mircobiology

classifying bacteria

Bacteria can be distinguished from one another by their size, shape, metabolism, antigenic features, genetics and by their staining characteristics.

shapes of bacteria:

• coccus - spherical

• bacillus - rod-shaped

• spirillum - spiral

Gram positive bacteria

a thick peptidoglycan layer which retains a crystal violet stain, so after Gram staining they appear purple down the microscope.

Gram negative bacteria

have a thin peptidoglycan layer and have extra layers of lipopolysaccharide, alcohol used in the procedure will wash the crystal violet out. The counter staining with safranin stains the cells red.

gram stain

1. Stain using crystal violet stain

2. Fix the stain with iodine

3. Decolourise with alcohol

4. Counterstain with safranin.

Gram positive cells are purple

Gram negative are red.

nutritional requirements of bacterial reproduction and growth

• temp - optimum temp for human pathogen is around 37˚C, temp of the human body

• nutrients - nutrient agar, usually glucose for respiration and nitrogen (nitrate ions)for amino acid and nucleic acid synthesis

• pH- bacteria slightly alkaline conditions

• oxygen requirement

Obligate aerobes

Obligate aerobes must have oxygen in order to carry out metabolism and reproduce

Obligate anaerobes

Obligate anaerobes can only survive and metabolise in absence of oxygen

Facultative anaerobes

Facultative anaerobes can metabolise and reproduce in the presence or absence of oxygen

aseptic technique.

avoid introducing contaminating microorganisms into the culture

before starting culturing microorganisms

• sterilise all media and equipements

• bunsen burner to create convection current

• disinfect work bench

during transfer

• flame the neck of culture bottle before opening and closing

• lift the lid of agar dish to 45 degree

• sterilise before and after uses of inoculating loop

after inoculating

• seal agar dish with adhesive tape (discourages the growth of human pathogens, and allows oxygen into the culture)

• plates should be incubated upside down at 25˚C for 24 hours

sterilisation using an autoclave

• glassware and metal equipment

• heated to 121˚C in steam

• under high pressure for 15 minutes

sterilisation using gamma irradiation

• plastic equipment e.g petri dish

total cell count

methods that count all the cells present but cannot distinguish between live and dead cells

viable cell count

only counts the cells that are capable of reproducing (forming colonies) and are therefore alive.

serial dilution

This dilutes the sample of bacteria so that there are not too many to count; this can also mean that there is not simply a continuous lawn of overlapping colonies on the agar plate.

serial dilution is under diluted

colonies might merge and counting may be inaccurate resulting in an underestimating of cell number

serial dilution is over diluted

there will be too few colonies on each plate to count to be statistically sound (leading to inaccuracy )