Bacterial growth & division Flashcards
What does media provide?
what are macronutrients for all life? what are some macros for some life?
micronutrients?
how do heterotrophic bacteria get carbon?
autotrophic bacteria?
what 2 ways can these bacteria get energy?
what are the inorganic forms of nitrogen, phosphorus & sulphur?
nutrients for growth & multiplication
carbon, hydrogen, oxygen, nitrogen, phosphorus, sulphur
- K, Mg, Ca, Na, Fe (micromolar)
nanomolar: Co, Zn, Mn, Cu, Ni, Se, W
organic (fixed) carbon so from glucose, sugars, acids etc = energy for biosynthesis
inorganic carbon = CO2
phototroph = light chemotroph = oxidation of electron donors e.g H2S, NH3
NH4+, NO3-, N2
PO4 3-
SO4 2-, H2S, S
Some bacteria need specific growth factors like vitamins & amino acids. How do they get these now & what was it before?
What is defined media?
what is undefined/complex media?
on which medium is it easier to grow bacteria on?
what is the great plate count anomaly? why is it caused?
environment- used to have biosynthetic pathways to make them
defined = medium made from specific list of chemicals- must know biochem of organism
undefined = contain nutrients most bacteria require
undefined/complex- don’t know all growth factors required
taking samples from environment & counting bacteria and then plating them to see how many colonies form.
- more cells seen in environment than colonies on plates as some bacteria don’t grow on pure culture as require different nutrients
what does bacterial growth lead to?
what is it accompanied by? 2
what are the 2 procedures to binary fission? what species do this?
what is filamentous growth?
how do cocci divide?
can you characterise bacteria based on how they divide?
what is the mass of 1 bacterium?
increase in biomass
binary fission (divides cell into 2 new daughters = clones) or budding (cell buds off mother cell)
cell division & separation of cells- rods & cocci
cells divide but dont separate = cell filaments
different planes (1D, 2D or 3D)
yes
10^-13g
How would you monitor/quantitate the growth of cells in batch culture?
what would you plot
what is the beginning lag phase?
log/exponential phase?
stationary phase?
death phase?
what is the phase after?
Inoculate small quantity culture in medium & incubate
plot log cell number (y) vs time (x)
no increase in cell number- as adapts to new conditions as bacteria are in stress state. reprogram cells to synthesise new cellular components
fastest rate achieved of growth & divide as fast as possible
growth rate plateaus- nutrients & oxygen exhausted & waste builds so growth & death rate balanced
build up of waste & toxins means cells die, death rate exceeds growth rate, starvation, some cells survive & are resistant
phase of prolonged decline- slowly all cells die & resistant ones might too
What is diauxic growth?
what happens when glucose runs out?
what does it look like on a logN vs time graph?
what is tgen?
growing bacteria in 2 different types of carbon (e.g glucose & lactose)
re-enter lag phase- remodel for lactose & then have 2nd log phase
very large exponential growth line bit
time takes for N to be 2N (doubling time = mean generation time) or how long takes generation to double in exponential/log phase
What method can you use to count cells of a dense broth?
what do you need?
if the depth is normally 0.02mm, and the dimensions are 1mm x 1mm. there are 25 squares each with 28 bacteria inside, how many are in 1ml?
what are the disadvantages?
use small volume on a glass slide & count with counting chamber & haemocytometer
counting chamber
haemocytometer
depth (to calculate volume)
28 x 25 x 50 (to make up 0.02mm to 1mm) x 1000 (mm3 to cm3)
tedious
counts live & dead cells
briefly what is the serial dilution & plating method for counting bacteria?
what are the steps?
what do you do with the number of colonies? (calc)
what should you avoid?
diluting the original culture 10x fold each time
- take original culture & dilute it 10 fold how ever many times
- plate 0.1 ml of your diluted sample & spread on plate & incubate
- count the number of colonies
conc of original culture = (dilution factor x number of colonies) / volume plated
so if have 200 colonies & diluted it 5 times
10^5 x 210 / 0.1
too few colonies <100 = large margin for error
too many >100 = hard to count
what 2 methods can you use to grow discrete colonies?
how does the 2nd method produce a discrete system?
there is a margin of error with counting colonies & converting to number of bacterial cells- why?
what units can you use instead? why is this better? why is it long?
- dilute sample plated with pipette & use glass spreader for even distribution
- add sample & molten agar together & mix & incubate
- end up with cells throughout media
aerobic bacteria will grow on the surface = larger colonies there
not each colony arises from 1 cell as some don’t divide
colony-forming units (cfu) per ml- more accurate & counts live cells- slow & depends on bacteria growing at reasonable rate & if they are culturable
What way can you measure number of cells without counting them individually?
What are the pros & cons?
how can you quantify this?
what is the graph axis?
what does an algem twin photobioreactor do?
Optical density- using a spectrophotometer to measure light scattering
pros = very quick cons = dense cultures only
optical density at specific wavelength is proportional to cell concentration with a calibration curve- so prepare standard curve with cell number to find out number of cells in 1 absorption unit
x = time y = klett units & OD (calibrated)
uses OD to automatically plot growth in 2 different chambers- used as a proxy to measure growth performance
what is coulter counter? how does it count number of cells?
what are some problems?
how do you measure biomass of cell culture?
what is the most probable number MPN? what is it used for?
how is electrical impedance used to measure number of cells? what is it used for?
cell suspension forced through a small hole with electrodes either side so when cell passes the current drops- count number of times current drops
less accurate with bacteria as smaller
can’t distinguish between living or dead
debris & filaments interfere
harvest cells (centrifuge & filter) & measure weight of wet or dry pellet
dilute sample & score growth or no growth in dilutions- compare with statistical tables to estimate likely cell number - water industry
microbe growth in sample means decreases electrical impedance due to ionic metabolites produced - food industry (quality)
in what can you grow bacteria as a continuous culture?
how are they held?
what happens constantly?
chemostat
optimum growth rates/steady state for log phase
flow of fresh medium & tapping out of bacterial broth
