6.1 Microbial Technique Flashcards
Aseptic technique
Alcohol
Moist heat (autoclave)
Dry heat
UV
Ionising radiation
Filtration
Chemical agents
Alcohol
65-80%
Disinfect hands and equipment
Solubilize lipids and denature proteins
Moist heat (autoclave)
Sterilise liquid cultures
Denature nucleic Acids, denature proteins and disrupt cell membrane
UV
Sterilises air and exposed surfaces
Damages DNA
Ionising radiation
Sterilises antibiotic hormones, sutures and disposable supplies
Damages DNA and oxidises cell content
Filtration
Cleaning liquid cultures though sterilised filtration machine
0.2 micro meter pore size strains out bacteria in small volumes
Chemical agents
Sterilising work bench and equipment
Stop it slow bacteria growth
Media: selective inhibition
Adding dyes
Antibiotics if specific inhibitors
Couture needs carvin for energy (respiration) and nitrogen to grow (protein)
Liquid culture
Method: conical flash allows large SA in contact with the air. Aerobic bacteria cultured sterile air. Brit is sterile and agitated.
Pros:
Culture does not die
Metabolic products can be harvested
Measuring growth: direct count
Haemocytometer
Dilution plating
Total or viable used
Measuring growth: indirect count
Dry mass
Optical methods (turbidity)
Streak plate
Measuring growth: haemocytometer
Direct count
Cell count in a specific volume
NW rule
Viability due (methylene blue)
Limitations:
Needs stain for viability
Cells must be killed/imobilised
. suspension of low density hard to see
Measuring growth: dilution plating
Cell count in solid mass.
Diluted and x by dilation factor
Check with haemocytometer
Pros:
Viable cell count
Simple equipment
Con:
Time taken to dilute
need to keep track of volume and dilution
Can’t be used for predictions
Direct count
Indirect count
Turbidimetry (optical)
Rapid
measures growth- calibration curve
Done with spectrophotometer
Area of fungi
Radius if mycelium in different conditions
Indirect count
Mass of fungi/bacteria
Indirect
Sample of broth removed at intervals
Separated by centrifuge or filtration
Dried and weighed
Lag phase
Cell active- not reproducing
Adjusting to surroundings (taking in water, growing, making MRNA enzymes etc)
Length depends on conditions
Log phase
Bacteria divide by binary fission (max rate)
Size of population doubles at each division (cell growth>cell death)
Most susceptible to antibiotics
Length of time depends on species
Stationary phase
Deplete growth medium and release waste products - pH change
Cell growth= cell death
Carrying capacity of an environment in an open system
Death phase
Die at an exponential rate
Growth<death
Dead cells become nutrients for growing cells
Most cellular activity stops
In a closed system this causes extinction
Causes of inaccuracy with haemocytometer
Can’t distinguish between living and dead cells
Cells are not evenly distributed
Har to tell is cells are touching the side
