LAB NO 8LAB NO 8

Environmental Factors Environmental Factors Affecting Microbial Affecting Microbial

growthgrowth

Environmental Factors Affecting Environmental Factors Affecting Microbial growthMicrobial growth

There are many factors in the environment of the microorganism that can dramatically affect the rate and the extent of the microbial growth.

1. Hydrogen Ion Concentration (PH).2. Moisture content.3. Solute Concentration (osmotic

pressure).4. Oxygen.5. Temperature.

11..Moisture contentMoisture 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.

22 . .Hydrogen Ion Concentration Hydrogen Ion Concentration (pH)(pH)

The enzymes, electron and nutrient The enzymes, electron and nutrient

transport system found in the cell transport system found in the cell

membrane are sensitive to the membrane are sensitive to the

concentration of hydrogen ions (Hconcentration of hydrogen ions (H++).).

The optimum pH for most bacteria is 6.5 - The optimum pH for most bacteria is 6.5 -

7.57.5

Fungi & acidophilic bacteria [Fungi & acidophilic bacteria [e.g; e.g;

Lactobacillus spLactobacillus sp.] can tolerate acidic pH up .] can tolerate acidic pH up

to 4.to 4.

while basophilic bacteria [e.g;while basophilic bacteria [e.g;VibrioVibrio

choleracholera] can grow at pH up to 9.] can grow at pH up to 9.

※ That’s why certain food are not That’s why certain food are not

easily contaminated with bacteria easily contaminated with bacteria

due to their acidity like citrus fruits.due to their acidity like citrus fruits.

3. Solute Concentration 3. Solute Concentration (osmotic pressure)(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.

Most microorganisms can grow well Most microorganisms can grow well

at isotonic environment. at isotonic environment.

In Hypertonic environment, cells have In Hypertonic environment, cells have

the tendency to lose water to their the tendency to lose water to their

environment, as the conc. outside the environment, as the conc. outside the

cell is greater than inside the cell.cell is greater than inside the cell.

3. Solute Concentration (cont.)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.

3. Solute Concentration 3. Solute Concentration (cont.)(cont.)

In general microorganisms are In general microorganisms are

classified into two major groups classified into two major groups according to their oxygen according to their oxygen requirement:requirement:

1. Aerobes: • In which oxygen is absolutely

necessary for growth.2. Anaerobes: • They can reproduce in the absence of

oxygen. In fact oxygen inhibit their growth & in most cases kill them.

4.Oxygen 4.Oxygen

55..TemperatureTemperature Microorganisms can be classified Microorganisms can be classified

according to the optimum according to the optimum temperature for growth into:temperature for growth into:

a)a) Psychrophilic Psychrophilic ((cold-loving);cold-loving); Which can grow best at Which can grow best at

temperature between temperature between 0 - 20ºc.0 - 20ºc.b)b) MesophillicMesophillic (middle-loving);(middle-loving); which can grow best at which can grow best at

temperature between temperature between 20 - 40ºc.20 - 40ºc.c)c) ThermophilicThermophilic (heat-loving);(heat-loving); which can grow best at which can grow best at

temperature between temperature between 40 – 80ºc.40 – 80ºc.

Temperature (cont.)Temperature (cont.)

The lethal effect of high temperature on The lethal effect of high temperature on

microorganisms can be attributed to:microorganisms can be attributed to:

1.1. Irreversible denaturation of intracellular Irreversible denaturation of intracellular

proteins such as enzymes and other vital proteins such as enzymes and other vital

components of the cell.components of the cell.

2.2. Water loss due to evaporation leads to Water loss due to evaporation leads to

increase concentration of electrolytes increase concentration of electrolytes

within the cell causing toxic effect. within the cell causing toxic effect.

Temperature (cont.)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.)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.

DeterminationDetermination ofof ThermalThermal DeathDeath TimeTime

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.

DeterminationDetermination ofof ThermalThermal DeathDeath TimeTime

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.

DeterminationDetermination ofof ThermalThermal DeathDeath TimeTime

55 1010

15152020

B B

E E

5 min5 min

10 min10 min

50 50 ºcºc

7070ºcºc

95 95 ºcºc

Invert & incubate for 48 hrsInvert & incubate for 48 hrs

Result

1.E. coli1.E. coli

Temp. time

5min 10min 15min 20min

50℃70℃95℃

2- B. subtilis;2- B. subtilis;

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 thetest 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:

45°c

S

o.2 ml

AB 1 2 3 4

1 ml 1 ml 1 ml 1 ml

1 ml sterile water

4

Incubate the plate Uninverted at 37°c for 24h

3

21

43

2 1

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 MethodResults:

AB conc

Log conc

Zone diameter

Well diameter

X X²

100

50

25

12.5

X = X = Zone diameter – Well diameter Zone diameter – Well diameter 22

If Original Antibiotic Conc. = 200 µg/ml

x²x²

Log concLog conc

Anti-log = MICAnti-log = MIC

x

x

x

x

concconc

MICMIC

If use semi-log paper, then No need to calculate Log conc.

x²x²

x

x

x

x

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