Lecture 8: Bacterial Growth in the Lab Flashcards
Where can bacteria grow in a lab?
- agar plates
- broth
- nutrient rich media
Growth in nutrient rich media takes on
the form of ________ growth
logarithmic growth
Logarithmic growth
- Binary fission - 1, 2, 4, 8, 16…
Growth rate can be expressed as doubling
time
E. coli has a doubling time in rich broth culture of 20 min
More formally, N=N0e^kt
How to measure microbial growth?
- perform measurements on liquid cultures only
- plate counts
- optical density (turbidity)
- Microscopic visualization
Plate counts
count number of colony forming units (CFU) on plate
Optical density (turbidity)
direct measure the ability to absorb light at a specific wavelength
microscopic visualization
count bacteria in a given amount of culture directly
how to conduct plate counts?
Dilute culture in an appropriate buffer/
medium (serial dilutions)
If sample is very dilute, may use filtration method to concentrate instead
Plate onto nutrient agar plates
Count colonies (CFUs) after right amount of time (24h for standard
organisms)
Advantages and disadvantages of plate counts
advantage- only counts bacteria capable of dividing (CFU)
Disadvantage- takes time to do; subject to experimenter error in dilution
How to do optical density (turbidity)
Use a spectrophotometer
Shines light at a specific
wavelength through the culture, measures the amount that passes
through (transmittance or its converse, absorbance)
Advantages and disadvantages of turbidity
- Advantage- quick, accurate
method for determining density of
culture
Disadvantage- may count dead cells
How to do microscopy?
Take direct sample, count cells (use
hemocytometer or Petroff-Hauser cell counter)
Advantage and disadvantage of microscopy
Advantage-quick, precise
Disadvantage- may vary greatly from field to field, so need to observe many fields to get statistically relevant numbers
Disadvantage- doesn’t distinguish live from dead cells
Disadvantage- intensive compared to other two techniques
This is not commonly used for cell counting
Logarithmic growth phases
bacterial culture growth has 4 phases
- lag
- log phase
- stationary phase
- death phase
Lag phase
when inoculating
a culture, cells take
time to adjust to new
environs
Log phase
maximal growth rate
N=N0e^kt
Calculate doubling time
(N=2N0)
Stationary phase
number of cells is
steady
Death phase
rate of cell death exceeds division rate
Solve N=Noe^kt
No=100, N =3200, t=300
3200 = 100e^k(300)
32 = e^300k
ln32 = 300k
ln32/300 = k
k = doubled
tD is when N = 2No
2No = Noe^ktD
2 = e^ktD
ln2 = ktD
tD = ln2/k
Solve N=Noe^kt
No=300, N =600, t= 60
then plug into tD equation
600= 300e^k(300-240)
2= e^k(60)
ln2 = 60k
ln2/60 = k
tD = ln2/(ln2/60)
tD = 60
What kind of culture does logarithmic growth curve describe?
batch cultures
What is batch culture?
The microbes are exposed to the same media, even as they utilize the nutrients and release various products
Where is continuous culture done?
in a chemostat
What is continuous culture?
The spent media (containing bacteria) is removed, and fresh
media added, at a constant flow rate
Chemostat culture graph
D = f/V
- at low dilution rates, growth rate proportional to D (Monod)
maintenance energy
high dilution rates - washout
What environmental factors affect growth rate?
- temperature
- pH
- water activity
- oxygen
psychrophilic
cold loving
thermophile
heat loving
mesophile
moderate temp. loving
hyperthermophile
high temp. loving
Why does temperature curves have that asymmetrical shape?
after you reach optimal temp. the cells immediately inhibit growth and eventually denature at the higher temperatures.
What do cardinal temperatures reflect?
reflect niche where normally found
What bacterium are found in skin (can tell by their cardinal temperatures 30-37)?
- listeria monocytogenes
- staphylococcus aureus
acidophile
acidic condition loving
neutrophile
neutral pH loving
alkalophile
high pH loving
How does chemiosmosis work?
uses proton gradient to generate ATP across a membrane
compatible solutes
a molecule that doesn’t interfere with the biochemistry happening in a cell, but pulls water into the cell ( or holds water in the cell)
- modulate osmolyte concentration so it can quickly change whether to release or retain water.
how does bacteria make compatible solutes?
- manipulate ion flows across a membrane
- make derivative compounds of simple ones they use a lot, to use them as a compatible solute temporarily (ex: trehalose)
osmotic pressure
solutes
- salts
halophile
salty conditions
matric pressure
absorption to solids
- desiccation
water activity
available water for organisms
- proportion of water available for biochemistry
low water activity
makes life difficult
formula for water activity
aw = Psoln / Pwater
desiccation
loss of water? no water?
What protects against modest changes in water activity?
the cell wall
What happens at low water activity?
DNA disordering
What is botulism? What food is a known botulism carrier? Who is at risk of it?
Clostridium botulinum
- a rare illness caused by a toxin that attacks the body’s nerves and causes difficulty breathing, muscle paralysis, and even death
- honey
- babies, not adults because we already have bacteria in our gut so clostridium botulinum cant plant itself in our gut.
staphylococci
facultative anaerobes?
(use oxygen when its there, doesn’t use it when its not)
streptococci
aerotolerant anaerobe
(ignore oxygen completely)
- never aerobes
- always anaerobes
Other factors on microbial growth
- radiation
- pressure
Radiation
UV, ionizing
DNA damage T-T
dimers
Deinococcus
radiodurans
Endospores
barophilic
high pressure
barotolerant
can survive high or normal pressures
piezophilic
- deep sea
lives under elevated hydrostatic pressure
Liebig’s law of the minimum
total biomass of organism determined by nutrient present at lowest concentration
Shelford’s law of tolerance
above or below certain environmental limits, a microorganism will not grow, regardless of the nutrient supply
Nutrient stress (oligotrophy) May result in ___________ in cells
physiological changes
Viable but non-culturable (VBNC)
Can be identified by staining protocols
Postgate microviability
Morphological changes at single cell level
Many bacteria studied show capacity
All organisms need ____ and energy to survive
carbon
Carbon sources
- autotrophy
- heterotrophy
autotrophy
fixation of carbon dioxide (gets it from the environment?)
heterotrophy
use of organic carbon
energy sources
- prototrophy
- chemotrophy
phototrophy
energy from light
chemotrophy
energy from chemical reactions
photoautotroph
light and fix CO2
photoheterotroph
light and uses organic carbon
chemoautotroph
chemical rx and fix CO2
chemoheterotroph
chem rxs and uses organic carbon
Redox reactions generate…
cell energy
Where is the energy cells need stored?
in chemical bonds
what forms bonds in a molecule?
electrons
______ are moved between molecules by ____ reactions
- electrons
- redox
In biological systems, oxidation of the nutrient is coupled with….
reduction of an electron carrier
What does a carrier do to electrons?
can transfer electrons from the nutrient molecule to other systems, where it is converted to ATP
OILRIG
- oxidation is loss
- reduction is gain
