L30 - bacterial growth & replication Flashcards
outlines
- how bacteria exist in the environment
- factors affecting bacterial growth (nutrients, (iron)
oxygen, temp) - how bacteria can be cultured
- describe and calculate bacterial growth in batch culture
- 2 ways to measure bacterial growth
what can bacteria exist as?
planktonic cells (free living cells)
where do bacteria usually live in?
biofilms (preferred way)
where do bacteria form biofilms and how do they form them?
- surfaces with moisture
- bacteria attach to surface and grow
- become developed in an extra cellular matrix (ECM)
- ECM mostly composed of polysaccharide, proteins, DNA
5 stages of biofilm formation?
- initial attachment
- irreversible attachment
- maturation 1
- maturation 2
5 dispersal
what is initial attachment? (biofilm formation)
individual bacteria attach weakly to a surface
what is irreversible attachment?
attachment becomes irreversible using fimbrae and pilli. bacteria then multiply and attract other microbes to attach.
what is maturation 1?
- bacteria secrete a sticky, protective extra cellular matrix (ECM)- polysaccharides, protein and DNA.
- bacteria continue to join and multiply
what is maturation 2?
biofilm grows in size and structure- form large 3D colony.
what is dispersal?
sections of the biofilm break off.
the cells can go and colonise new areas
bacteria are how many times more resistant to antibiotics in a biofilm?
1000x
why is living in a biofilm a successful way of living?
protects against phagocytosis, antibiotics, disinfectants
why are biofilms a huge issue in healthcare?
- growth on medical devices and implants
- e.g catheters, hip replacements, heart valve, stents
what is E.coli a common cause of?
catheter associated urinary tract infection
what is endocarditis?
infection of the heart valve caused by Enterococcus sp
what is cystic fibrosis?
Cystic fibrosis patients.
Opportunistic infection by
Pseudomonas aeruginosa
what are nutrients required for?
cellular biosynthesis and energy generation
macroelements
C, H, O, N, S, P, K, Ca, Mg, Fe, water
trace elements
Mn, Zn, Co, Mo, Ni, Cu, vitamins, growth factors
how do bacteria use nutrients?
bacteria colonising/ infecting out bodies derive nutrients from their host
what are the factors affecting growth of bacteria?
- nutrients
- iron
- oxygen
- temp
how does iron affect bacteria growth?
used for energy generation
is iron in body available?
NO
in mammalian cells what is iron stored as?
- 90% in ferritin or as a haem group
- 8% stored in other cellular proteins, e.g myoglobin
what is iron stored as outside a mammalian cell?
- 1-2% attached to transporters
- e.g transferring in serum, lactoferrin in mucosal secretions
- non complexed iron exists as Fe3+: insoluble, not absorbed into blood
what are siderophores?
bacterial iron transport system
describe siderophores
- low molecular weight with high affinity for iron
- high energy strategy
- produced + exported from bacteria when conc of iron is low
- blind iron & allow uptake into the cell
- remove iron complexed with transferrin
- enable uptake into bacterial cell
how does oxygen affect bacteria growth (for aerobes)?
- aerobes require oxygen for growth (e.g Mycobacterium tuberculosis)
- some bacteria are microaerophile: can grow in low concentrations of oxygen
how does oxygen affect bacteria growth (for anaerobes)?
- anaerobes do not require oxygen for growth
- obligate anaerobes: cannot growth in presence of oxygen
- facultative anaerobes: can grow in oxygen if it is available
why is C.difficile a problem in hospital?
form spores, spores hard to remove as hard to disinfect them
how does temperature affect growth of bacteria?
- mesophiles (common): live art 20-40oC.
- psychrophiles live at 40-20 (optimum is 15), e.g listeria monocytogenes
- thermophiles live at 45 to 100, e.g thermophiles aquatics
how does pH affect bacteria growth?
most organism are neutrophils which grow best at pH 6.5-7.5
how can bacteria culture be grown?
suspension or colony
what is a suspension?
bacteria grown in complex liquid media as batch culture
what is a colony?
bacteria grown on complex media solidified with agar
what are suspensions used for?
to determine growth rate/ effect of antimicrobials
what are colonies used for?
- to obtain pure culture
- perform viable count
- assess diversity
- aid identification
what do bacteria replicate by?
binary fission
what is binary fission?
ONE bacterial cell grows and divides into TWO identical daughter cells
describe the process of binary fission
- elongates to approx double length
- cell about to divide copies its chromosomes which go to opposite ends of the cell
- septum begins to form
- 2 copies of the chromosome are pulled apart
- septum formation continues until 2 daughter cells are pinches off
binary fission continues until?
- until nutrients depleted
- or conditions become unfavourable
- cells can potentially divide forever
what is exponential growth?
bacteria multiply by doubling as fast as conditions will allow
what is generation time?
time taken for bacteria to divide
what is the rat of cell division determined by?
- time needed for DNA replication
- conditions
what is the lag phase?
what is the exponential growth phase?
- cells act in constant predicate way
- generation time is constant
- straight upwards line of graph
- ideal phase to use bacteria for research
what is the stationary phase?
- population running out of resources
- no increase or decrease in cell numbers
- some cell dividing, some dying
- cells behaving unpredictably
what is the death phase?
- less resources
- so decline in cell numbers
- some cells persister cells which are dormant
- persister cells are viable but non-culturable cells
how to calculate generation time (g)?
- read from exponential phase of growth curve
- generation time (g) = Time (T) / no. of generations (n)
how to calculate number of generations (n)?
n = Log10Nt – Log10N0 / Log10 2
what is Nt?
number of cells present at time T
what is N0?
number of cells initially present
what is n?
number of generations
A population of Escherichia coli increases from 103 to 109 cells in 10 hr.
What is the generation time?
- N0 = 10^3
- Nt = 10^9
- so number of generations = 9-3 / 0.301 =19.93
- g = 10/20 =0.5
how to get a viable count?
- 1 bacterium produces 1 colony (CFU) on agar plate which can be counted
1. dilute sample, spread on agar, incubate overnight @ 37 degrees, count colonies as colony forming units / ml(CFU/ml), use standard form
OR
- best way: measure number actively dividing cells, except for clumps/ chains of cells, optimum conditions, overnight culture
indirect measurement of bacterial number
- optical density (OD)
- OD increases with increasing cell number overtime
- cell number directly related to OD
- read off cell number from standard curve of OD vs cell number
- CELLS MUST BE IN EXPONENTIAL PHASE for OD to represent number of DIVING cells
