Bacterial growth and the cell division cycle L3 Flashcards
What is the rate of bacterial cell division and what did this allow
fast rate allowing
- spread of antibiotic resistance: neg
- use in lab: pos
how can bacteria be used in labs
Recombinant E. coli can be used to express proteins for research, medical or commercial purposes (e.g. production of Insulin)
Fast growth rate allows lots of protein to be made rapidly
define growth
An increase in the number of cells
define generation time
the time needed for one cell to divide and form two cells
what is the term given to the growth of bacteria in the right conditions
exponential growth
doubles each generation
example of exponential growth of bacteria
This species has a 30 min generation time. The number of cells doubles every 30 mins during exponential growth.
Starting with only 1 cell…
after 2 hours there are 16 cells
after 4 hours there are 256 cells
after only 10 hours there are 1,048,576 cells!!!
when do bacteria stop dividing exponentially
when conditions are unfavourable
- run out of nutrients
what are the 4 stages of a typical batch growth cycle
- nutrients run out
- lag phase
- exponential phase
- stationary phase
- death phase
describe lag phase
slow growth
cells adjust to new conditions, synthesise required metabolic enzymes and metabolites
describe exponential phase
exponential growth
– optimal growth with regular doubling in cell numbers
describe stationary phase
number of dying cells equal to number of new cells
growth limited by nutrient depletion or accumulation of toxic metabolites. Rate of new cell production balanced with rate of cell death so no overall growth in the culture
describe death phase
more cells die creating toxic condition, more cells dying than being produced
complex gradual loss of viability but with some cell turnover
what are the 4 ways of measuring bacterial growth
Plating methods
Turbidity
Direct microscopic counting
Flow cytometry
define total cell count are viable cell count
- total cell count
- total number of bacterial cells - viable cell count
- number of living bacterial cells
describe plating method
serial dilution carried out- diluted 10X every time
grow culture
first plates will have too many colonies, last plates will have too little colonies
middle plates will have good number of colonies
one colony is assumed to be from one bacteria
- if there are 17 colonies on plate then started with 17 bacteria
to work out original, times number of colonies by 10 to the power of the serial dilution number
what are positives of plating methods
- highly sensitive
- growth conditions can be customised so only bacteria of interest grow
- only measures alive cells
what are negatives of plating methods
Underestimates for cells in chains or clusters
describe Turbidity method
use spectrometer to read absorbance
higher the absorbance reading, more bacteria
what are positives of turbidity methods
- simple and convenient
- non-destructive and can be done continuously
what are negatives of turbidity methods
- Measures all particles, including any dead cells
- Low sensitivity (>106 cells per ml lower limit)
- Culture turbidity has to be within a certain range to be accurate (not too low, not too high)
describe direct counting method
Use microscopes and grid on slide
Know how big is grid and volume of culture can sit under grid
Count how many bacteria are in grid
what are the positives of direct counting
- can account for chained bacteria
what are negatives of direct counting
Doesn’t discriminate live / dead
-But staining methods are available
Laborious
-But can be automated
describe Flow cytometry
bacteria culture placed in machine similar to spectrophotometer but uses laser not light
bacteria are in narrow tube under high pressure causing them to flow
when laser hits bacteria, a count is taken
what are positives of flow cytometry
Highly automated
describe FACS
Fluorescence Activated Cell Sorting
add florescence to bacteria, changing wave length of laser allows them to be separated by fluorescence
Requires the right equipment, reagents and expertise
what do most bacteria divide by
binary fission
what are the steps in binary fission
Steps in the division cycle
Chromosome replication and segregation
Importance of the Z-ring
Regulation of Z-ring formation
what are the steps in cell division
- Cell grows
Cell structures duplicated (cell wall, membrane, ribosome etc.)
Chromosome replicated - Daughter chromosomes segregate to different ends of the cell
3.
Septum forms at mid cell as Z-ring constricts
New cell poles synthesised as ring constricts
4.
Cell division occurs at mid-cell
Results in two identical daughter cells
describe DNA replication
the replisome unwinds DNA into two strands to create replication forks
Using chromosomal DNA as a template, replisome moves 5’ to 3’ direction synthesising two new DNA strands
Product is two copies of the original DNA
describe bacterial chromosome replication
bidirectional- 2 replication forks
starts at oriC and ends at terC
give the steps of chromosome replication
1.replisome binds to oriC
2. bidirectional replication at 2 replication forks
3. As DNA is being replicated, more replisomes bind both old, and new DNA
4. Initial replication complete, chromosomes segregate and cells divide
- Replication already halfway complete in daughter cells
how does cell ensure both daughter cells get same amount of DNA
oriC localised to the middle of the cell
“Left” and “right” arms of the circular chromosome are carefully positioned
During chromosome replication, daughter oriCs localised to quarter distance along the cell (1/4 and ¾)
Following division each cell has one copy of the chromosome
What is a divisome
bacterial cell division machinery which is a contractile Z ring (FtsZ protein)
what does contraction Z ring cause
Contraction of the Z-ring causes septum formation, new cell pole synthesis and ultimately division
how does cell division in Anabaena (cyanobacteria) differ
this bacteria is able to photosynthesise due to vegitive cells
these cells can not nitrogen fix
some vegetative cells undergo asymmetric division to form heterocytes or akinete
heterocytes can fix nitrogen
akinete store nutrients and are needed for survival
- they will survive during periods of starvation
how does division in Bdellovibrio differ
it grows inside other bacteria
attaches to prey (E.Coli) and divides in periplasm evertually causing E.coli to burst
how does division in Myxococcus differ
eats other bacteria then makes complex fruiting bodies
- Has various life cycles depending on where it is
- When no food, they form spores via asymmetric cell division
