Bacterial growth and the cell division cycle L3

Question 1

What is the rate of bacterial cell division and what did this allow

Answer 1

fast rate allowing
- spread of antibiotic resistance: neg
- use in lab: pos

Question 2

how can bacteria be used in labs

Answer 2

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

Question 3

define growth

Answer 3

An increase in the number of cells

Question 4

define generation time

Answer 4

the time needed for one cell to divide and form two cells

Question 5

what is the term given to the growth of bacteria in the right conditions

Answer 5

exponential growth
doubles each generation

Question 6

example of exponential growth of bacteria

Answer 6

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!!!

Question 7

when do bacteria stop dividing exponentially

Answer 7

when conditions are unfavourable
- run out of nutrients

Question 8

what are the 4 stages of a typical batch growth cycle
- nutrients run out

Answer 8

1. lag phase
2. exponential phase
3. stationary phase
4. death phase

Question 9

describe lag phase

Answer 9

slow growth
cells adjust to new conditions, synthesise required metabolic enzymes and metabolites

Question 10

describe exponential phase

Answer 10

exponential growth
– optimal growth with regular doubling in cell numbers

Question 11

describe stationary phase

Answer 11

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

Question 12

describe death phase

Answer 12

more cells die creating toxic condition, more cells dying than being produced
complex gradual loss of viability but with some cell turnover

Question 13

what are the 4 ways of measuring bacterial growth

Answer 13

Plating methods
Turbidity
Direct microscopic counting
Flow cytometry

Question 14

define total cell count are viable cell count

Answer 14

1. total cell count
   - total number of bacterial cells
2. viable cell count
   - number of living bacterial cells

Question 15

describe plating method

Answer 15

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

Question 16

what are positives of plating methods

Answer 16

1. highly sensitive
2. growth conditions can be customised so only bacteria of interest grow
3. only measures alive cells

Question 17

what are negatives of plating methods

Answer 17

Underestimates for cells in chains or clusters

Question 18

describe Turbidity method

Answer 18

use spectrometer to read absorbance
higher the absorbance reading, more bacteria

Question 19

what are positives of turbidity methods

Answer 19

1. simple and convenient
2. non-destructive and can be done continuously

Question 20

what are negatives of turbidity methods

Answer 20

1. Measures all particles, including any dead cells
2. Low sensitivity (>106 cells per ml lower limit)
3. Culture turbidity has to be within a certain range to be accurate (not too low, not too high)

Question 21

describe direct counting method

Answer 21

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

Question 22

what are the positives of direct counting

Answer 22

1. can account for chained bacteria

Question 23

what are negatives of direct counting

Answer 23

Doesn’t discriminate live / dead
-But staining methods are available
Laborious
-But can be automated

Question 24

describe Flow cytometry

Answer 24

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

Question 25

what are positives of flow cytometry

Answer 25

Highly automated

Question 26

describe FACS

Answer 26

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

Question 27

what do most bacteria divide by

Answer 27

binary fission

Question 28

what are the steps in binary fission

Answer 28

Steps in the division cycle
Chromosome replication and segregation
Importance of the Z-ring
Regulation of Z-ring formation

Question 29

what are the steps in cell division

Answer 29

1. Cell grows
   Cell structures duplicated (cell wall, membrane, ribosome etc.)
   Chromosome replicated
2. 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

Question 30

describe DNA replication

Answer 30

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

Question 31

describe bacterial chromosome replication

Answer 31

bidirectional- 2 replication forks
starts at oriC and ends at terC

Question 32

give the steps of chromosome replication

Answer 32

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

Question 33

how does cell ensure both daughter cells get same amount of DNA

Answer 33

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

Question 34

What is a divisome

Answer 34

bacterial cell division machinery which is a contractile Z ring (FtsZ protein)

Question 35

what does contraction Z ring cause

Answer 35

Contraction of the Z-ring causes septum formation, new cell pole synthesis and ultimately division

Question 36

how does cell division in Anabaena (cyanobacteria) differ

Answer 36

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

Question 37

how does division in Bdellovibrio differ

Answer 37

it grows inside other bacteria
attaches to prey (E.Coli) and divides in periplasm evertually causing E.coli to burst

Question 38

how does division in Myxococcus differ

Answer 38

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