Download Nutrition and Growth

Nutrition and Growth:
• Nutritional Classification
– Energy source
– Carbon source
• Requirements for Growth
– Temperature
– Salts
– Nutrients
- pH
- Oxygen
- Light
• Laboratory Cultivation
– Media Types
– Growth Curves
– Measuring Growth
Nutritional Classification:
This is
us!
What does it mean to use water
to reduce carbon dioxide? How
does this relate to oxygen
generation or not?
Carbon Source:
CO2 Only
6 cycles for the 2
GP needed to
make 1 glucose.
• Autotrophs don’t feed on
organic matter to acquire the
small organic compounds
needed for cell growth.
• Instead they use energy and
electrons to reduce 6 carbon
dioxide to 1 glucose, which is
then converted to all the small
organic compounds needed
for cell growth. They do it all
themselves; hence “auto”.
• The metabolic pathway
responsible for “CO2 fixation”
is the Calvin Benson Cycle.
Reduction
step
(adding e-)
Light for Energy: Phototrophy
This is called Cyclic
Photophosphorylation;
because electrons cycle
back to chlorophyll.
Photoheterotrophs do this!
• Light excites chlorophyll (photopigment) electrons to a higher energy state.
• As with electrons from NADH in respiration; these light excited electrons transport
through an ETC; energy released pumps protons across a membrane creating PMF.
• Like in oxidative phosphorylation of respiration, the PMF drives ATP synthesis by
phosphorylating ADP; because light and not NADH oxidation is the source of excited
electrons, we call ATP production by phototrophy, photophosphorylation.
Non-cyclic Photophosphorylation:
Excited electrons leave chlorophyll and end up in the form of the electron
carrier, NADPH; chlorophyll needs to get new electrons from somewhere.
Electrons can come from water results
in O2 production. All plants, algae,
and cyanobacteria do this; oxygenic
photosynthesis.
Some bacteria can not tolerate
oxygen, nor can they get electrons
from water to supply chlorophyll. They
may use other compounds like H2S;
anoxygenic photosynthesis.
Requirements for Growth:
• An organism will grow and reproduce when it’s in an
environment where it can tolerate all physical and chemical
conditions. (Shelford’s Law of Tolerance)
– E.g. nutrients, oxygen, salts, light and pH levels are optimum for
growth, but if temperature is above that cell’s tolerance range it
won’t grow.
– What might you conclude if you could not isolate a marine bacterium
on agar that was made without salts, but all other conditions were
optimum?
• Growth will be slower when one or more condition is not
optimum, yet within the range of tolerance.
• Different microbes have different tolerance ranges for
different physical and chemical conditions.
Temperature: Peaks in these plots represent the optimum
growth temperature for that group of temperature tolerance. Growth rate
decreases at temperature values higher and lower than the optimum
until there is no growth (maximum and minimum growth temperatures).
Food Storage Considerations
15-50ºC = Danger Zone
Rate of cooling will be
different depending on
the surface to volume
ratio of your stored food.
Many psychrotrophs are
involved in food storage,
even at refrigerator
temperatures (4ºC).
Osmotic Pressure Tolerance
• Hypertonic (solute concentration higher outside of cell) can cause plasmolysis.
• Some bacteria are osmophiles; if the solute is salt we call these halophiles.
• Some bacteria are more osmotolerant than others (make compatible solutes).
• pH:
– Acidophiles (pH 0 – 5) acid mine drainage, vinegar
– Neutrophiles (pH 5.5 – 8) pure water, blood
– Alkalophiles (pH 8.5 – 10.5): soap, ammonia
• Light:
– Visible Light (380-760 nm; phototrophs need it)
– Ultraviolet Light (130-380nm; DNA damage)
• Nutrients:
– Carbon (quality of organics for heterotrophs)
– Macronutrients (nitrogen, phosphorous, sulfur)
– Trace elements (micronutrients; iron, copper, zinc…)
Oxygen Requirements / Tolerance
Anaerobic Growth Techniques:
Media Types
• Complex versus Defined:
– When every chemical and its amount in the media is known, we call it a
defined media.
– When one or more ingredient is of uncertain composition (e.g. yeast
extract), we call this an undefined or complex media.
• Non-selective versus Selective:
– Media designed to favor the growth of as many different bacteria as
possible is called a non-selective media.
– Media with special ingredients or sources of organic matter to enrich the
growth of a specific group of bacteria over others is called a selective
media.
– (E.g. lactose broth for coliform bacteria enrichment).
• Differential:
– Often these are selective media in the solid (agar) form that have had
additional ingredients added so particular kinds of selected bacteria can
be differentiated from other growing bacteria.
– (E.g. coliform bacteria on ENDO agar will be a darker red color with a
metalic sheen to the colonies, as compared to other bacteria on the
same media.)
Prokaryote Exponential
n
Growth (N = 2 )
• Every time bacteria in a culture divide by binary fission the
population number increases 2-fold.
• The time it takes for the bacteria to divide, and double the
population, is called the generation time.
• With each generation (n) the total population (N) increases by a
power of 2.
• Intrinsic growth rate (µ) is the number of generations per hour (h-1).
Plotting Growth on a Logarithmic Scale:
Bacterial Growth Curve
(For a “closed” culture; e.g. a tube of broth media)
Growth Curve Phase Summary:
• Lag Phase:
– Bacteria make adjustments to new conditions.
– Takes time to “express genes” for new protein types (like enzymes)
now needed.
• Log Phase:
– Physiologically adjust for maximum growth for the environmental
conditions (note log scale in previous growth curve plot).
• Stationary Phase:
– Death and growth balance, i.e. they are in equilibrium.
– Growth slows due to some resource becoming limiting (used up).
– Growth may also slow due to a change in the environment that
exceeds the bacterium’s tolerance range (e.g., pH decrease).
• Death Phase:
– Conditions promote death faster than growth.
– Death rate or decline is logarithmic; more and more cells die for
each hour of decline (note log scale in previous growth curve plot).
Viable Plate
Counts:
• There are two methods for
evenly spreading bacteria
across an agar plate for
counting:
- Pour Plate (We did this!)
- Spread Plate
• Each method requires a
series of quantitative
dilutions to reduce cell
density so that we can see
well isolated (separated)
colony forming units (CFU)
for counting.
• Here they performed a series of 1:10 dilutions and plated the same volume (1 mL)
using the pour plate technique.
• After incubation, look for the plate with between 30 to 300 CFU; count it.
• Bacteria Density = (CFU * dilution factor) = 32 CFU * 10,000/mL = 320,000 CFU/mL
Direct Microscopy Count
Only works well for very
large cells; not very good
for most bacteria!
Growth Monitored by Turbidity
This is a rapid assay for broth culture growth, as is does not require incubation.
How can you convert turbidity to bacteria cell number per milliliter (i.e., CFU/mL)?
Multiple Tube Test for
Most
Probable Numbers (MPN)
As the inoculum volume decreases, the probability of there being growth of bacteria
also decreases. More positive growth tubes having received the smallest inoculum
volume means a greater number of bacteria in the inoculum (sample). Every
combination of growth results has a statistical probability of being due to a particular,
or most probable, number of bacteria in the inoculum (sample).
Reading the MPN Table:
We will use this in lab this week to determine MPN for coliform bacteria.