6.1 Microbial Techniques Flashcards
What do microorganisms need to grow?
- nutrients
The correct level of: - pH
- temperature
- oxygen
Why is it important to take care when culturing microorganisms
- there is always a risk of a mutant strain arising that may be pathogenic
- there is a risk of contamination from pathogenic organisms
- when growing a pure strain - there is a risk of contamination from any other microorganism
Describe the safety precautions that take pace when culturing a microorganism
- all of equipment must be sterile before use
- once the culture has grown, it should not b left in the lab, it should be disposed of in plastic bags and sterilised at 121° for 15 mins
Explain how to investigate rate of growth of microorganisms in liquid culture with a colorimeter
- Wash hands with soap, disinfect bench area with Virkon, light a Bunsen burner, in working area, wear safety goggles
PRODUCE A SINGLE CULTURE FLASK - Add 250 cm3 of 0.5% glucose solution
- Stopper with cotton wool and cover with aluminium foil
- Add 1.25g yeast, swirl to mix
- Place on magnetic stirrer and stir continuously
USING A COLORIMETER
3. Fill a cuvette with culture medium (no microorganism), zero the colorimeter
4. Measure 3cm cubed of suspension using a measuring cylinder then into a cuvette
5. Measure absorbance and record time
6. Repeat steps 3-5 at least 5 times over the next 12 hours
What is a selective medium, and where is it useful
- a growth medium with a specific mixture of nutrients, so only a particular type of microorganism will grow on it
Describe how to isolate an individual species from a mixed culture using streak plating
day 1
1. Wash hands with soap, disinfect bench area with Virkon, leave to soak for 10 minutes, light a Bunsen burner in working area
2. Label bottom of agar jelly plate with name and date
3. Loosen cap of culture tube slightly
4. Sterilise inoculating loop, hold it in flame
5. Allow loop to cool, do not put it down
6. Briefly pass the neck of the culture tube through the flame at an angle
7. Dip loop into mixed culture, lightly make streaks on agar jelly plate, making multiple at different angles
8.flame loop before placing down
9. Place lid on Petri dish, add 2 pieces of tape on opposite sides, turn Petri dish upside down and incubate at 25° for 24 hours
day 2
10. observe plate and sketch what has grown
11. obtain two nutrient agar plates and label them white and yellow
12. flame inoculating loop using bunsen burner
13. allow to cool
14. touch the loop on an only yellow colony then streak onto plate labelled yellow
15. flame loop before putting it down
16. place a piece of tape on each side of petri dish lid and incubate at 25 C for 24 hours
day 3
17. keeping lids on, observe what has grown and make a sketch
18. disinfect bench area and return plates for sterilisation
how do you carry out a cell count using a hemocytometer
- the sample of nutrient broth is diluted by half. with an equal volume of trypan blue (stains dead cells)
- cells are viewed using a microscope and counted
- the number of cells in each set of the 14 squares is counted, and the mean is calculated
- if the cells are touching the edge of two out of four sides of the grid then they are counted, you need to pick two sides that count
describe how to carry out dilution plating to find the total viable cell count
- start off with sample and add 1 cm3 to test tube and add 9 cm3
- take 1 cm3 of the new diluted sample and add add itg to a different test tube (0.1 dilution)
- then add 9 cm3 of water (0.01 dilution)
- after adding a drop to a microscope slide and counting the number of cells, take 1cm3 of the new sample and add to a new test tube, add 9 cm3 of water again (0.001)
- continue these steps until you have gotten to a point where you can count the number of cells on the slide
- make the appropriate calculations to find the amount of microorganisms in the original sample
describe how to measure the growth of bacterial cultures using optical methods
- fill a cuvette with only the culture medium and use it to zero the colorimeter
- use aseptic technique to measure 3 cm3 sample and immediately transfer into a cuvette
- measure absorbance and record the time
- return the stoppered yeast culture to the stirrer
- repeat steps 1-4 at least 4 times over the next 12 hours of culturing
describe the different phases of bacteria
- when the number of bacterial cells in a sample are measured over time, the results may be explained by these phases
1. lag phase - they have to adapt to their environment before they can reproduce at their maximum raye
2. log phase (exponential phase) - rate is close to the theoretical maximum, increasing
3. stationary phase - growth rate is 0, flat line, the number of new cells formed by binary fission is equivalent to the amount of cells dying
4. death phase - reproduction has almost ceased and the death rate is increasing
why does the rate of bacterial growth eventually slow down
- reduction in the amount of nutrients available. As the amount of microorganisms multiply in the log phase, its used up
- build up of waste products. The amount is minimal at the beginning, but as cell number increases, the amount of toxic material becomes sufficient to inhibit growth and poison and kill the culture
- CO2 produced lowers pH to the point where bacteria can no longer grow
how do bacteria cause disease
- the invade and destroy host tissues and produce toxins as a product of their metabolic processes, these are what cause the symptoms of disease
what pathogenic effects can be produced by exotoxins
- a toxin released to the surroundings
- their effects are more widespread than endotoxins, as they act at sites far from their place of infection
- some damage cell membranes, some cause cell break down or internal bleeding
- some act as competitive inhibitors to neurotransmitters
- rarely cause fevers
give an example of an exotoxin
staphylococcus spp.
give an example of an endo toxin
salmonella spp.
