microbiology Flashcards
How are bacteria classified
- classified according to their shape, cell wall structure and metabolic,antigenic and genetic features
What are the three shapes of bacteria
- coccus - spherical
- bacillus - rod shaped
- spirillium - spiral
What does the name of the bacteria mean
reflects the name of the disease
How can bacteria be further classified
using gram staining technique
Describe how the antibiotic pencillin work
- prevents the cross links from forming within the peptidoglycan layer and so weakens the cell wall in newly divided bacteria
- gram positive bacteria are most affected as they are then subject to osmotic lysis when water enters the bacterial cell causing the cell to burst
describe the differences between gram positive bacteria and gram negative bacteria
gram positive bacteria
* thicker cell wall
* thick layer of peptidoglycan
* no lipopolysaccharide layer (LPS) so vulnerable to penicillin and lysozyme action
* peptidoglycan layer retains cyrstal violet stain so stains purple
* staphylococcus and streptococcus
gram negative bacteria
* thinner cell wall
* thin layer of peptidoglycn
* lipopolyaccharide layer (LPS) protects against penicillin and lysozyme action
* lipopolysaccharide layer prevents uptake of cyrstal violet stain so only stains red once LPS removed and a counter stain used
* salmonella and e coli
Describe the gram staining technique
- transfers a small sample of bacteria to a glass microscope slide using an inoculating loop. pass the slide through a bunsen flame a few times to fix bacteria to the slide (also kills them)
- add a few drops of crystal violet stain and leave for 30 seconds
- rinse excess using water
- add grams iodine for 1 minute to fix stain
- bacteria which stain purple are gram positive
to stain remaining bacteria: - wash with acohol for 30 seconds to dissolve lipids in lipopolysaccharide layer and expose inner peptidoglycan layer
- re stain using another stain eg safranin which stains unstained bacteria red
obligate aerobes
microbes that require oxygen for growth
facultative anaerobes
microbes that grow better with oxygen but can grow without it
obligate anaerobes
- microbes that cannot survive in the prescence of oxygen
Describe conditions necessary for bacterial growth
- nutrients- a source of carbon for respiration eg glucose, nitrogen for synthesis of nucleotides and proteins and vitamines and mineral salts
- water
- suitable temperature - 25-45 degrees celcius for most bacteria 37 degrees is optimum for mammalian pathogens. thermophiles can survive at 90 degrees evolved in hot springs
- suitable pH- optimum is slightly alklaine for most bacteria some can survive acidic conditions
- oxygen may or not be required depending upon the mode of repiration
aseptic technique / sterile technique
good laboratory practice that maintain sterile conditions and prevents contamination
pathogen
a diease causing microorganism
Why is aseptic technique used
to ensure that the desired bacterium is grown and that you don’t contaminate yourself or the environment
how is equipment and media sterilised for aseptic technique
- heat at 121 degrees celcius for 15 minutes in an autoclave or pressure cooker or by passing the equipment through a bunsen flame for 2-3 seconds until it glows red eg inoculating loop. this works for inanimate objects (non living)
- irradiation works well for heat labile plastics
- benches cannot be sterilised but can be disinfected eg 3% lysol which reduces the numbers of microbes but not fungal spores
- living tissues cannot be safely sterilised without kiling them so antiseptics are used which kill or inhibit microbes on the outside of living tissues only
Why do we grow bacteria at 25 instead of 37 degree celcius
- so that pathogenic microorganims aren’t grown
what happens with all material after doing aseptic technique
- safely disposed or afterward by sterlising in an autoclave
What are the four phases of measuring bacterial growth
- lag phase
- logarithmic phase
- stationary phase
- death phase
why do we use log numbers with bactieral growth
this is as the range of number is too large
What happens during lag phase
- population number increases very slowly because time is needed for enzyme synthesis
Describe what happens during the log/exponential/growth phase
- there are plenty of nutrients and few toxic by products so there are no limiting factors
- allow rapid reproduction
Describe what occurs during stationary phase
- cells are reproducing but population is relativley constant fluctuating around the carrying capacity due to cell production equalling cell death
- the population has reached its carrying capactiy because reduced resources (eg nutrients/space/toxic waste products) are now limiting factors
Describe the death phase
- more cells are dying than are being produced so the population decreases
- death of cells is due to lack of nutrients, lack of oxygen or increases toxicity of the medium
How is growth measured
- directly where the total number of cells is calculated
- indirectly by measuring the turbidity (cloudiness) of a culture
What are the types of direct counts
- direct counts can either be viable counts where only living cells are counted or total counts where both living and dead cells are counted
- by using a haemocytometer originally developed to count bloos cells
Describe the method of viable cell count and what they are
- this counts the number of living cells and is particularly useful in medical and food hygeine applications
- even in small cultures the total viable cell count can exceed several million per cm3 so a serial dilution must be performed first
- this is often done in tenfold steps 1 in 10 but higher dilutions can be performed 1 in 100
- for 1 in 10 dilution 1 cm3 is added to 9cm3 of sterile medium and mixed and repeated until a range of dilutions is obtained
- plate out each dilution and incubate the plates at 25 degrees celciius for 24-48 hours to allow bacteria to grow
- plates are examines and a plate is chosen to count
How do you know the best plate to count
- 20 - 100 colonies
- more than 100 is difficult to count
- fewer than 10 has increased error due to small numbers involved
How is viable count calculated and what does it assume
multiplying the dilution factor by the number of colonies
assume each colony originated from a single bacterium that divided asexually
