6.4.7 microorganism cultures
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2 types of growth mediums used for microorganisms
• broth - kept in bottles/tubes • agar - melted & poured into petri dishes
what does typical nutrient agar contain
• peptones • yeast extract • salts • water • may contain glucose/blood
standard 6-step procedure of aseptic techniques
• wash hands • disinfect working area • have bunsen burner nearby to heat air = causes air to rise which prevents air-borne microorganisms settling & creates area of sterile air • pass neck of bottle over flame once opened/when closing to prevent bacteria entering bottle • don’t lift lid of petri dish off completely - only open enough to introduce desired microorganism • glassware/metal equipment passed through flame before/after contact with desired microorganisms
3 main steps involved when growing microorganisms on agar plates
• sterilisation • inoculation • incubation
how is the nutrient agar medium sterilised
• heating autoclave at 121 degrees for 15 minutes • kills all living organisms eg. bacteria, fungal spores • when medium cooled sufficiently, it’s poured into sterile petri dishes & left to set • lid kept on petri dish to prevent infection
different ways of inoculation
• streaking = wire inoculating loop transfers drop of liquid medium onto surface of agar & drawn into streak • seeding = sterile pipette transfers small drop of liquid medium onto surface of agar/petri dish before agar poured in • spreading = sterile glass spreader used to spread inoculated drop over agar surface • small cotton swab/bud moistened with distilled water & collects microorganisms from surface to carefully wipe over agar medium
how is the petri dish incubated
• label & tape top to bottom using 2 strips of adhesive tape (don’t seal dish completely) • placed in suitable warm environment eg. incubator • placed upside down = prevents condensation falling onto agar & agar drying out too quickly • temperatures depend on organism being grown
define closed culture
population where all conditions set at start & no exchange with external environment
4 stages of closed culture growth curve
• lag phase • exponential phase • stationary phase • dead/decline phase
describe the lag phase
• population doesn’t grow quickly • population is small/adjusting to environement • may involve: taking up water, cell growth, activating genes, synthesising specific proteins (enzymes) etc.
describe the log (exponential phase)
• organisms adjusted • have enzymes needed • all have sufficient nutrients/space to grow rapidly & reproduce • population doubles in size with every generation (some microorganisms this is once every 20-30 minutes)
describe the stationary phase
• increasing numbers of microorganisms eventually use up nutrients & produce increasing waste products (eg. carbon dioxide, other metabolites) • rate of population growth declines & number of individuals dying increases until rate of reproduction equals death rate • no population growth
describe the death phase
• nutrients run out • concentration of waste products may become lethal • more organsims die than being produced = population begins to fall • eventually all organisms die
when are primary metabolites made
produced during normal activities of microorganism & collected from fermenter during log phase
when are secondary metabolites made
• produced in stationary phase • population must be kept in close culture & metabolites can be collected at end of stationary phase/during decline phase
what can a liquid broth be used for & how
= to measure growth rate of microorganism population • sterile broth inoculated & population size measured at regular intervals in incubation • population size measured by transferring small sample to agar plate & incubating agar culture = each individual microorganism produces visible colony