Microbial Growth

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20 Φεβ 2013 (πριν από 4 χρόνια και 8 μήνες)

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Microbial Growth



I. The Growth Curve


Closed system = Batch Culture


Closed culture vessel


One batch of culture medium


Different from continuous culture (see below)


Nutrients used up, culture eventually dies


Four stages of bacteria growth in batch
culture



A period of apparent inactivity in which the cells are
adapting to a new environment and preparing for
reproductive growth, usually by synthesizing new cell
components


ATP


Ribosomal proteins


rRNA


tRNA


Co
-
factors


Enzymes

Lag Phase


Varies in length depending upon
the condition of the microorganisms
and the nature of the medium


Assessment of medium


Receptors


DNA synthesized


initiation of cell
division


Exponential phase (Log Phase)


Optimal growth rate and cell division dependent
on medium, O
2
, temperature, pH, genetic
composition


Regular, constant cell division (logarithmically)


Smooth curve


division not synchronous


Most useful phase for biochemical, physiological
and DNA replication studies


Biotechnology applications


competent cells


uptake
of plasmid DNA


Late log = optimal plasmid concentration


The population is most uniform in terms of
chemical and physical properties during this period


Stationary Phase


When the population reaches ~10
9
/ml (10
6
for
protozoan and algal cultures), cell division = cell
death (stasis)


Nutrients become scarce


O
2

is depleted


Toxic waste accumulates


The number of viable microorganisms remains
constant either because metabolically active
cells stop reproducing or because the
reproductive rate is balanced by the rate of cell
death

Death Phase


Viable cell mass decreases


Often logarithmic


Cells not viable when inoculated into
fresh medium


Cells have reached the carrying capacity of
their environment



The mathematics of growth
-
microbial
growth can be described by certain
mathematical terms:


Mean generation

(doubling) time
(g)

is
the time required for the population to
double


Mean growth rate constant

is the
number of generations per unit time,
often expressed as generations per hour


Generation times vary markedly with
the species of microorganism and
environmental conditions


they can range from 10 minutes for a few
bacteria to several days with some
eukaryotic microorganisms


Population size = 2
n

where n = the
number of generations



II. Measurement of
Microbial Growth

Counting cells directly (live and
dead)


Petroff
-
Hausser Counting Chamber


Slide with depressed etched grids (25 squares)


Covered with a coverslip


25 squares (area) = 1mm
2


Depth = 0.02mm


Volume = 2 x 10
-
5

ml in 25 squares


Determination of cell numbers:


20 cells in one square x 25 squares/2 x 10
-
5

ml = 2.5
x 10
7

cells/ml


Electronic Counter


Coulter counter


Measures electrical resistance as cells pass
single file through a thin stream


RBC and WBC are counted


Less accurate with small cells


High interference


Clumping

Counting only live cells


Plating techniques (spread plate, pour plate)
using serial dilutions


Colony forming units (CFU) usually arise from
one organism (but may be several if clumpy)


Membrane filtration assay


Membrane traps bacterial on the surface


Membrane transferred to an agar plate


Colonies grow


counted


Can use selective media (e.g. Endo agar for coliform
counts in contaminated water supplies)

Measurement of cell mass


Cell mass increases as cell number increases


Dry weight measurements


Growth, concentration, wash, dried, weighed


Spectrophotometric determination


Light is scattered and is proportional to cell number


Linear relationship between absorbance and cell
density


Often written as % transmittance (as absorbance
increases, transmittance decreases)


Requires cultures to be ~10
7
/ml and upwards (slight
turbidity)


III. Continuous Culture
Techniques


Used to maintain cells in the
exponential growth phase

at a
constant biomass concentration for
extended periods of time


Conditions are met by continual
provision of nutrients and removal of
wastes = OPEN SYSTEM


Constant conditions are maintained


Chemostat


A continuous culture device that maintains a
constant growth rate by:


supplying a medium containing a limited amount of
an essential nutrient at a fixed rate


removing medium that contains microorganisms at
the same rate


As fresh media is added to the chamber,
bacteria are removed


Limiting nutrients control growth rates


Cell density depends on nutrient concentration


Turbidostat


A continuous culture device that regulates
the flow rate of media through the vessel in
order to maintain a predetermined
turbidity
or cell density


There is no limiting nutrient


Absorbance is measured by a photocell (optical
sensing device)


The number of cells in culture controls the flow
rate and the rate of growth of culture adjusts to
this flow rate



Balanced and Unbalanced
Growth


Balanced

(exponential) growth occurs
when all cellular components are
synthesized at constant rates relative
to one another


Unbalanced

growth occurs when the
rates of synthesis of some components
change relative to the rates of
synthesis of other components.


This usually occurs when the
environmental conditions change


IV. Environmental Factors
Affecting Microbial Growth

Solutes and Water Activity


Osmotic concentrations affect microbes (e.g.
plasmolysis in hypertonic solutions)


Water activity (a
w
) = measurement of
availability of water in particular environments


A
w

= P
solution
/P
water
(P = vapor pressure)

= inversely related to osmotic pressure


If the solution has a high osmotic pressure (high
extracellular solute concentration), then its A
w

= low


Energy is required by microbes to tolerate
low a
w

because in order to keep water,
solute concentration inside of cells must
be kept high

= Osmotolerance


S. aureus can tolerate up to 3M NaCl


Archaebacteria halophiles tolerate 2.8
-
6.2M NaCl
(Great Salt Lake, Dead Sea)


Avoidance of plasmolysis


pH

is the negative logarithm of the
hydrogen ion concentration

pH (Log scale of 0


14; each pH
unit = 10x change)


Acidophiles

grow best between
pH 0 and 5.5


Neutrophiles

grow best between
pH 5.5 and 8.0


Alkalophiles

grow best between
pH 8.5 and 11.5



Extreme alkalophiles

grow best at pH 10.0 or
higher


Despite wide variations in habitat pH, the
internal pH of most microorganisms is
maintained near neutrality either by
proton/ion exchange or by internal buffering


Sudden pH changes can inactivate enzymes
and damage PMs


Reason for buffering culture medium, usually with
a weak acid/conjugate base pair (e.g.
KH
2
PO
4
/K
2
HPO
4



monobasic potassium/dibasic
potassium)


Microorganisms are sensitive to
temperature changes


Usually unicellular and poikilothermic


Enzymes have temperature optima


If temperature is too high, proteins denature,
including enzymes, carriers and structural
components


Temperature ranges are enormous (
-
20 to
100
o
C)

Temperature



Organisms exhibit distinct
cardinal
temperatures

(minimal, maximal, and optimal
growth temperatures)


If an organism has a limited growth
temperature range = stenothermal (e.g.
N.
gonorrhoeae)


If an organism has a wide growth temperature
range = eurythermal (
E. faecalis
)


Psychrophiles

can grow well at 0

C, have
optimal growth at 15

C or lower, and
usually will not grow above 20

C


Arctic/Antarctic ocean


Protein synthesis, enzymatic activity and
transport systems have evolved to function
at low temperatures


Cell walls contain high levels of unsaturated
fatty acids (semi
-
fluid when cold)


Psychrotrophs

(facultative psychrophiles)
can also grow at 0

C, but have growth
optima between 20

C and 30

C, and growth
maxima at about 35

C


Many are responsible for food spoilage in
refrigerators


Mesophiles

have growth minima of 15 to
20

C, optima of 20 to 45

C, and maxima of
about 45

C or lower


Majority of human pathogens



Thermophiles

have growth minima
around 45

C, and optima of 55 to 65

C


Hot springs, hot water pipes, compost heaps


Lipids in PM more saturated than
mesophiles (higher melting points)


Hyperthermophiles

have growth minima
around 55

C and optima of 80 to 110

C


Sea floor sulfur vents



Oxygen concentration


Obligate aerobes

are completely dependent on
atmospheric O
2

for growth


Oxygen is used as the terminal electron acceptor for
electron transport in aerobic respiration


Facultative anaerobes

do not require O
2

for
growth, but do grow better in its presence


Aerotolerant anaerobes

ignore O
2

and grow
equally well whether it is present or not


Obligate (strict) anaerobes

do not
tolerate O
2

and die in its presence


Microaerophiles

are damaged by the
normal atmospheric level of O
2

(20%)
but require lower levels (2 to 10%) for
growth


Oxygen tolerance is determined by an
organism’s ability to destroy toxic
oxidizing products of oxygen reduction


Remember, because oxygen has two unpaired
outer orbital electrons, it accepts electrons
readily



Toxic compounds


Superoxide radical:


O
2

+ e
-



O
2

-


Hydrogen peroxide:


O
2

-

+ e
-

+ 2H
+



H
2
O
2


Hydroxyl radical:


H
2
O
2

+ e
-

+ H
+



H
2
O + OH



These compounds are used deliberately by
phagocytic WBC to break down
intracellular microbes (respiratory burst)


Solution used by obligate aerobes and facultative
anaerobes:


Produce enzymes that convert these toxic oxidizing
products to non
-
toxic compounds


Superoxide dismutase

2O
2

-

+ 2H
+



O
2

+ H
2
O
2



Catalase

2H
2
O
2



2H
2
O + O
2



Aerotolerant microbes have SOD; Obligate anaerobes
lack SOD and catalase or have low concentrations


Laboratory considerations


Aerobic cultures


Shaken or sterile air introduced to medium


Anaerobic cultures


Remove oxygen


Include reducing agents in medium (e.g. thioglycollate
or cysteine


Dissolved oxygen is destroyed


Growth beneath surface


Replace oxygen with nitrogen gas and CO
2

gas


Gas
-
Pak jar


H2 + palladium catalyst + O
2



H
2
O


Bags or pouches


CaCO
3



CO
2

rich atmosphere




Pressure


Barotolerant

organisms are adversely
affected by increased pressure, but not
as severely as are nontolerant organisms


Barophilic

organisms require, or grow
more rapidly in the presence of,
increased pressure


Radiation


Ultraviolet radiation

damages cells by
causing the formation of thymine dimers
in DNA


Photoreactivation

repairs thymine
dimers by direct splitting when the
cells are exposed to blue light


Dark reactivation

repairs thymine
dimers by excision and replacement in
the absence of light


Ionizing radiation

such as X rays or
gamma rays are even more harmful to
microorganisms than ultraviolet
radiation


Low levels produce mutations and may
indirectly result in death


High levels are directly lethal by direct
damage to cellular macromolecules or
through the production of oxygen free
radicals