Topic 6: Microbiology and Pathogens Flashcards
What is a microbial culture?
A method of multiplying microbial organisms by letting them reproduce in a predetermined culture medium under controlled lab conditions
What factors must be controlled when making a microbial culture?
Nutrient levels
Oxygen
pH
Temperature
What are the main problems when culturing microorganisms?
1 - Harmless microorganisms might mutate to dangerous ones
2 - Pathogens enter and grow - can cause disease
3 - Contamination with unwanted microorganisms will ruin the investigation
What precautions should be taken when culturing microorganisms?
Use sterile equipment
Do not remove culture from lab
Dispose of culture safely
What does sterile mean?
Being sterile is to be free from living microorganisms and their spores
How do you dispose of microorganism cultures safely?
Seal in a plastic bag and sterilise at 120 degrees for 15 minutes under high pressure and then throw it away
Outline the method to culture microorganisms
- Choose a microorganism to culture
- Obtain a culture of the microorganism
- Select and make up the nutrient medium
- Innoculate the medium
What are the different types of nutrient medium?
Liquid broth, nutrient agar and selective medium
What is nutrient agar?
A solid nutrient elly extracted from seaweed used in Petri dishes
How do you make nutrient agar?
Pour nutrient broth and molten agar into a Petri dish and let it set at 50C
What is the advantage of agar
Sets at 50C but melts at 90C so you can keep it at a high temperature
What is a selective medium?
A growth medium with a specific combination of nutrients so only a certain type of microorganism grows on it
Define innoculation
The process by which microorganisms are transferred into a culture medium under sterile conditions
Explain how to innoculate a liquid nutrient medium
Innoculating loop scrapes bacteria from solid culture to liquid nutrient broth to form innoculating broth.
Flask stoppered with cotton wool
Incubate at suitable temp
Regularly mix
Why do you stopper a flask of liquid culture when culturing it?
To prevent contamination by microorganisms in the air
Why do you mix a flask of liquid culture regularly when culture?
To ensure the broth is aerated bc microorganisms need oxygen
Explain how to innoculate a solid nutrient medium
Sterilise the innoculating loop in a Bunsen Dip into bacteria suspension Streak across agar surface Replace lid, clse tape and label Turn upside down
What does aseptic mean?
Sterile - free from contamination from harmful bacteria
Why are aseptic techniques used when culturing bacteria?
To ensure the procedure is safe and to prevent the contamination of the culture.
Give examples of aseptic technique
Using sterile equipment
Using flamed equipment
Replacing the lid of the petri dish as soon as possible
What is a pure culture?
A culture containing only one type of microorganism
How can a pure culture be made?
Making conditions aerobic/anaerobic depending on organism
Making selective medium specific to it
Reinnoculate a plate with the microorganism on it (identified by using an indicator)
What information do we need to measure the growth of a culture?
The number of cells at different times
Name some methods used to measure the growth of cultures
Cell count
Dilution plating
Turbidimetry
Size/area
What is a cell count?
A method of measuring the growth of cultures of bacteria or single selled fungi
What equipment is needed for a cell count?
Haemocytometer
Microscope
What is a haemocytometer?
A thick glass microscope slife with a rectangular chamber that holds liquid. It has perpendicular lines
What is trypan blue?
A dye that stains dead cells blue and leaves living cells white
Why do we dilute cells with trypan blue before putting them into a haemocytometer?
To stain them so counting is easier
To separate the cells to make them easier to count
What is the rule for cells on lines when counting cells in a haemocytometer?
Count them if they’re on the top or right line
Don’t count them if they’re on the bottom or left
How is a haemocytometer used?
Put under a microscope and viewed under a low power so the cells can be seen
Why is it important to not move the haemocytometer when counting?
Will mve the cells so counting becomes incorrect
Air bubble may be introduced
What is turbidity?
The cloudiness of a liquid caused by a large number of individual particles in the solution
What is turbidimetry?
A method of measuring the concentration of a substance by measuring the amount of light that passes through it. A specialised form of colorimetry
What are calibration curves?
Graphs of known concentrations against their absorbances
How are calibration curves made?
Control culture is made and samples taken at regular intervals
Put into colorimeter and turbidity is measured
Do cell count of the same sample
Repeat over time
Plot graph of turbidity against cell count and compare unknown turbidities to get cell count directly
How is turbidity measured?
The absorbance of light of a sample is measured by a colorimeter
What is dilution plating?
A method used to obtain a culture plate with a countable number of bacterial colonies
What assumption is made during dilution plating?
One colony comes from one bacterial cell
Define total viable cell count
A measure of the number of cells that are alive in a given volume of culture.
Why is dilution plating needed?
Because directly counting the number of colonies from a concentrated solution is difficult bc there are so many so they clump together. By dilution you can get a smaller number of cells
What calculation can be done to get the total viable cell count?
number of colonies x dilution factor
How can you check the accuracy of dilution plating?
Doing a cell count with a haemocytometer
What do you do if there are two or more plates with enough colonies to count in dilution plating?
Count the number of colonies in each plate then calculate a mean
How can you find the mass of microorganisms/fungi in a culture?
Sample of broth centrifuged/filtered to remove bacteria from liquid
Material dried until mass stabilises
Measure the dry mass overtime
Increase in mass = increase in growth
By what process do bacteria reproduce?
Binary fission
Define generation time
The time between divisions of a bacteria
What kind of scale is used for bacterial growth and why?
A logarithmic scale because the numbers are huge bc bacteria reproduce hella fast
What equation can be used to find the number of bacteria in a population?
Nt = No x 2^kt Nt - no of organisms at time t No - number of organisms at the start k - exponentional growth constant t - time
What equation can be used to find the exponential growth constant?
k=(log10Nt - log10No)/(tlog10(2))
How can the growth of a bacterial population be shown
A graph with axis time (t) against log10Nt (y)
Name the phases in the bacterial growth curve
Lag phase
Log phase
Stationary phase
Death phase
Explain the lag phase of the bacterial growth phase
Line is flat because bacteria are adjusting to their environment so growth is slow
Enzymes and genes still activating
Explain the log phase of the bacterial growth phase
Bacteria growing really fast
Growth time depends on number of nutrients and amount of space
Explain the stationary phase of the bacterial growth phase
Growth rate is zero - new cells forming = cells dying
Nutrients being used up
Waste products starting to build up
Explain the death phase of the bacterial growth phase
No. of cells dying exceeds the no of new cells forming
Nutrients have run out
Waste products have built up too high
Why doesn’t the exponential growth of bacteria continue forever?
Nutrient exhaustion - nutrient levels can’t support growth
Build up of waste products - levels can become toxic and inhibits growth (eg CO2 lowers pH)
How can bacteria cause infection in the body?
Destroying host tissue
Releasing toxins
How does TB cause infection?
Destroys host tissue
Name ways bacteria can enter the body
Wounds
Natural openings
Name the ways in which bacteria can be transferred
Droplet infection
Vector (intermediate carrier)
Direct contact
Touching the surface someone infected has touched
Why do bacteria produce toxins?
By products of their metabolism
Name the different types of toxins
Endotoxins
Exotoxins
What are exotoxins?
Soluble proteins that are produced and released into the body outside of the bacterial cell
What effect do exotoxins have on the body and why?
Widespread effect because they can travel through body fluids so they have a range of effects
What are endotoxins?
Lipopolysaccharides in the membrance of Gram negative bacteria that may be released from the bacteria if it breaks down