of the microorganism being cultured.
- Contaminated cultures in laboratory experiments give impurities and may be hazardous to health.
- Contamination on an industrial scale can be very costly because entire cultures may have to be thrown away.
What are important aseptic techniques that you should follow when culturing microorganisms in the lab?
- Disinfect work surfaces.
- Work near a Bunsen flame. Hot air rises, so any microorganisms in the air should be drawn away from your culture.
- Sterilise the instrument used to transfer cultures before and after each use, e.g. sterilise a wire inoculation loop by passing it through a hot Bunsen burner flame for 5 seconds and neck of broth container.
- Minimise time agar plate is opened, work in inoculation cabinet. -> decrease chance of airborne microbes contaminating cultures.
How to inoculate broth?
- Make a suspension of the bacteria to be grown.
- Mix a known volume with the sterile nutrient broth in the flask.
- Stopper the flask with cotton wool to prevent contamination from the air.
- Incubate at a suitable temperature, shaking regularly to aerate the broth providing oxygen for the growing bacteria.
How to inoculate agar?
- Make suspension.
- Sterilise wire inoculating loop.
- Dip loop in bacterial suspension -> remove lid of Petri dish -> make zigzag streak across agar surface.
- Replace lid - hold down with taped, not completely sealed so O2 can enter -> prevents growth of anaerobic bacteria. Incubate at suitable temperature.
What does grown in a “closed culture” mean?
Microbes isolated from external environment = extra nutrients not added and no waste products removed.
What are the different stages of the growth curve in a closed system?
The lag phase -> bacteria adapting to new environment - growing, synthesising enzymes needed, not at max rate.
The exponential phase -> rate of bacterial reproduction is close or at its max rate.
The stationary phase -> growth rate = 0.
The decline/death stage -> death rate exceeds reproduction rate.
Limiting factors that prevent continued exponential growth?
- Nutrient availability.
- Oxygen levels.
- Temp.
- pH change.
- Build up the waste.
How do you investigate factors that affect growth of microbes?
- Grow bacteria on agar plates at different conditions e.g. effect of temp on bacterial growth.
What is the process of this?
1- Sample of bacteria in broth -> add set volume to agar plate.
2- Spread broth across surface.
3- Lid taped shut -> do this for 6 plates.
4- 3 plates 4 degrees Celsius, 3 25 degrees Celsius - incubator.
5- Agar plate with no bacteria - negative control.
6- Leave for set time and count colonies.
Number of colonies = number of bacteria on agar, use serial dilutions to see properly.
Choose plate where plenty colonies have grown, but too many to count.
On an industrial scale how are cultures grown?
In massive containers called fermenters.
