Chapter 7.

Control of Microbial Growth

1. Physical methods (sterilization, aseptic techniques, filtration, radiation)

2. Chemical methods (disinfectants, and antiseptics)
❖ In microbiology laboratories, many procedures applied to prevent contamination of
the work environment and specimen cultures, and subcultures.

❖ It is necessary to inhibit the growth of microbes (in hospitals, nursing home, restaurants,
kitchens, bathrooms, etc.), to avoid transmission of infectious diseases among people.

Contamination: is the growth of unwanted organisms (e.g., bacterial and fungal

spores) to land on the surface of the medium.

1. Sterilization: The complete destruction (killing) of all living organisms,

including spores and viruses
- Commercial Sterilization: Heat treatment that kills endospores of the bacteria
Clostridium botulinum (the
causative agent of botulism) in canned food. It does not kill endospores of
thermophiles above 45oC.

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Contaminated culture media

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2. Disinfection: The elimination of most or all pathogens (except bacterial

spores) from non-living objects

• Disinfectant: strong chemical substances used only on inanimate (non-
living) objects.
• Antiseptic: mild chemicals used to disinfect skin and other living tissues
• Sanitization: is the reduction of microbial population to levels considered
safe by public health standards.
Aseptic techniques are used to eliminate pathogens in surgical
instruments, medical personnel, and the patient during surgery
(Hand hygiene is a good example of aseptic technique)

“Cide” or “cidal” refers to killing. “Static” refers to inhibiting growth of

Microbicidal agents are disinfectants that
kill microbes
Bacteriocidal agents are disinfectants that
kill bacteria but not necessarily bacterial
endospores
Fungicidal agent used to kill fungi including
fungal spores
microbes.
Microbistatic agents are disinfectants that
inhibit growth of microbes
Bacteriostatic agents are disinfectants that
inhibit the growth of bacteria
Fungistatic agent used to inhibit the growth
of fungi 3
Aseptic techniques practices in microbiology laboratories

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 Physical Methods of Microbial Control
Heat: is the most common type of sterilization for non-living objects could to withstand high temperature.
* “Susceptibility of microorganisms to heat is variable”
* “The higher the temperature the shorter the time required to kill microorganisms”.
• Heat is a practical, efficient, and inexpensive method of sterilization of inanimate objects; can tolerate
high temperatures.
• Two factors: temperature and time determine effectiveness of the sterilization.
A) Dry Heat:
1. Flaming loops and needles.
2. Incineration (Burning): to destroy contaminated disposable materials (dressings, and biological waste).
3. Hot Air (Oven) Sterilization: effective sterilization for metals, glassware, oil, wax requires 1 hours at 170
oC for sterilization.
B) Moist Heat: In general, moist heat is much more effective than dry heat.
Boiling: Heat to 100 oC at sea level. Kills vegetative forms of bacterial pathogens,
almost all viruses, and fungi and their spores within 10 minutes or less. Endospores and
some viruses are not destroyed this quickly.

Boiling is Not a sterilization method

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 Autoclave: large metal pressure cooker with temperature 121 oC and
pressure 15 psi for 20 minutes. (preferred method of sterilization)
All organisms killed within 15 minutes even bacterial endospores

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Aseptic techniques and hospital precautions

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Pasteurization: is a method of disinfecting liquids (by heat) to eliminate pathogens from
milk and other beverages.
It is a preservation methods. Not sterilization procedure.
Types of pasteurization methods:

❖ Classic Method of Pasteurization: Milk was exposed to 63 oC for 30 minutes.

❖ High Temperature Short Time Pasteurization (HTST): Used today.
Milk is exposed to 72 oC for 15 seconds.
❖ Higher-Heat Shorter Time HHST: Milk is treated at 89 oC for 1 second.
❖ Ultra High Temperature Pasteurization (UHT): Milk is treated at (140 oC for 3
seconds or 72 oC for 15 seconds) and then cooled very quickly in a vacuum
chamber.
Advantage of UHT: Milk can be stored at room temperature for several months

Low Temperature: It is a preservation method

Refrigeration: Temperatures from 0 to 7 oC. Bacteriostatic effect. Reduces
metabolic rate of most microbes so they cannot reproduce or produce toxins.
Freezing: Temperatures below 0 oC. Over a third of vegetative bacteria may
survive 1 year
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Milk pasteurizer
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Desiccation (drying): In the absence of water, microbes cannot grow or
reproduce, but some may remain viable for years. After water becomes
available, they start growing again. Drying is a preservation method

Lyophilization (freeze-drying): is a process that combines dehydration

(drying) and freezing; used to preserve food, antibiotics and microorganisms

Osmotic Pressure: The use of high concentrations of salts and sugars in

foods is used to increase the osmotic pressure and create a hypertonic
environment. In Plasmolysis: As water leaves the cell, plasma membrane
shrinks away from cell wall. Preservation (Not sterilization)
Filtration: Removal of microbes by
passage of a liquid or gas through a screen
like material with small pores. Used to
sterilize heat sensitive materials like
vaccines, enzymes, antibiotics, and some
culture media.
High Efficiency Particulate Air Filters
(HEPA): Used in operating rooms and
burn units to remove bacteria from air. 11
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Radiation:

1. Ionizing Radiation:
* Gamma rays, X rays, electron beams, or higher energy rays.
* Cause mutations in DNA and produce peroxides.
* Used to sterilize pharmaceuticals and disposable medical supplies. Food
industry is interested in using ionizing radiation.
** Disadvantages: Penetrates human tissues. May cause genetic mutations in
humans.

2. Ultraviolet light (Non-ionizing Radiation):

* Damages DNA by producing thymine dimers, which cause mutations.
* Used to disinfect operating rooms, nurseries, cafeterias.
** Disadvantages: Damages skin, eyes. Doesn’t penetrate paper, glass, and
cloth.

3. Microwave Radiation:
* Heat is absorbed by water molecules. May kill vegetative cells in moist
foods.
* Bacterial endospores, which do not contain water, are not damaged by
microwave
radiation. Solid foods are unevenly penetrated by microwaves.

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UV sterilizers

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 Chemical Methods of Microbial Control
Some Types of Disinfectants
1. Phenols Rarely used today because it is a skin irritant and has strong odor.
❖Used in some throat sprays and lozenges.

2. Halogens: Effective alone or in compounds.

A) Iodine: used as skin antiseptic. Not effective against bacterial endospores
B) Chlorine: When mixed in water forms hypochloric acid:
❖ Used to disinfect drinking water, pools, and sewage.
❖Sodium hypochlorite (Na O Cl): Is active ingredient of bleach.

3. Alcohols

Kill bacteria, fungi, but not endospores or naked viruses.

Act by denaturing proteins and disrupting cell membranes.
Used to mechanically wipe microbes off skin before injections or blood drawing.
Not good for open wounds, because cause proteins to coagulate.
Ethanol: Drinking alcohol. Optimum concentration is 70%.
Isopropanol: Rubbing alcohol. Better disinfectant than ethanol, less volatile.

4. Quaternary Ammonium Compounds (Quats):

Widely used surface active agents.

5. Chlorhexidine: is a disinfectant and antiseptic that is used for skin disinfection before
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surgery and to sterilize surgical instruments.
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6. Aldehydes: Include some of the most effective antimicrobials.
A. Formaldehyde gas: Excellent disinfectant.
Commonly used as formalin, a 37% aqueous solution.
Formalin was used extensively to preserve biological specimens and inactivate viruses and
bacteria in vaccines. Also used in mortuaries for embalming.

B. Glutaraldehyde:
One of the few chemical disinfectants that is a sterilizing agent (liquid).
Commonly used to disinfect hospital instruments.
Also used in mortuaries for embalming.

7. Ethylene Oxide: A sterilizing gas

Kills all microbes and endospores, but requires exposure of 4 to 18 hours.
Toxic and explosive in pure form, Highly penetrating.
Most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment.

8. Peroxygens (Oxidizing Agents):

A) Ozone: Highly reactive form of oxygen.
*Used along with chlorine to disinfect water.

B. Hydrogen Peroxide: Used as an antiseptic.

* Not good for open wounds because quickly broken down by catalase present in human cells.
* Effective in disinfection of inanimate objects.
* Used by food industry and to disinfect contact lenses. 17