Topic 6.1 Microbial techniques Flashcards
Why are aseptic techniques important when culturing microorganisms?
To produce uncontaminated culture so results are reliable and repeatable
List the basic aseptic techniques
- Wipe surfaces with antibacterial cleaner
- Set up bunsen burner nearby. Convection currents prevent microbes from enetering culture
- Flame innoculating loop and neck of bottles before use
- Minimise time that vessels containing bacteria are open
- Sterilise all equipment e.g. using an autoclave
- Wear protective clothing
Outline how to culture microorganisms
- Transfer bacteria to agar plate using sterile inoculating loop or pipette
- Tape lid on at 2 ends then invert the dish and incubate. In school lab conditions, ensure dish is not airtight and do not incubate above 25oC to avoid growth of pathogens
Explain the difference between a spread plate and a streak plate
Spread plate: ditrubute microorganisms evenly with a sterile spreader
Streak plate: aim to obtain single colonies by rotating the plate to build layer of the culture on at least 3 separate streaks
Describe 3 types of nutrient medium
Usually contain nitrogen carbons and minerals. Often enriched with protein from extract of yeat, blood or meat. May be liquid broth or solid agar.
Selective mediums only contain highly specific nutrient balance. Only certain microorganisms can grow.
Give the advantages of using a broth medium
- Can provide anoxic and oxic conditoins depending on depth, which helps to identify microbes/ determine thier optimum conditions.
- Can grow a very large volume of bacteria.
Give the advantages of using a broth medium
- Can provide anoxic and oxic conditoins depending on depth, which helps to identify microbes/ determine thier optimum conditions.
- Can grow a very large volume of bacteria.
Give the advantage of using agar as the medium
Can obtain a single discrete pure colony for study.
Name the 4 phases of a bacterial growth curve
- Lag phase
- Log phase
- Stationary phase
- Death phase
What happens during the lag phase?
Microorganisms need to adjust to the enviroment before reproducing so population size only increases slowly.
What happens durong the log phase?
After every round of division population size doubles (exponential growth).
What happens during the stationary phase?
Reproduction rate = death rate, so population size stabilises at its maximum.
What happens during the death phase?
Microorganisms die due to buildup of toxic waste and lack of nutrients.
Name three methods to estimate the growth of a bacterial culture
- Cell count (using a haemocytometer)
- Turbidity meaurement (type of colorimetry to measure opacity)
- Dilution plating
Dry mass could also be used
Explain how to conduct a cell count
- Dilute broth sample with equal volume of trypan blue stain to stain dead cells blue
- Use a calibrated haemocytometer with volume 0.1mm3. Count the cells in each of the sets of squares and calculate mean
- Number of bacterial cells = number counted x 104 per cm3
