eduqas A level biology Microbiology

How do bacteria reproduce?

asexually by binary fission

whats the layer between the cell wall and cell membrane is bacterial cells

capsule

What are the 3 shapes of bacteria?

coccus (sphere)
bacillus (rod shaped)
spirillum (corkscrew)

step 1 of gram stain test

apply crystal violet

step 2 of gram stain test

apply grams iodine solution

step 3 of gram stain test

alcohol wash

step 4 of gram stain test

apply safranin

what colour is gram positive at the end of gram stain test

purple

what colour is gram negative at the end of gram stain test

red

describe the cell wall of gam positive

has a thick peptidoglycan cell wall

why is gram positive purple after gram stain test

the crystal violet remains when bacteria is washed with alcohol

Describe a gram negative cell wall

outer lipopolysaccharide membrane with thin peptidoglycan cell wall

why is gram negative red

outer lipopolysaccharide layer lost with the alcohol wash so crystal violet stain no longer there so can take up safranin stain

what type of bacteria is not susceptible to some antibiotics eg penicillin

gram negative

what are the conditions microorganisms require for growth

nutrients
temp
pH
oxygen requirement

how are microorganisms supplied with nutrients

nutrient media on nutrient agar or liquid

what nutrients do microorganisms need

carbon from glucose and nitrogen from nitrate ions

whats the temperature usually needed for microorganism growth

25-45 'C
37'C in mammalian pathogens

whats the pH required for microorganism growth

usually slightly alkaline for bacteria (fungi grow better in slightly acidic or neutral)

What's an obligate aerobe?

can only survive and metabloise in the presence of oxygen

What's an obligate anaerobe?

can only survive and metabolise in the absence of oxygen

What's a facultative anaerobe?

prefer to metabolise in the presence but can survive and metabolise without it

What's the lag phase?

no/little cell division
intense metabolic activity like enzyme synthesis

What's the log phase?

rapid increase in numbers no limiting factors

What's the stationary phase?

limiting factors prevent further growth of the population

in what phase is the carrying capacity reached

stationary phase

What's the death phase?

limiting factors cause the population size to decrease

why might the death phase occur

build up of toxic waste or no glucose left in the medium

how can you prevent contamination of pure culture from the environment

sterilise media and equipment
flame neck of culture before opening and closing
disinfect work benches

how can you prevent contamination of the environment by cultures

sterilise work surface before and after
lift lid of agar to 45'
seal agar all the way around
flame neck of culture bottle

what temperature and for how long do you inoculate an agar dish

25-30'C 24-48h

conditions to sterilise using an autoclave

121'C
high pressure
autoclave bag
15mins

what equipment do you sterilise in a autoclave bag

glass and metal

what equipment do you sterilise before using by using gamma irradiation

plastic

how do you dispose plastic equipment

biohazard waste bin

how do you dispose of plastic equipment if there's no biohazard waste bin

autoclave biohazard bag
autoclaved for 15mins 121'C high pressure and discard safely in a bin

whats the total cell count

living and dead cells in a bacterial sample

whats the total viable count

living cells in a known volume of liquid medium

serial dilution: how much distilled water is initially put into each test tube

9cm3

serial dilution: how much of the original bacterial culture is put into the 1st test tube

1cm3

serial dilution: what is put into the 2nd test tube

1cm3 from the first test tube

serial dilution: what is put into the 3rd test tube

1cm3 from the 2nd test tube

serial dilution: what do you do once all the test tubes have had some bacteria in it

transfer 1cm3 of each sample onto a sterile nutrient agar plate and spread it using a spreader

serial dilution: what is dont so a mean can be calculated

repeat the whole sterile dilution 3 times so theres 3 plates per dilution factor

serial dilution: what happens to the agar plates

taped (not all the way around) and incubated at 25'C for 24-48h

serial dilution: what happens after incubation

look for the dilution that shows distinct colonies without merging and count the number of colonies on each plate for that dilution

serial dilution: what do you do once you've counted the colonies

multiply the number of colonies by the dilution factor to give the number of bacteria in original 1cm3 bacterial culture sample

what inaccuracies may occur when carrying out serial dilution of original bacterial culture is under diluted

colonies might merge and counting may be inaccurate resulting in an UNDER estimate of cell numbers

what inaccuracies may occur when carrying out serial dilution of original bacterial culture is over diluted

there will be too few colonies in each plate to count to be statistically sound

0.1 cm3 original bacterial culture put into test tube with 9.9cm3 of distilled water. 0.1cm3 from test tube 1 put into test tube 2 ect ect
there are 4 bacteria in test tube 5.
Whats the dilution factor in test tube 5 and how many bacteria were in original culture

10 (to the power) -10
1:10000000000
4x10(to the power of) 10
(40,000,000,000)

how can you indirectly count cells

measuring turbidity using a colorimeter

how does a colorimeter 'count' the number of bacteria

finds the absorbance of the suspension then reading from a standard graph of light absorbance plotted against the number of cells

whats the kingdom bacterial cells are in

prokaryote

whats meant by aseptic technique

process aperatus and equipment are kept free of microorganisms

whats a hemocytometer

microscope slide used to calculate the number of microbes in a sample

what shape is coccus

spherical

What shape is bacillus?

rod shaped

what shape is spirillum

corkscrew

Name three ways we can classify bacteria

gram stain, shape, oxygen requirements

How does penicillin kill gram positive bacteria?

No outer polysaccharide layer, interferes with with production of cell walls and causes bacterial lysis.

What do we use to kill gram negative bacteria?

Antibiotics like polymyxins, which target the outer lipopolysaccharide layer.

What is the difference between a defined and undefined culture medium?

defined - has known components w/ conc specified
undefined - exact components and conc not known

What is a complete medium?

contains all necessary nutrients for growth of microorganisms, e.g. glucose, nitrogen