LEARNING UNIT 1 - AN OVERVIEW OF MICROBIOLOGY

LECTURE 1 - Brief history and personalities involved in the development of microbiology

LECTURE 2 - The scope, characterization and classification of microorganisms
LECTURE 3 - Major structures differences between prokaryotes and eukaryotes and their
functions

LEARNING UNIT 2 - MAJOR GROUPS OF MICROORGANISMS

LECTURE 4 - Virus
Mycoplasmas
Prokaryotes: characteristics of 2 major groups of bacteria (Eubacteria and
Archaebacteria)
LECTURE 5 - Eukaryotes: Fungi - main features, classification and some species of special
interest.
LECTURE 6 - Eukaryotes: Algae, protozoa and helminths - main features, classification and
some spices of special interest.
LEARNING UNIT 3 –CONTROL OF MICROORGANISMS
LECTURE 7 - Physical agents and processes in controlling growth of microorganisms
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Microbial Growth Control – Terminologies

• Sepsis refers to microbial contamination.
• Asepsis is the absence of significant contamination
- Aseptic surgery techniques prevent microbial contamination of wounds.
• Antisepsis - removing pathogens from living tissue
• Sterilization - removing all microbial life.
- Commercial sterilization: killing C. botulinum endospores.
• Disinfection - removing pathogens
• Degerming - removing microbes from a limited area.
• Sanitization - lowering microbial counts on eating utensils.
• Biocide/germicide - killing microbes.
• Bacteriostasis - inhibiting, not killing, microbes.
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Microbial Growth Control - Introduction

• Microbes present a constant threat to health, jeopardize our food supplies and
destroy many useful materials.
• Fortunately, microbes can be eliminated or reduced in number through the use
physical or chemical methods.
• These protective measures are particularly important in the hospital, where high
concentrations of potentially pathogenic organisms are found.
• Types of microbial growth control:
1.Inhibition:
Microbes or their undesired activities are inhibited by bacteriostatic compounds
or techniques.
2.Sterilization:
Microbes themselves are killed by bacteriocidal compounds or techniques.
• Considerations involved when dealing with microbial control:
1. Areas of treatment or application.
2. Types of control/treatment to be utilized.
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Microbial Growth Control - Introduction

1.Areas of treatment or application.
• Treatment of inanimate (table top/working surfaces) objects.
1.Decontamination removes or kills most microbes, making an object or
surface safe.
2.Disinfection removes most or all pathogens from an object or surface.
3.Sterilization removes or kills all microbes.
• Treatment of living organisms or tissues.
1.Agents used to treat living organisms or tissues must be nontoxic as well as
antimicrobial.
2.Antiseptic compounds can be applied to tissues.
3.Antimicrobial agents can be taken internally.
2.Types of control/treatment to be utilized
• Basically, there are three principles involved in the control of microbial control:
1. Physical methods. This lecture will discuss the physical methods in
2. Chemical methods. the control of microbial growth.
3. Antibiotics.
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Microbial Growth Control - Physical Methods

• Physical methods of microbial growth control can be radiation
heat method
method
ultrasonic method
divided into four types :
• Heat – most widely used
• Filtration
• Radiation
• Ultrasonic vibrations
• Heat is mostly used because :
• Useful for inanimate objects, effective and least a filtration set
expensive.
• Lethal effects of heat on microbes may be due to:
• Protein denaturation (coagulates) and enzymes.
• Effects on membrane fluidity.
• Denaturation of other macromolecules.
• Two types of heat sterilization:
• Dry heat methods. autoclave system
• Moist heat methods. moist heat method
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods - Dry Heat Methods

a. Direct flaming direct flaming
• Example: inoculation loop.
b. Incineration
• Burning to ashes/oxidation.
• Must be complete.
• Used to destroy disposable items, soiled dressings,
tissue specimens.
• Example: Hospital disposables and specimens.
c. Hot-air sterilization
• Baking in oven.
• Very effective method of sterilization, but
requires temperature of 170 ºC for ~ 2 - 3 hr.
• Causes oxidation of microbes, sterilizes.
• Used when moisture is undesirable:
example - glassware, metals, powders or laboratory oven for dry heat
petroleum-based products (oils). sterilization
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods - Moist Heat Methods

a. Boiling
• 98 -100ºC for 10 min.
• Inactivates most vegetative cells, not heat-resistant forms
(i.e. endospores, some viruses & bacterial toxins).
• e.g. drinking water, canning jars, etc.
b. Tyndallization
• A sterilization process used for foods, especially in home canning.
• Foods are sealed and heated to boiling (this kills microbial cells, but
generally not endospores).
• Food is then cooled and stored for 1 day at room temperature
(endospores germinate to vegetative cells).
• The food is reheated (100 ºC) to kill those cells.
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods - Moist Heat Methods

c. Pasteurization
• Heat material, holding at specific temperature for specific length of time, cool
rapidly.
• Low temperature long term (holding) method: 63ºC for 30 min.
• High temperature short term method: 72ºC for 15 sec.
• Inactivates pathogens, reduces total microbial population, but does not
sterilize.
• i.e. Dairy products, wine, beer, etc.
d. Autoclave
• Autoclaves uses steam under pressure (increases
the steam temperature) for moist sterilization.
• Destroys all lifeforms – sterilization.
• Coagulate proteins and causes hydrolysis.
• The chamber contains 100% steam during
the lid must be secured
sterilization process. toautoclave
produce system
the high
table-top autoclave
moistpressure
heat method
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods - Moist Heat Methods

 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods - Moist Heat Methods

d. Autoclave
• During autoclave, the typical parameters are :
• Pressure : 15 - 20 psi
• Temperature : 115 - 121ºC
• Time duration : 15 - 20 min.
• Application : microbiological media, surgical instruments, etc.
• Autoclave cycle :
• After the door is sealed and sterilization begins, the autoclave pressurizes
and the temperature of the sample rises.
• Large samples warm more slowly than small ones (large autoclave system
may require hours for heating).
• Aqueous samples boil during depressurization to cool (heat of vaporization)
to 100ºC (cooling must be slow to avoid boiling over).
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Physical Methods - Filtration

• Liquids can be sterilized by syringe
filtering out particles. type
• Solids physically separated from
liquids by passage through filters
with extremely small pores (i.e.
porcelain, ground glass,
diatomaceous earth, asbestos,
sand, membrane filters). basic/typical filtration set
• Does not sterilize unless pore size
small enough to trap all
organisms
• Membrane filtration removes
microbes >0.22 µm.
• HEPA removes microbes >0.3 µm.
membrane before membrane after filtering
filtering (notice the bacteria cells)
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods - Filtration

• Liquids mechanically forced or
pulled through (using a vacuum)
filters which trap microbes.
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods - Filtration

• Filtration methods are used widely for :
• Heat-sensitive materials (media, medications) that can't be heated.
• Beer and wine.
• Swimming pools and spas.
• Sewage, air, testing water or air for organisms or allergens.
• Disadvantages of filtration method :
• Not suitable for liquids such as milk (not a solution).
• Limitations in size of organisms to be removed (sand filters in water treatment
remove only protozoal cysts and helminth eggs).
• Even nucleopore filters cannot remove viruses.
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods - Radiation

• Radiation cause lethal changes in DNA, denatures proteins (produces hyperactive
ions and free radicals).
• Two types of radiation treatments : UV radiation is used to sterilize
a. Non-ionizing rays (UV light - UV, 260 nm) laminar flows
• UV radiation is often used to sterilize surfaces.
• Causes DNA damage including breakage of
the dna backbone by the formation of
thymine dimers.
• Limit - cannot penetrate materials (cloth,
glass, paper, etc).
b. Ionizing rays (X-rays, gamma rays, electron beams).
• Ionizing radiation is more effective and can penetrate materials.
• Ions (loss of electrons) are formed when they pass through molecules.
• Causes dna damage and production of toxic reactant products.
• Used to sterilize plastics, medications, foods (retards spoilage).
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods – Ultrasonic Vibration

• Ultrasonic vibrations uses high frequency sound waves from a machine called a
sonicator.
• Killing microbes by shock waves that disintegrates cell wall and membranes.

Different models of sonicators

 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods – Preservation Methods

a. Increased osmotic pressure.
• High concentrations of salt, sugar.
• Dehydrates cells, more effective against bacteria than fungi.
• Application : food preservation.
b. Desiccation
• Drying, removal of water.
• Retards growth, but does not always kill.
• More effective against bacteria than fungi.
• Application : food preservation.
 LEARNING UNIT 3 –CONTROL OF MICROORGANISMS

Physical Methods – Preservation Methods

c. Acid, Alkaline pH
• Inactivates enzymes, inhibits growth.
• Does not always destroy microbes.
• Uses: acid pH (benzoic, sorbic, propionic acids) - food preservation.
d. Decreased temperatures
• Refrigeration.
• 5 - 10ºC.
• retards growth, does not prevent growth.
• Freezing
• below 0ºC (-10ºC).
• prevents growth, does not kill all organisms.