3.4 Microbiology Flashcards
Cocci
Spherical
Bacillus
Rod shaped
Spirillum
Corkscrew
Gram positive stain
Purple
Gram negative stain
Red
Gram positive contain
-Thick outer wall of peptidoglycan
-No lipopolysaccharide layer
-Crystal violet can remain
Gram negative contain
-Thin peptidoglycan wall
-Outer lipopolysaccharide membrane
-Alcohol removes lipopolysaccharide layer
-Thin peptidoglycan layer allows stain to be washed away
Gram staining steps
Fixation
Stain with Crystal violet
Iodine
Decolorisation
Safranin
Needed for growth of bacteria
-Carbon source (glucose or lactose)
-Nitrogen source (ammonia or amino acids)
-Sulphur and phosphorus
-Vitamins and minerals
-Suitable pH
-Suitable temperature
Obligate aerobe
Needs oxygen
Obligate anaerobe
No oxygen
Facultative anaerobe
Grows best with oxygen but can respire anaerobically if needed
Aseptic technique prevents
-Contamination of the environment by the microorganism being handled
-Contamination of the bacterial culture by environment
How to sterilise tools
Pass through blue Bunsen burner flame until it glows red
How to serialise glassware
High pressure and temperature in autoclave for 15 min
Pouring a sterile agar plate
-Open bottle using little finger and do not pace on bench
-Flame neck of bottle
-Work close to flame as updraft prevents contamination
-Open sterile Petri dish at an angle
-Pour and close immediately
-Swirl to move air bubbles
-Secure lid with tape
Inoculating nutrient agar plate
-Sterilise inoculating loop in blue flame until glows red
-Dip into milk
-Hold Petri dish lid at an angle to reduce contamination
-Spread milk across surface of agar in zig zag while rotating
-Tap lid shut
-Do not completely seal as anaerobic conditions encourage pathogenic bacteria
-Incubate at 25c and not 37c as human pathogen
Staining
-Crystal violet is absorbed by the peptidoglycan as binds to it in the cell wall
-Add iodine which crystallises the purple stain
-Add alcohol which dissolves the lipopolysaccharide layer of the gram negative and dehydrates the peptidoglycan
-Gram positive is purple
-Gram negative is colourless as purple is washed away
-Safranin stains the gram negative pink/red and is not seen in the positive
What does safranin do
Stain gram negative red
Does not stain gram positive
What alcohol does
Dissolves the lipopolysaccharide layer of the gram negative and dehydrates the peptidoglycan
What iodine does
Crystallises the violet
2 categories for counting bacteria
-Viable count
-Total count
Viable count
Counts only living bacteria
E.g serial dilutions
Total count
-Counts living and dead bacteria
Bacteria in a liquid can be counted
Directly
Indirectly (cloudiness)
Viable count method
-Sterile water
-1cm3 of sample into tube (10^-1)
-Mix and then take 1ml and place into second tube
-This is 10^-2
-Repeat until 10^-4
To estimate the number of bacteria present in original sample
Multiply the number of colonies counted by the dilution factor
Why is count an estimate
-It does not include dead or noon viable bacteria
-We cannot be sure that each colony has grown from a single bacterium
Why is log scale used
Allows a rapidly growing population to be accurately plotted on a graph
Lag phase
-Initial exposure to the environment
-Little growth but the cells are taking up water and carrying out protein synthesis and producing enzymes
Stationary phase
-Carrying capacity of the environment
-Dying at same rate as they are reproduced
Death phase
Build up of toxic waste products in cells or due to lack of oxygen or nutrients
Log phase
-Population increases rapidly
