Microbiology Flashcards
estimating the number of bacteria present calculation
number of colonies counted x dilution factor x 2 (to estimate in 1cm3 if counted in 0.5cm3)
how correct is the estimate and why
underestimate - does not include non-viable/dead bacteria and cannot be sure each colony has grown from a single bacteria (clumps)
what is the assumption made during dilution plating
each colony has arisen from a single bacterium - may not be the case due to clumps
colony
cluster of cells from a single bacterium
pathogen
microorganism that causes disease in its host
aseptic technique
lab practice that maintains sterile equipment and prevents contamination
how do you count bacteria directly
in liquid culture (nutrient broth)
by counting each cell
how do you count bacteria indirectly
measuring turbidity (cloudiness of culture medium) with a colorimeter
total counts
living and dead cells
viable counts
only living or actively growing cells which underestimates the number
when is serial dilution/dilution plating used
when population density of the sample is too much to count
serial dilution method
- fill 5 test tubes with 9cm3 water
- add 1cm3 sample to first tube (1 in 10 dilution)
- mix dilution and pipette 1ml into second tube (1 in 100 dilution)
- repeat the process until 1 in 10000 dilution
- plate each on agar
why do you count the plate with a middle amount of bacteria
plates with lots of bacteria (1/10 and 1/100) is impossible to distinguish individual colonies and cannot count
plate 1/1000 cannot count reliably
plate 1/10000 has enough and easy to count * most reliable
plate 1/100000 does not have enough for reliable
3 types of bacteria
spherical coccus
rod shaped bacillus
spiral spirillum
what colour does gram positive bacteria stain and why
purple - thicker peptidoglycan cell wall which retains the crystal violet stain and becomes purple
what colour does gram negative bacteria stain and why
pink - thinner peptidoglycan cell wall and extra lipopolysaccharide membrane. when treating with alcohol, the extra layer is lost and purple stain is washed away so remain colourless, but stain pink after counterstaining with safranin
gram stain procedure
fixation
crystal violet stain
treat with iodine
decolourisation
counterstain with safranin
what can bacteria be cultured on
nutrient rich agar jelly or in a nutrient broth
growth media for bacteria must include
carbon source - glucose or lactose
nitrogen source - amino acids or ammonium
source of sulphur and phosphorus
vitamins, minerals, growth factors
suitable pH
suitable temperature - 25 in school
obligate aerobe
growth is inhibited in absence of oxygen
obligate anaerobe
growth is inhibited in presence of oxygen
facultative anaerobes
grow best in presence of oxygen but can respire anaerobically
aseptic techniques are used to prevent
contamination of environment by microorganisms being handled
contamination of bacterial cultures by unwanted microorganisms
examples of sterilisation used
passing metal through flame
pre-sterilised petri dishes
sterilising glassware in high temps
heating agar to sterilise before pouring in plate
how to pour a sterile agar plate using aseptic techniques
open culture bottle slightly
flame neck of bottle
work close to flame to prevent contamination
open dish at angle
pour in and close immediately and swirl to remove air bubbles
secure with tape
inoculating set nutrient agar plate
sterilise inoculating loop in flame
dip loop in milk
spread milk across agar while rotating
tape lid shut (not completely)
incubate at 25
