Flashcards in Microbiology Deck (13):
How is bacteria classified?
According to their shape, cell wall structure, antigenic, metabolic and genetic features. They vary in sizes (0.4 to 700 um).
Three typical shapes?
Spherical = Coccus, in groups (staphylococi), in pairs (diplococci)
Rod-shaped = Bacillus
Spiral = Spirillum
Gram positive bacteria
Thicker cell wall (peptidoglycan), No lipopolysaccharide layer so vulnerable to penicillin and lysosomes action.
Peptidogylcan layer retains crystal violet stain so stains purple.
Gram negative bacteria
Thinner cell wall (peptidoglycan), lipopolysaccharide layer protects against penicillin and so prevents uptake of crystal violet stain only stains pink/red once LPS removed and a counter stain used.
How to carry out a gram stain?
Bacteria put onto glass slide using inoculating loop.
Pass slide through Bunsen flame to fix to slide.
Add few drops of crystal violet.
Rinse excess with water.
Add Grams iodine for 1 min to fix stain.
Bacteria which stain purple = gram positive.
If not stained:
Wash with alcohol to dissolve lipids in LPS, expose peptidoglycan layer.
Re-stain using counter stain which will dye negative red.
Conditions necessary for bacterial growth?
Nutrients- Carbon (resp), nitrogen (AA), vits, salts.
Suitable temperatures- 25 to 45, most 37.
Suitable pH- optimum is slightly alkaline (7.4).
O2 may or not be required. (If need= obligate aerobe).
Some grow better with but don't need it= facultative anaerobes.
Those that cannot grow in presence of O2= obligate anaerobes.
Why use aseptic technique?
When culturing bacteria use this technique to ensure that only desired bacterium is grown, and that you don't contaminate yourself or the environment.
How to carry out an aseptic technique?
Heat at 121 for 15 mins in pressure cooker or pass through Bunsen burner until glowing red.
Benches cannot be sterilised but can be disinfected.
Living tissue cannot be sterilised without killing them so antiseptics are used to kill on outside only.
It is important to grow bacteria at 25 rather than 37 so that no pathogenic microorganisms grow, dish secured with tape and all material safely disposed of.
Measuring bacterial growth
Lag= population>, time needed for enzyme synthesis.
Log= No limiting factors, rapid reproduction occurs.
Stationary= (production=death) population moves around carrying capacity, reached it as reduced resources.
Death=more cells dying than produced, pop
Two ways of measuring growth
Directly= total number of cells is calculated. (viable- only living cells or total cells counted)
Indirectly= measuring the turbidity (cloudiness) of a culture.
Viable cell counts
This counts the number of living cells and is particularly useful in medical and food hygiene applications.
How to carry out a serial dilution?
Done in tenfold steps (1 in 10), but can be 1 in 100.
For 1 in 10 dilution= 1cm3 id added to 9cm3 of sterile medium and mixed, repeated till range of dilutions.
Now, plate out each dilution and incubate at 25 for 48 hours- allow bacteria to grow.
Plates examined, chose one with 20 to 100 colonies (more=difficult to count, less=> error due to small no)