EXERCISE 20. EFFECT OF
TEMPERATURE ON GROWTH OF
MICROORGANISMS
EFFECT OF TEMPERATURE ON GROWTH OF MICROORGANISMS
Cardinal temperatures
Minimum temperature: : It is the lowest temperature
which will support the growth of the microorganism.
Below the minimum temperature the microorganisms
survive for a long time and such low temperatures may
be used for their storage.
Optimal temperature: It is the temperature, or more
usually the range of temperatures, which supports the
fastest growth rate.
Maximum temperature: It is the highest temperature
which will support the growth of the microorganism.
Above the maximum temperature the microorganism
is usually killed and such high temperatures may be
used in sterilization.
Types of microorganisms classified by
optimum temperature:
•
•
•
•
•
•
Psychrophiles:
Psychrotrophs (facultative psychrophiles):
Mesophiles:
Thermophiles:
Thermotolerant and thermoduric microorganisms:
Hyperthermophiles:
Types of microorganisms classified by
optimum temperature:
Adaptations to cold
temperatures
• Membrane composition
(increased membrane fluidity)
– Increased number of double
bonds in membrane fatty acids
– Increased amount of hopanoids
(in bacteria) or cholesterol (in
eukarya) in membrane
– Decreased fatty acid chain
length
• Enzymes
– Must function efficiently at
lower temperatures
Adaptations to high
temperatures
• Membrane composition
– Decreased number of double bonds in membrane
fatty acids
– Decreased amount of hopanoids or cholesterol
– Increased fatty acid chain length
– Archaea – use ether linkages instead of ester
linkages – greater stability at high
temperatures.
• Some archaea use tetraether lipids that form a
monolayer instead of a lipid bilayer.
• Proteins and enzymes
– More cross-linking or altered composition (change
the hydrophilic and hydrophobic
interactions, increased charge interactions between
amino acids) for greater protein
stability
• Ribosomes and other molecular machines need to
be very stable
Method
Serratia marcescens and
Escherichia coli
1. Label the tubes as follows
Slants: label one 25oC (RT), and
the other 37oC,
Broths: 5oC, 25oC (RT), and 42oC
2. Inoculate the tubes as follows
Slants: one with E. coli, other one
with S. marcescens
Broths: E. coli or S. marcescens
3. Place the tubes in racks and
incubate at proper
temperatures for 24 hours.
Evaluation:
Slants: Compare the nutrient agar slants of
S. marcescens. Using colored pencils,
draw
the appearance of the growths on the
laboratory report.
Broths:
1. Calibrate the spectrophotometer with
sterile nutrient broth according to the
instructor’s direction.
2. Shake each culture sufficiently to
completely disperse the organisms and
pour 2
ml into cuvette in given order.
3. Measure the absorbance (O.D.) of your
cultures and record these values on the
laboratory report.
4. Plot the O.D. values on the graph-paper.
EXERCISE 21. LETHAL EFFECT OF
TEMPERATURE
The factors affecting the heat
resistance of a microorganism:
• The species and strain,
• How it is grown,
• The medium in which it is heated (food vs.
buffer),
• The environment in which it will recover after
heating.
Measurements used in order to compare the
susceptibility of different organisms to increased
temperatures
• Thermal Death Point (TDP): lowest temperature
required to kill all microbes in liquid suspension in 10
minutes.
• Thermal Death Time (TDT): Minimal time required to
kill all microorganisms keeping temperature (or
bacteriocidal agent) constant; i.e. at a specific
temperature.
• Decimal Reduction Time (DRT or D value): Used for
heat resistant bacteria; Time required to kill 90% of the
population at a given temperature (Log growth
and log death concept).
* Moisture, pH, composition of medium and age of cells will greatly influence these values
Materials:
1. 24 h nutrient broth culture
of the following organisms:
a. Escherichia coli DH5α
b. Bacillus thuringiensis
2. Nutrient agar plates (five for
each group)
Method:
•
Label five agar plates as; control, 10 min., 20 min., 30min.,
and 40 min.,
respectively.
•
Each group is assigned one culture of organisms to be
heated at a specific temperature (55-65-75-85 oC)
•
Shake the culture of organisms and transfer 0.1 ml of the
organism with a 1 ml
pipette to control plate.
•
Place the culture of your group into the water bath.
•
As soon as the temperature reaches the desired one,
record the time. Watch the temperature carefully to make
sure it does not vary appreciably.
•
After ten minutes have elapsed, transfer 0.1 ml from the
culture to the 10 min. plate with a fresh 1 ml pipette.
Repeat this operation at ten minute intervals until
all the plates that have been inoculated. Use fresh pipettes
each time and be sure to shake the culture.
•
Spread the culture over the medium by rotating the glass
spreader.
•
Incubate at 37oC for 24 – 48 hours.
Evaluation
EXERCISE 22. OSMOTIC PRESSURE
AND BACTERIAL GROWTH
• Osmosis: the net movement of H2O across a
semipermeable membrane (i.e. cell membrane).
• Osmotic pressure: the force that drives osmosis.
It is caused by different solute concentrations in
the cell and its environment.
Osmophiles
– Love high osmotic pressure
– Salts or sugars
Saccharophiles:
– Osmophiles which love high
sugar
Halophiles:
– Osmophiles which love high salt
Xerophiles:
– Osmophiles which love very dry
environments
Material
1. 24 h nutrient broth cultures of
following organisms:
a. Escherichia coli
b. Staphylococcus aureus
c. Bacillus subtilis
2. Following plates for each group:
• 1 nutrient agar plate (0.5 % NaCl)
• 1 nutrient agar plate (5 % NaCl)
• 1 nutrient agar plate (10 % NaCl)
• 1 nutrient agar plate (15 % NaCl)
Method
1. Mark the bottoms of the four Petri plates as
indicated in below
2. Streak each organism in a straight line on
the media, using a wire loop.
3. Incubate all the plates at 37oC for 48 h
Evaluation