Transcript lab8

Environmental Factors
Affecting Microbial growth
LAB NO 8
Environmental Factors Affecting Microbial
growth

1.
2.
3.
4.
5.
There are many factors in the environment of
the microorganism that can dramatically affect
the rate and the extent of the microbial growth.
Hydrogen Ion Concentration (PH).
Moisture content.
Solute Concentration (osmotic pressure).
Oxygen.
Temperature.
1.Moisture content


⁂
Humidity is absolutely necessary for metabolic
reactions and multiplication of the microorganism.
However, drying does not kill microorganism, but
only prevent them from further multiplication.
generally, endospores and relatively small cells
are more resistant to drying than vegetative and
large cells.
2. Hydrogen Ion Concentration (pH)

The enzymes, electron and nutrient transport system
found in the cell membrane are sensitive to the
concentration of hydrogen ions (H+).

The optimum pH for most bacteria is 6.5 - 7.5

Fungi & acidophilic bacteria [e.g; Lactobacillus sp.] can
tolerate acidic pH up to 4.

while basophilic bacteria [e.g;Vibrio cholera] can
grow at pH up to 9.
※
That’s
why
certain
food
are
not
easily
contaminated with bacteria due to their acidity like
citrus fruits.
3. Solute Concentration
(osmotic pressure)

The conc. of solutes dissolved in the aqueous
environment of the bacterial cell can influence the
passage of water and nutrient into the cell.

Some microorganism inhibited or killed by different
concentration of solute such as NaCl or sugars as a
result of their effect on the osmotic pressure of the
medium.
3. Solute Concentration (cont.)

Most microorganisms can grow well at isotonic
environment.

In Hypertonic environment, cells have the tendency
to lose water to their environment, as the conc.
outside the cell is greater than inside the cell.
3. Solute Concentration
(cont.)

Under
this
condition,
most
bacteria
can’t
reproduce due to dehydration of the cells
leading to concentration of solute & electrolytes
within the cells which cause toxic effects.
4.Oxygen

1.
•
2.
•
In general microorganisms are classified into
two major groups according to their oxygen
requirement:
Aerobes:
In which oxygen is absolutely necessary for
growth.
Anaerobes:
They can reproduce in the absence of oxygen.
In fact oxygen inhibit their growth & in most
cases kill them.
5.Temperature

a)

b)

c)

Microorganisms can be classified according to
the optimum temperature for growth into:
Psychrophilic (cold-loving);
Which can grow best at temperature
between 0 - 20ºc.
Mesophillic (middle-loving);
which can grow best at temperature
between 20 - 40ºc.
Thermophilic (heat-loving);
which can grow best at temperature
between 40 – 80ºc.
Temperature (cont.)

The lethal effect of high temperature on
microorganisms can be attributed to:
1.
Irreversible denaturation of intracellular proteins such
as enzymes and other vital components of the cell.
2.
Water loss due to evaporation leads to increase
concentration of electrolytes within the cell causing
toxic effect.
Temperature (cont.)

Generally, bacterial endospores are more resistant to
adverse environmental conditions such as drying or high
temperature due to:
A. Low water content of spores and low metabolic activities.
B. Low permeability due to thick spores coat.
C. Presence of dipicolinic acid and dicarboxylic acid in the
spore coat which are complex with divalent ions
particularly calcium to give a heat resistant complex.
Temperature (cont.)
NOTICE:
 Bacterial endospores are heat and chemical
resistant as they serve a protective function.

However, the spores of yeast and molds are not
resistant to environmental conditions as they
serve a reproductive function.
Determination of Thermal Death Time




Thermal death time:
of a given species defined as the time required to
sterilize (kill) a standardized pure culture of that
species at a specified temperature.
Thermal death point:
of a given species defined as the lowest
temperature required to sterilize (kill) a
standardized pure culture of that species at a
specified time.
Determination of Thermal Death Time


Determination of thermal death time is useful
in sterilization processes, preparation of heat
killed vaccines, canning of food and many
other application.
Materials: Culture
of Bacillus subtilis and E. coli.
 Nutrient agar plate.
 Thermometer and water bath.
Determination of Thermal Death Time
50 ºc
5 min
70ºc
10 min
95 ºc
E
5
20
10
15
B
Invert & incubate for 48 hrs
Result
1.E. coli
2- B. subtilis;
Temp.
time
5min
10min 15min
20min
50℃
70℃
95℃
Compare the result of spore forming microorganism against non-spore forming one.
give your comment.
MIC:
It is the lowest concentration of the
antimicrobial agent that inhibits the
growth of the test organism but not
necessarily kills it.
MBC(minimum bactericidal conc.)
It is the lowest concentration of the
antimicrobial agent that kills the
test organism.
Bactericidal drugs have MBC 2-4 times
MIC.
Bacteriostatic drugs have MBC much
higher than MIC.
The breakpoint of an
antimicrobial agent:
It is the concentration of the
drug which can be achieved in
the serum with optimal dose.
Susceptible organisms :
are those with MICs at/below the breakpoint.
Resistant organisms:
are those with MICs above the breakpoint.
Material:
 Culture of staph. aureus
 Augmentin
 Saline
 Melted nutrient agar bottle
 Sterile peri dish
 4 sterile vials
 2 sterile pipette 1ml
 1 sterile pipette 5 ml
Procedure: 
o.2 ml
S
45°c
AB
1 ml
1 ml
1 ml
1
2
3
1 ml sterile water
1 ml
4
4
3
3
2
2
4
1
1
Incubate the plate Uninverted at 37°c for 24h
Results:
Measure the diameter of each inhibition zone 
The diameter of the inhibition zones are directly
proportional to the conc. of the antibiotic.
Zone diameter
Well diameter
Agar Diffusion Method
Results:

If Original Antibiotic Conc. = 200 µg/ml
AB conc
Log
conc
Zone
diameter
Well
diameter
100
50
25
12.5
X = Zone diameter – Well diameter
2
X
X²
x²
x
x
x
x
Log conc
Anti-log = MIC
If use semi-log paper, then No need
to calculate Log conc.
x²
x
x
x
x
conc
MIC
THANK YOU