Lecture 11 - bacterial growth and nutrition Flashcards
How can you directly measure cell numbers?
Direct microscopic counting
Viable counts
What is the process of direct microscopic counting?
dilution of cells from a sample
-‘a total cell count’
-doesn’t specify if cells are dead or alive -> could stain cells to idicate if dead or alive
-sample added to ridged gridded slide
-work out: space between coverslip and slide (0.02mm) X area of the grid (1mm^2) = volume
Must multiply up volume to calculate how many cells per ml
-only works for densities of >5X10^6
What is the process of a viable cell count?
- determines how many cells are alive
- Method: spread bacterial culture onto agar plates to determine how many colonies present in the sample, use serial dilutions to acheive a countable number of colonies (30-300cfu)
- measured in cfu (colony forming units)
- assumes that each cell in culture can form a colony on the agar, may not be true if cells are clumped
- must also prove got pure culture by doing 100μm of culture onto a plate and incubating
- if contains O2 sensitive bacteria use pan plate method, bacteria incorperated into agar
- no of colonies X dilution factor = cells per mg of original sample (cfu per ml)
What indirect method can be used to measure cell mass?
Measure turbitity
What is the process and features of measuring turbidity?
- less time consuming that a direct measurement
- spectrophotometer to detect the amount of unscattered light that passes through a curvette containing a suspension of the culture
- light scattering is a function of: cell volume (more bacteria more light scattered), cell size (larger cells scatter more light), wavelength (more light scattered at a shorter wavelength) - must calibrate for each organism at a particular wavelength
- measure ODxnm (x= particular wavelength)
- provides instant measure of biomass
- if standard curve of OD verses cell numbers has been determined prev, then v quick method of calculating no of cells
- light scattering is not a linear function of cell mass, at higher concs less light is scattered per unit of cell mass (use LOG)
What is the fastest rate of e.coli growth and how does it acheive this?
- grows fasted via aerobic resporation with glucose
- on a rich media and aerobic, doubling time = 20mins
- 1 glucose in mitochrondria -> 38 ATP (however in E.coli, 1 glucose = 30ATP)
1. glucose can be completely oxidised to CO2 resulting in production of NADH from NAD
2. only v small amount of ATP is made this way
3. NADH is then reoxidised via the aerobic respriritoy chain which terminates in the reduction of O2 to H2O
4. protons are pumped across the cytoplasmic membrane to generate a membrane potential (pumped outside the cell)
5. this is used by the F1F0-ATPase to covert ADP and Pi to ATP (where more of ATP is made)
How can E.coli grow by fermentation?
- only if must do so when there is no O2
- O2 missing as final e- acceptor, alternative used is CO3 or NO2
- uses mixed acid fermenation
- glucose is converted to pyruvate
- made 2ATP and 2NADH
- cells try to make as much ATP as possible by using substrate level phosphorylation whilst reoxidising NADH (redox balancing)
- process wastes carbon
What are the features of batch growth in the lab?
- produces a batch growth curve
- initally has an adaptation phase (lag)
- once adapted exponential growth curve is shown, but doesn’t last long
- followed by stationary phase, variable in diffememt microorgaisms (cell division AND cell death) [turbidimetry would be useless (dead cells??)]
- turns on secondary metabolism (production of secondary metabolites, antibiotics)
- followed by death phase, variable for different organisms
How can you derive quantitative growth data from batch growth?
- generation time = how long it takes for the population to double (mass or numbers), determine using a semi-log plot of exponential growth
- once generation time has been estimated can use a formula to describe how many cell are present at a certain time
Nt=No2^n
=no of cells at time t =number of cells at time 0 multipled by 2 raised to the power of the number of doubling times (generation)
How have simple growth experiments revealed the function of genes, and give an example
- many genes in bacteria required for growth on a particular energy/nutrient source
- if these genes are disrupted can look for growth defects
E.g.
- nanT transporter gene is needed for growth on sialic acid as the sole carbon source but not for growth on glucose
- if transporter deleted then no growth at all on sialic acid (or v low)
- most organisms have multiple growth systems - rare to totally stop growth - therefore this gene has a v imp role
- if add back in a copy of nanT in a plasmid, growth is recued (ensures that stopping growth was the cause of the loss of this transporter)
Outline the ‘feast and famine’ model of bacterial growth and what this means for experiments
- most of the time bacteria are starving
- lab does not normally represent these conditions, in reality batch growth curve is rare
- tend to ‘survive’ only until conditions improve then grow v quickly in good conditions
- v. flexible about what is good
What occurs molecularly to describe the action of bacteria in a ‘feast and famine’ environment?
- bacteria have high affinity uptake systems to be able to use low concentration of nutrients available (e.g. in aquatic environments only 50μm of free C is available)
- these systems are dependent on substrate binding proteins e.g. ABC transporters, TTTs, TRAP
- have multiple copies of each, which all likely recognise a different small molecule
Give examples of competeition between bacteria in nutrient rich environmetns and what means for the evoltuon of the microorganisms present
Gut
- nutrients are very abundant, but there is huge competition for the limited resources available
- relatively small changes in efficiency rapidly lead to the selection of a species in the community
- microorganisms tend to secrete enzymes to break down plant derive polysaccharides (oligosaccharides, monosaccarides (then used for growth)) - but secrete enzymes to cleave sugars from the cell surfaces when polysaccharides etc. run out
- bacteroids have systems to utilise polysaccharides e.g. B. thetaiotaomicron has 88 polysaccharide utilisation loci (PULs)
How do E.coli (and other bacteria) adapt to famine?
E.coli in stationary phase -induces around 100 genes via sigmaS Prepare the cell for starvation: -metabolism of the cell is decreased -cells become more resisitant to a variety of stresses (pH, osmotic, temp) -cell becomes shorter
Other bacteria
- make secondary metabolites e.g. streptomyces makes antibiotics
- undergo physical change e.g. B.subtilis sporulates
What is the long term stationary phase?
In E.coli
-in this phase, cells are dying but other cells are using their remains to grow i.e. the rate of cell death equals the rate of growth - can continue for many generations without the addition of fresh nutients
