Chapter
6
Microbial
Growth
Growth: an increase in cellular constituents which
may lead to a rise in cell number when MO reproduce by budding or binary binary
fission:
Ø Portions of the parental cells envelope are
shared with the progeny cells.
Ø Cells enlarge and divide to yield two
daughter cells of equal size.
Microbiologists
usually study growth by following the change in population number.
Population Growth is
studied by the Growth Curve of a
microbial culture.
Batch culture
Ø MO are grown in a liquid medium
Ø Closed
system:
§
MO incubated in a
closed culture vessel with a single batch of medium where no fresh medium in
introduced
§
nutrient
concentration declines
§
concentrations of wastes increases.
Growth Curve:
Ø
growth of MO
reproduce by binary fission can be plotted as the logarithm of the number of
viable cells verses the incubation time.
Four Phases:
1. Lag Phase
Ø When MOs are inoculated into fresh growth
medium, there is usually no immediate increase in cell numbers.
§
Yet the cell is
synthesizing new components
Ø Reasoning
for Lag Phase:
§
Old cell depleted
of ATP, essential cofactors and
ribosomes (All necessary before cell growth)
§
Different medium,
may mean new enzymes needed to use different nutrients
§
Possible injured
MOs require time to recover
§
Eventually, cells
retool, replicate DNA, begin to increase in mass and finally divide
2. Exponential Phase or Log
Phase
Ø
MOs are
growing and dividing at the maximal rate possible, genetic potential, nature of
medium, and conditions under which they are growing.
§
Rate of growth is
constant (MOs are dividing and doubling in number)
§
Population is
most uniform in terms of chemical and physiological properties
§
Balanced growth: all cellular components are
manufactured at constant rates relative to each other.
§
Unbalanced growth: if
nutrient levels or other environmental conditions
change
n
growth: rate of synthesis of cell
components varies until a new balanced is reached.
n
Shift-up experiments: bacteria are transferred from a nutrient
poor to a rich medium.
-
cells construct new ribosomes for protein
synthesis,
-
increase in protein and DNA synthesis
-
Rise in reproductive rates
n
Unbalanced growth also results from a Shift-down from a rich medium to a poor one.
-
When introduced into a nutrient inadequate
medium, Mos need time to make enzymes required for biosynthesis of unavailable
nutrients.
-
After Shift-down:
§
Cell division and DNA replication continue,
but net protein and RNA synthesis slow
§
Cells become smaller and reorganize
metabolically until they are able to grow.
3. Stationary Phase
Ø Population growth ceases and the growth
curve become horizontal
§
Bacteria at
population level of 109 cells/ml
§
Protozoan and
algal cultures reach population level of 106
§
Total number of
viable Mos remain constant – balance between cell division and cell death or
population may cease to divide though remaining metabolically active.
Mos enter
stationary phase:
n Oxygen in not very soluble and may be
depleted quickly resulting in adequate oxygen for only the surface of the
culture to grow.
n
Only if culture
id shaken or aerated will the cells beneath the surface growth
n limiting factor for many anaerobic cultures.
n Streptococci à
lactic acidà from sugar
fermentation à medium becomes acidic
and growth is inhibited.
n produce starving proteins which makes cell
more resistant to damage:
·
Increase
peptidoglycan cross-linkage and cell wall strength
·
Dps protein
protects DNA
·
Chaperon prevents
protein denaturation and renature
damaged protein
·
Resulting in
the starved cell becoming harder to kill
4. Death Phase
Ø Nutrient depletion and buildup of toxic
waste lead to the decline in the number of viable cells
n Death of microbial population is
logarithmic (constant proportion of
cells die every hour.
n Death:
the irreversible loss of the ability to reproduce.
During exponential
phase each Mo is dividing at constant intervals; thus population will double in
number during a specific length of time -
generation time or doubling time.
Measurement of Microbial
Growth:
Direct microscopic
counting: Sample
can be liquid or dried
n Petroff-Hausser counting chamber used for PC.
-- bacteria in several of squares are counted
-- average number of bacteria in these squares is used to calculate the concentrations of cells in the original sample.
-- there are 25 squares covering an area of 1 mm2:
--The total number of bacteria in 1mm2 of the chamber is
(number/square) (25 squares).
-- Chamber is 0.02 mm deep which can be converted 1/50 mm2
-- Chamber’s volume and any dilutions
Bacteria/mm3 =
(bacteria/square) (25 squares) (50)(103)
= (28) (25) (50) (103)
= 3.5 X 107
Limitations of this
method:
(1). Dead cells are counted b/c
they are not distinguished form
live cells.
(2)
Small cells are
difficult to see under the microscope and can be missed
(3)
Percision is
difficult
(4)
A phase contrast
microscope is required when cells are not stained.
(5)
Not suitable for
cell suspension of low density.
o Microbial numbers can be determined by from counts of colonies growing on special membrane filters.
§ Sample drawn thru a special membrane filter (with different pore sizes small enough to trap bateria)
§ Filter is placed on agar medium
§ Incubated until each colony forming unit forms a separate colony
Spread Plate
Pour Plate
Increase in total cell
mass accompany cell growth.
Most Direct Approach
is Determination of:
Ø
Microbial Dry
Weight
§
Cells growing in
liquid medium
§
Collected by
centrifugation
§
Washed
§
Dried in an oven
§
Weighed
§
Used for Fungi,
time consuming and is not very sensitive
Turbidity and Microbial Measurement
§
More rapid and
sensitive techniques – microbial cells scatter light that strikes them.
§
B/c microbial
cells in a population are constant size, the amount of scattering is directly
proportional to the biomass of cells present and indirectly related to cell
number.
§
107 cells
per ml population- medium appears cloudy.
§
The extent of
light scattering can be measured by a spectrophotometer and is almost linearly
related to bacterial concentration at low absorbance levels.
The Continuous Culture of
Microorganisms:
n
A microbial population of can be maintained in the
exponential growth phase and at a constant biomass concentration for extended
periods.
n
Open System: system with constant environmental conditions maintained through continual provision of
nutrients and removal of wastes .
Two
Common Major Types of Continuous Culture Systems:
(1). Chemostats
Ø
Sterile medium is fed into the culture vessel at the
same rate as the media containing the Mos is removed.
Ø
Two important factors that controls the chemostat
(1). Concentration of the limit nutrient:
§
Medium possess an essential nutrient (AA) in
limiting quantities. B/c of the
limiting nutrient, the growth rate is determined by the rate at which the
medium is fed into the growth
chamber. The final cell density depends
on the concentration of the limiting nutrient.
(2). Dilution rate:
§
The rate of nutrient exchange is expressed as the dilution
rate: the rate at which medium
flows through the culture vessel relative to the vessel volume.
D =f/V
D – Dilution rate
F – flow rate (ml/hr)
V – vessel volume
(2). Turbidostats
Ø Photocell that measures the absorbance or
turbidity of the culture in the growth vessel.
§
The flow rate of
media through the vessel is automatically regulated to maintain a predetermined
turbidity or cell density.
Difference of
Turbidostat and Chemostat:
Environmental Factors on
Growth:
The growth of Mos
are effected by Chemical and Physical surroundings:
Live
1.5 miles below the earth’s surface, w/o oxygen, and below 60° C.
Temperature
Ø
Microbial cell
temperature directly reflects that of the cell’s surrounding.
§
Most bacteria can
grow over a temperature range of
about 30˚ or more but have a
narrow range for optimal growth.
§
As we decrease
the temperature below the optimum, we see a decline in growth rate that is
consistent with enzymatic activity, but
then it becomes very steep, giving rise to a fairly well defined minimal growth
temperature.
§
Above the optimum
temperature, we see the growth rate decline very steeply, which gives rise to a
sharply defined maximum growth temperature
It is not known what sets the upper and lower temperature although they are thought to
§
reflect
properties of the membrane lipids,
§
effects on
protein conformation, and/or initiation of protein synthesis.
Temperature
Sensitivity of Enzyme-Catalyzed Reactions:
§
A temp rise,
increases the growth rate due to the velocity of an enzyme-catalyzed reaction.
§
Velocity will
double for every 10º C rise in temperature.
§
As rate increase,
the metabolism is more active at higher temp, Mo grow fater.
§
Example: 10
-- 30, Velocity is 15 What is
the velocity of the cell at 50º C?
High
Temperatures:
§ Damage
MOs by denaturing enzymes, transport carriers, and other proteins
§ Membranes are disrupted, lipid bilayer simply melts and disintergrates.
Low
Temperatures:
§
membranes
solidify and enzymes don’t work properly.
The temperature range of an organism can be used as a
classifying characteristic.
Cardinal Temperatures Growth Temperatures:
§
Minimum
§
Optimum
§
Maximum
The major factor determining the growth
range is water.
Pc can grow at much higher Temp than EC
Psychrophiles can grow at temperatures between 0-20º,
optima growth is 15º
§
Frequently found
in naturally cold waters and soils.
Such
as the Artic and Antarctic.
§
Examples include
the Pseudomonads and Bacillus,
§ Enzymes, transport systems and protein
synthetic mechanisms function well as low temp.
§ Cell membrane have high levels of
unsaturated fatty acid and remain semifluid when cold.
Psychrotrophs
or Facultative Psychrophiles can
grow at 0 to 7 º C.
§ Optima
20-30 º C
§ Maxima 35 º C
§ Psychrotrophic bacteria and fungi are important in spoilage of refrigerated foods
Most bacteria are Mesophiles and grow between 20-45 ºC.
§
Those that are
found in the mammalian body have an optimum temperature of 37-44 º C, Maxima is 45 º C.
§ Those found in the environment have an
optimum of about 30 degrees C
§ Almost all human pathogens are mesophiles,
env. is around 37ºC.
Thermophiles grow at
temperature of 55º C or higher.
Minimum of 45 ºC and
optima between 55 and 65 º C.
§
Majority of
prokaryotes
§
Flourish in
composts, self-heating hay stacks, hot water lines, and hot springs.
§
More heat sable
enzymes and protein synthesis systems / funct at higher temp.
§
Membranes lipids
more saturated and have higher melting points causing membrane to remain in
tact at higher temp.
§
These organisms
are extremely useful in that they serve as sources for exceptionally stable
forms of enzymes (i.e. bacillus stearothermophilus)
Hyperthermophiles are
thermophiles that can grow at 90º C or above,
§
Procayotes gowth
optima between 80 and 113 º C.
§
Do not grow well
below 55 º C.