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INTRODUCTORY MEDICAL
MICROBIOLOGY
CODE: MDID021
Medical Microbiology is a branch of
medicine concerned primarily with the
prevention, diagnosis and treatment of
infectious diseases.
Nutritional Microbiology is the study
of microorganisms that contaminate
food products and cause Food-Bourne
diseases.
What are infectious diseases?
Communicable diseases?
The profession of Medical Microbiology
consists primarily of four major spheres
of activity:
• Scientific and administrative direction of a clinical
microbiology laboratory.
• The establishment and direction of a hospital
infection control program.
• Provision of clinical consultations on the
investigation, diagnosis and treatment of patients
suffering from infectious diseases.
• Public Health and communicable disease
prevention and epidemiology
MICROORGANISM/MICROBES
• Living organisms (such as bacteria,
fungi, parasites and viruses) that are
too small to be seen with naked eye
but visible under a microscope and
special staining techniques used to
see them.
BACTERIA
• Microscopic, single-celled organisms,
inhabit virtually all environments,
including soil, water, organic matter,
and the bodies of multicellular
animals.
• Bacteria are distinguished in part by
their morphological and genetic
features; for instance, they may have
spherical, rod-like, or spiral shapes.
• They also can be divided into two
main groups, gram-positive or gramnegative, based on the structure of
their cell wall and their reaction to the
Gram stain.
• Many bacteria swim by means of
flagella (flagellum).
• The DNA of most bacteria is found in
a single circular chromosome and is
distributed throughout the cytoplasm
rather than contained within a
membrane-enclosed nucleus.
• Though some bacteria can cause food
poisoning and infectious diseases in
humans, most are harmless and many
are beneficial.
• They are used in various industrial
processes, especially in the food
industry (e.g., the production of
yogurt, cheeses, and pickles).
VIRUSES
• A microorganism smaller than bacteria,
which cannot grow or reproduce apart
from a living cell.
• They are acellular
• A virus invades living cells and uses their
chemical machinery to keep itself alive
and to replicate itself.
• It may reproduce with fidelity or with
errors (mutations)-this ability to mutate is
responsible for the ability of some
viruses to change slightly in each
infected person, making treatment more
difficult.
• Viruses may contain either DNA or
RNA as their genetic material.
• Herpes simplex virus and the hepatitisB virus are DNA viruses.
• RNA viruses have an enzyme called
reverse transcriptase that permits the
usual sequence of DNA-to-RNA to be
reversed so the virus can make a DNA
version of itself.
• RNA viruses include HIV and the
hepatitis C virus. (even Corona virus)
• Researchers have grouped
viruses together into several major
families, based on their shape,
behavior, and other
characteristics.
• These include the herpesviruses,
adenoviruses, papovaviruses
(papilloma viruses),
hepadnaviruses, poxviruses, and
parvoviruses among the DNA
viruses
• On the RNA virus side, major families
include the picornaviruses (including
the rhinoviruses), calciviruses,
paramyxoviruses, orthomyxoviruses,
rhabdoviruses, filoviruses,
bornaviruses, and retroviruses.
• There are dozens of smaller virus
families within these major
classifications. Many viruses are hostspecific, causing disease in humans or
specific animals only.
THE STAGES OF THE LIFE CYCLE OF HUMAN IMMUNEDEFFECIENCY VIRUS (HIV-1) cause of ACQUIRED IMMUNODEFFICIENCY SYNDROME (AIDS)
FUNGI
• Any of about 80,000 known species of
organisms belonging to the kingdom
Fungi, including yeasts, rusts, smuts,
molds, mushrooms, and mildews.
• Though formerly classified as plants,
fungi lack chlorophyll and the organized
plant structures of stems, roots, and
leaves.
• The thallus, or body, of a typical fungus
consists of a mycelium through which
cytoplasm flows.
• The mycelium generally reproduces by
forming spores, either directly or in
special fruiting bodies that make up the
visible part of a fungus.
• The soil provides an ideal habitat for
many species, although fungi can also
live in the air and water and on plants
and animals.
• Fungi are found in all regions of the
world that have sufficient moisture to
enable them to grow.
• Lacking chlorophyll, fungi are unable to
carry out photosynthesis and must
obtain nutrients by secreting enzymes
onto the surface on which they are
growing.
• These enzymes digest organic matter,
forming solutions of nutrients that can
be absorbed through the mycelium.
• Essential to many food and industrial
processes, fungi are used in the
production of enzymes, organic acids,
vitamins, and antibiotics.
• Parasitic fungi invade living
organisms, often causing disease and
death certain insects.
PARASITES
• An organism that lives on or in a different
kind of organism (the host) from which it
gets some or all of its nourishment.
• Parasites are generally harmful to their
hosts, although the damage they do
ranges widely from minor inconvenience
to debilitating or fatal disease.
• A parasite that lives or feeds on the outer
surface of the host's body, such as a
louse, tick, or leech, is called
Ectoparasites.
• Ectoparasites do not usually cause
disease themselves although they are
frequently a vector of disease, as in the
case of ticks, which can transmit the
organisms that cause such diseases as
Rocky Mountain spotted fever and Lyme
disease.
• A parasite that lives inside the body of its
host is called an Endoparasite.
• Endoparasites include organisms such
as tapeworms, hookworms, and
trypanosomes that live within the host's
organs or tissues, as well as organisms
such as sporozoans that invade the
host's cells.
Parasitic diseases include infections by
protozoa, helminths, and arthropods:
• Protozoa -- Malaria is caused by
plasmodium, protozoa, a single-cell
organism that can only divide within its host
organism.
• Helminths -- Schistosomiasis, another set
of very important parasitic diseases, is
caused by a helminth (a worm).
• Arthropods -- The arthropods include
insects and arachnids (spiders, etc.), a
number of which can act as vectors
(carriers) of parasitic diseases.
BACTERIAL CLASSIFICATION IS BASED ON
SEVERAL MAJOR PROPERTIES
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Gram staining (and other stains).
Morphology.
Metabolic behaviour.
Infection patterns.
Intracellular vs extracellular.
Antigenic composition.
DNA sequence.
Binomial (scientific) nomenclature
Gives each microbe 2 names:
Genus - noun, always capitalized
species - adjective, lowercase
Both italicized or underlined
Staphylococcus aureus (S. aureus)
Bacillus subtilis (B. subtilis)
Escherichia coli (E. coli)
BACTERIA: MORE ON MORPHOLOGY
BACTERIAL SHAPES
BRIEF HISTORY OF MICROBIOLOGY
Old times…
• Humans knew how to deal with germs before even
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knowing about their existence.
Discovery of Microorganisms
• Antony van Leeuwenhoek
Microbiology, a Dutchman.
(1632-1723),
the
father
of
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A hobbyist microscopist
microorganisms accurately.
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Accurate shape, detailed movement.
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Subjects were most possibly bacteria and protozoa and he called
them “animalcules“, also reported spermatozoa, blood cells
first
to
observe
and
describe
The Conflict over Spontaneous Generation
 spontaneous generation
 living organisms can develop from nonliving or decomposing matter
 publically a common sense vision with social and cultural roots
(almost 2000 years ago)
 Jan Baptista Van Helmont believed in spontaneous generation
 Louis Pasteur (1822-1895) was the founder of modern microbiology
 He developed methods for bacteria, and propagated the virus of
rabies in animals
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Pasteur Refutes the Spontaneous Generation.
Ambient germs are necessary for promoting growth.
Principle of sterility and aseptic work is important. Specific aseptic
techniques are employed to avoid microbial contamination (e.g.,
Pasteur's experiments.
He developed Pasteurization (Method of prevention of spoilage of
liquid foodstuffs (milk, wine, beer) which utilizes heating)
Germ theory: Microorganisms Cause Disease
 There is a relationship between Microorganisms and
Disease.
• The germ theory of disease states that some
diseases are caused by microorganisms.
• These small organisms, too small to see without
magnification, invade humans, animals, and other
living hosts.
• Their growth and reproduction within their hosts can
cause a disease.
• "Germ" may refer to not just a bacterium, but also a
protist, fungus, virus, prion, or viroid.
• Microorganisms that cause disease are called
pathogens, and the diseases they cause are called
infectious diseases.
• Even when a pathogen is the principal cause of a
disease, environmental and hereditary factors often
influence the severity of the disease, and whether a
particular host individual becomes infected when
exposed to the pathogen.
 The germ theory was proposed by Girolamo
Fracastoro in 1546, but scientific evidence in
support of this accumulated slowly and,
 Galen's miasma theory remained dominant
among scientists and doctors.
 A transitional period began in the late 1850s
as the work of Louis Pasteur and Robert
Koch provided convincing evidence; by
1880, miasma theory was still competing
with the germ theory of disease.
 Eventually, a "golden era" of bacteriology
ensued, in which the theory quickly led to the
identification of the actual organisms that
cause many diseases.
• Viruses were discovered in the 1890s.
 Louis Pasteur (1860s), a noted chemist, took up the challenge and
utilized broths allowing air but disallowing microbes.
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Grew broths at different altitudes and in a dusty cellar.
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Used broths with cotton to show the germs accumulated on cotton.
Did the Swan neck
Pasteur went to work on chicken cholera and discovered one could
attenuate cultures and produce artificial vaccines.
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 Pasteur solved the riddle of rancid wines in France's vinyards.
 Recommended sterile technique and Pasteurization (applied to milk
and became a central method for controling TB, Diptheria, and other
diseases).
 Pasteur wanted to isolate a bacterium in pure culture that caused
disease. Began working with Anthrax.
 Anti-spontaneous generation experiments:
 Pasteur definitively demonstrated that microorganisms are
present in air but not created by air.
 Vaccine discoverer.
 Pasteurization.
 Semmelweis, Ignaz (1840s)
• Hand washing/childbirth fever:
• Demonstrated that hand washing prevented the spread of
childbirth fever.
• Ignaz Semmelweis, an eastern European physician working in a
Vienna Hospital, noticed the wards where delivery occurred by
midwives had 10X less Puerperal Fever and deaths than those
tended to by doctors.
• He showed he could dramatically decrease "Childbed Fever" by
having doctors wash hands in chlorine water after dissection of
cadavers and between patients.
• Was fired for blaming deaths on doctors who didn't wash hands.
• Oliver Wendel Holmes wrote on the Contagiousness of Puerperal
Fever". Author, Physician, and Anatomy Professor. Late 19th
Century.
Lister, Joseph (1860s)
• Chemical inhibition of infection:
• Connected and applied Semmelweis' and
Pasteur's work to develop and popularize
the chemical inhibition of infection during
surgery.
• Washed surgical wounds with phenol
(a.k.a., carbolic acid)
• Lister is considered to be the father of
antiseptic surgery.
Koch, Robert (1870s)
• Developed Koch's Postulates which are a
sequence of experimental steps for directly
relating a specific microbe to a specific
disease.
• Koch discovered:
i. Bacillus anthracis
ii. Mycobacterium tuberculosis
iii.Vibrio cholera
• Technique developer:
• In addition to Koch's postulates, Koch
played an important role in the
development of the use of agar as solid
medium.
• Louis Pasteur along with Robert Koch developed
the germ theory of disease which states that "a
specific disease is caused by a specific type of
microorganism."
• In 1876, Robert Koch established an experimental
procedure to prove the germ theory of disease.
This scientific procedure is known as Koch's
postulates.
• Koch’s postulates not only proved the germ theory
but also gave a tremendous boost to the
development of microbiology by stressing a
laboratory
culture
and
identification
of
microorganisms.
Koch's postulates:
 The causative agent must be present in every case of
the disease and must not be present in healthy
animals.
 The pathogen must be isolated from the diseased host
animal and must be grown in pure culture.
 The same disease must be produced when microbes
from the pure culture are inoculated into healthy
susceptible animals.
 The same pathogen must be recoverable once again
from this artificially infected animal and it must be able
to be grown in pure culture.
 Koch’s postulates not only proved the germ theory but
also gave a tremendous boost to the development of
microbiology by stressing a laboratory culture and
identification of microorganisms.
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Fleming, Alexander (1920s)
Penicillin/first antibiotic:
Fleming discovered that a mould accidentally
growing on one of his petri dishes had antibacterial activity.
The mould was producing penicillin.
This was the first antibiotic discovered.
 Microbial diseases changed history
• 14th century: Bubonic plague (Black death)
caused by Yersinia pestis in Europe.
• 19th century: Tuberculosis caused by
Mycobacterium tuberculosis
• Today: AIDS by the human
immunodeficiency virus (HIV).
THE NORMAL BACTERIAL FLORA OF HUMANS
• In a healthy animal, the internal tissues,
e.g. blood, brain, muscle, etc., are normally
free of microorganisms.
• However, the surface tissues, i.e., skin and
mucous membranes, are constantly in
contact with environmental organisms and
become readily colonized by various
microbial species.
• The mixture of organisms regularly found
at any anatomical site is referred to as the
normal flora, except by researchers in the
field who prefer the term "indigenous
microbiota".
THE NORMAL BODY FLORA
• Normal Flora: Also called normal biota, normal
microflora, and normal microbiota.
• Hundreds of species of bacteria and fungi live
symbiotically on the human body.
• The Normal flora, are bacteria, fungi, and protozoa
that live on or within the bodies of animals and
plants.
• These organisms are present on external surfaces
(skin and conjunctiva of the eye) and internal
surfaces (linings of the respiratory, digestive and
urogenital systems) of the body.
• Internal structures and organs (for
example, bone, heart, liver, kidneys) are
normally sterile Normal microflora may be
harmless, beneficial or disease causing.
• The spectrum of microorganisms changes
with growth and development of the
individual.
• The womb provides a sterile environment
for the developing fetus. Upon birth, the
newborn is colonized with numerous
different bacteria and fungi.
• During growth and maturation, different
microorganisms predominate at various
sites on the body.
Normal flora [normal microbiota]
Not disease-causing:
• Normal flora are those not-typically-diseasecausing microorganisms normally found in
and on healthy individuals.
• Also known as normal microbiota.
Very abundant:
• Normal flora are extremely abundant in
terms of absolute numbers.
• A normal human has approximately 1013
body cells and 1014 individual normal flora!
All found externally:
• Normal flora are found mostly:
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on the skin
in the eyes
in the nose
in the mouth
in the upper throat
in the lower urethra
in the lower intestine
especially in the large intestine
• Note that this list basically includes all
of the body surfaces exposed to the
external environment.
SIGNIFICANCE OF THE NORMAL FLORA
 We are covered in microorganisms – 10 times as many
prokaryotic cell (mainly bacteria) associate with our bodies.
 The normal flora substantially influences the well-being of the
host.
 Although the foregoing indicates that bacterial flora may be
undesirable, studies with antibiotic treated animals suggest
that the flora protects individuals from pathogens.
 The normal flora in humans usually develops in an orderly
sequence, or succession, after birth, leading to the stable
populations of bacteria that make up the normal adult flora.
 For example, an infant begins to contact organisms as it
moves through the birth canal. A Gram-positive population
(bifidobacteria arid lactobacilli) predominates in the
gastrointestinal tract early in life if the infant is breast-fed.
 This bacterial population is reduced and displaced somewhat
by a Gram-negative flora (Enterobacteriaceae) when the
baby is bottle-fed. The type of liquid diet provided to the infant
is the principal instrument of this flora control;
immunoglobulins and, perhaps, other elements in breast milk
may also be important.
What, then, is the significance of the normal
flora?
 Animal and some human studies suggest that
the flora influences human anatomy,
physiology, lifespan, and, ultimately, cause of
death.
 The human body provides many unique
environments
for
different
bacterial
communities to live and is referred to as the
host.
 A positive host-microbe relationship is usually
described
as
either
mutualistic
or
commensalistic.
 In mutualism both the host and the microbe
benefit.
Which
is
in
contract
to
commensalisms, where one partner of the
relationship benefits (usually the microbe) and
the other partner (usually the host) is neither
benefited nor harmed.
The benefits of normal flora
 Normal flora synthesize and excrete vitamins in excess of
their own needs, which can be absorbed as nutrients in the
host. For example, E. coli secretes Vitamin K and certain Bvitamins.
 The normal flora prevent colonization by pathogens by
competing for attachment sites or for essential nutrients.
 The normal flora may antagonize other bacteria through the
production of substances which inhibit or kill nonindigenous
species.
 The normal flora stimulate the development of certain tissues,
i.e., the caecum and certain lymphatic tissues in the GI tract.
 The normal flora stimulate the production of cross-reactive
antibodies. It is known that the normal flora behave as
antigens in an animal and therefore induce immunological
responses.
Harmful Effects Of Normal Flora
 Clinical problems from normal flora arise in the following
conditions:
 When the organisms are displaced from their normal site in
the body to an abnormal site, for e.g. the introduction of the
normal skin bacterium S. epidermidis into the blood stream
where it can colonize catheters and heart valves.
 When potential pathogens gain a competitive advantage due
to diminished populations of harmless competitors for e.g.
when normal bowel flora is depleted by antibiotic therapy.
 When harmless commonly ingested food substances are
converted into carcinogenic derivatives by bacteria in the
colon.
 When an individual is immunocompromised normal flora can
overgrow and become pathogenic.
 Their metabolism allows them to attack a broad variety of
compounds in our diets, some of which they can convert to
compounds that are harmful to us
 They can be opportunistic pathogens whenever
luck or injury allows them into regions of the
bodies where they are not wanted.
 Opportunistic
infection:
Clinically
apparent
infection with a microorganism that usually causes
in apparent infection or no infection at all. When
someone is weakened by illness, injury,
malnutrition, or other factors, opportunistic
infections may occur.
 Individuals with decreased resistance to infection
are called compromised hosts
 Nosocomial infection: infections caused by
medical treatment, usually in a hospital or clinic.
Hospitals are full of sick people, many of whom
are compromised hosts.
 These people often acquire opportunistic
infections from hospital personnel or medical
equipment.
HOST DEFENCES
• Host defences include both
physiologic barriers and
immunological responses.
• Some defences are non-specific;
others are highly specific.
• Host defences can vary considerably
due to many factors including
alcohol, drugs, nutrition, immunologic
disorders, etc.
Skin and mucous membranes
provide the first line of defence
through:
• Mechanical factors: physical barrier
to penetration.
• Chemical factors: gastric acidity,
unsaturated fatty acids, lysozyme.
• Microbial factors: antagonism by
normal flora.
Phagocytic
cells
provide
a
secondary line of defense by
consuming invaders and secreting
substance that produce immune
responses:
• Mononuclear cells: monocytes
(blood) and macrophages (tissue).
• Neutrophils: polymorphonuclear
(PMN) granulocytes.
P.T.O
• Inflammation: immune response
producing dilation of blood vessels,
increased vascular permeability and
diapedesis of monocytes.
• Phagocytosis: cells destroy invaders
by i) utilizing specific membrane
receptors for attachment (e.g.
immunoglobulin Fc and complement
C3b receptors) and ii) ingesting
invaders into phagocytic vacuoles,
leading to intracellular killing by fusion
with lysosome-like granules containing
lowered pH, peroxide, enzymes, etc.
Humoral factors: antibody
mediated defences include:
• Antitoxins: specific antibodies that
bind certain exotoxins.
• Bacteriolytic antibodies: antibodies
plus complement can directly lyse
Gram-negative cells.
• Opsonizing antibodies: coat cell
surface and enhance phagocytosis
(Fc receptors).
Cell-mediated factors: cellmediated defences include:
• Cytotoxic T-lymphocytes: specific
cells capable of destroying altered
host cells.
• K and NK cells: lyse altered or
transplanted host cells.
• Activated macrophages: phagocytes
that possess a greatly enhanced
capacity for intracellular destruction of
ingested microorganisms.
TRANSMISSION OF DISEASE
Communicability: infectious disease can be
transmitted either directly (e.g. person to
person) or indirectly (e.g. contaminated
water). Factors involved in the
communicability of an infectious agent
include:
• Source, including dormant or latent infections
(carriers).
• Number of infectious agents released from a
host.
• Capability of surviving transit from host to host.
• Percentage of the host population that is
susceptible to the agent
MECHANISMS OF PATHOGENESIS
• Pathogenic properties of bacteria:
microorganisms cause disease by two basic
mechanisms: 1) invasion of tissue and 2)
production of toxins.
 Invasiveness: the ability to invade host
tissues.
 Toxigenicity: the production of toxins.
 Virulence: the combination of invasiveness
and Toxigenicity producing the ability to
overcome host defences.
CONTROL OF MICROORGANISMS
The control of microorganisms is
desirable in several environments
including:
• Microbiology for prevention of
contamination by extraneous
organisms.
• In surgery for maintenance of asepsis
(absence of bacteria, viruses, other
microorganisms).
• In food and drug manufacture for
ensuring safety from contaminating
organisms.
• Decontamination is the process of
cleansing an object or substance to
remove contaminants such as microorganisms or hazardous materials,
including chemicals, radioactive
substances, and infectious diseases.
• The control of microorganisms is
achieved through the decontamination
processes of Disinfection and
Sterilization.
• Disinfection: It is a process in which
most or nearly all microorganisms (not
including their spores) capable of
giving rise to infection are eliminated.
• Sterilization: It is a process by which
objects are freed of all microorganisms
(harmful or not) both vegetative and
spore state.
• Sanitation: Reduction of the microbial
population to levels considered safe by
public health standards
• Antiseptic: A mild disinfectant agent
suitable for use on skin surfaces
• -cidal: A suffix meaning that “the
agent kills.” For example, a
bactericidal agent kills bacteria and
fungicidal kills fungi.
• -static: A suffix that means “the agent
inhibits growth.” For example, a
fungistatic agent inhibits the growth of
fungi, but doesn’t necessarily kill it and
bacteriostatic inhibits bacteria growth,
but doesn’t necessarily kill.it.
Basically two Methods of
Sterilization: Physical Methods
and Chemical Methods
PHYSICAL METHODS
1. Heat:
• most reliable and preferred method
• is non-selective (all organisms are
susceptible including spores).
• Is able to reach organisms protected
from action of chemicals and radiation.
• Lethal action of heat is due to
denaturation of proteins and nucleic acid
(DNA and RNA).
• There is variation in heat resistance in
different microbes.
Moist Heat sterilization
• Is more rapidly lethal than dry heat.
• Requires lower working
temperature and shorter periods of
exposure.
• Mechanism of killing is a
combination of protein/nucleic acid
denaturation and membrane
disruption.
Measurements of killing by Moist Heat:
• Thermal death point (TDP): refers to the
lowest temperature at which a microbial
suspension is killed in 10 minutes at pH 7.
• Thermal death time (TDT): Shortest time
needed to kill all organisms in a
suspension at a specified temperature
under specific conditions.
• Decimal reduction time (D value): Time
required reducing a bacterial population
by 90% (a 10-fold, or one decimal,
reduction) at a specified temperature and
specified conditions.
• Z-value: The change in temperature, in “oC”,
necessary to cause a 10-fold change in the D
value of an organism under specified
conditions.
• F-value: The time in minutes at a specified
temperature (usually 121.1oC) needed to kill a
population of cells or spores.
• Example of calculations using D value:
• Given that the Clostridium botulinum spores
are suspended in phosphate buffer, and the
D121=0.204 minutes (Note 121oC).
• How long would it take to reduce a population
of C. botulinum spores in phosphate buffer
from 1012 spores to 100 spores (1 spore) at
121oC?
• Answer: Since 1012 to 100 is 12 decimal
reductions, then the time required is
12x0.204minutes = 2.45minutes
Methods of Moist Heat:
Boiling method (100oC)
• Kills vegetative forms of bacteria, fungi, and
many viruses within 10 minutes.
• However, bacterial spores and some viruses can
survive.
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Autoclaving/ pressure canning
Most effective method of moist heat sterilization.
Is carried out in an autoclave where steam under
pressure is used.
Most procedures use 121.1 oC, achieved at
approximately 15psi pressure, with 1530minutes autoclave time to ensure sterilization.
Sterilization in autoclave in biomedical or clinical
laboratory must periodically validated by testing
with spores of Clostridium or Bacillus
stearothemophilus.
An example of an Autoclave
Pasteurization
• Used to reduce microbial numbers in
milk and other beverages while retaining
flavour and food quality of the beverage.
• Retards spoilage but does not sterilize.
• Traditional treatment of milk, 63oC for 30
minutes.
• Flash pasteurization (high-temperature
short term pasteurization); quick heating
to about 720C for 15 seconds, then
rapidly cooling.
Ultrahigh-temperature (UHT) sterilization
• Milk and similar products heated to
140-150oC for 1-3 seconds.
• Very quickly sterilizes the milk while
keeping its flavour and quality.
• Used to produce the packaged “shelf
milk” that does not require
refrigeration.
Dry Heat Sterilization
Incineration
• Burner flames: direct flaming of
inoculating loop.
• Electric loop incinerators.
• Incineration of infected carcasses,
laboratory animals and infected
material using controlled air, excess
air or rotary kiln incinerators.
Hot air oven sterilization
• Hot air oven sterilization
• Used to sterilize glassware and heatresistant metal equipment.
• Objects are exposed for 2 hours at
160oC–180oC in an electric or gas
oven.
• Treatment denatures proteins and
cell membranes desiccate cytoplasm
and oxidize cellular components.
2. Filtration Method
• Used for physically removing microbes
and dust particles of heat labile liquids
e.g. urea, vitamin.
• Liquid is filtered through sterile
membrane filters capable of retaining
microbes e.g. 0.45µm; 0.22 µm pore
diameter.
• Examples of filters:
• Membrane filters e.g. Nitrocellulose
nylon, polyvinylidene difluoride.
• HEPA filters: High efficiency particulate
air filters used in laminar flow biological
safety cabinets
3. Radiation Method
• Has varying effects on cells
depending on its wavelength, intensity
and duration.
a) Ionizing Radiation
• Example: x-rays (0.001-0.1nanometre
(nm): Gamma rays (0.12-100nm)
b) Non-Ionizing Radiation
• Example: Ultraviolet (UV) radiation
(10- about 400nm).
4. Low Temperatures
a) Refrigerator:
• around 4°C
• inhibits growth of mesophiles or
thermophiles; psychrophiles will grow
b) Freezer:
• ordinary” freezer around -10 to -20°C
• “ultra cold” laboratory freezer typically 80°C
• Generally, inhibits all growth; many
bacteria and other microbes may survive
freezing temperatures
CHEMICAL METHODS
• Chemical agents are used to sterilize
body surfaces and inanimate objects.
• Most do not achieve sterility but
reduce microbial population to safe
levels.
Characteristics of an ideal
Disinfectant:
• Acts quickly.
• Attack all or wide range of microbes.
• Is able to penetrate thoroughly the material that is
contaminated.
• Readily mixes with water to form a stable solution.
• Is not hampered (reduced activity) by organic
matter on the substance that is to be disinfected.
• Is not likely to decompose and lose its activity after
exposure to light, heat or unfavorable weather.
• Does not stain, corrode, or destroy the object being
disinfected.
• Is not toxic to man and animals if it is to be used as
an antiseptic.
Types of Chemical agents
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Phenols and Phenolics
Alcohols
Halogens
Aldehydes
Sterilizing Gases
Heavy metals and their compounds
Antimicrobial Chemotherapeutic
agents
Antimicrobial
Chemotherapeutic agents
These are drugs which have lethal
(Bactericidal) or inhibitory effects
(bacteriostatic) on the microbes.
Ideal characteristics of antimicrobial drugs:
• Should have selective toxicity i.e. should
be toxic for the microorganism and not
for the host.
• Should not produce hypersensitivity in
most host e.g.. Sensitivity to penicillin in
form of skin rash.
• Must have solubility in body fluids so that
it can readily or rapidly penetrate body
tissues.
• Should not readily lead to development of
drug resistance by microorganisms.
• The drug should not eliminate the normal
flora of the host where opportunistic
pathogen e.g. Pseudomonas, Candida
albicans cause superinfections.
• Should have broad spectrum activity i.e.
being effective against a large number of
Gram positive and Gram negative species
of bacteria. However, the problem is that
broad spectrum drugs can also kill normal
flora.
Four categories of antimicrobial
drugs namely:
• Antibacterial
• Antiviral
• Antiparasitic
• Antifungal
i) Antibacterial/antibiotic drugs
Classes of Antibiotics
target organisms
Penicillins: penicillin G, ampicillin, methicillin,
Gram +ve bacteria
oxacillin, carbenicillin
Cephalosporins: cephalothin,cefazolin, cefuroxime,
Gram +ve bacteria
ceftriaxone, ceftazidine
Moderate against gram –ve bacteria
Aminoglycosides: streptomycin, neomycin,
Gram -ve bacteria
kanamycin, gentamycin.
doxycycline
Gram +ve and Gram -ve bacteria;
rickettsia, chlamydias
Erythromycins/Macrolides: erythromycin,
Gram +ve bacteria
Tetracyclines: tetracycline, chlortetracycline,
spiramycin, clindamycin
Polypeptides: bacitracin, polymyxin
Gram +ve bacteria
Some Gram -ve bacteria
MODE OF ACTION OF ANTIBIOTICS
They interfere chemically with synthesis of function of vital
components of microorganisms. Cellular structures and
functions of prokaryotic cells (bacteria) differ from those of
eukaryotic cells of human body and provides basis for selective
toxicity.
Inhibition of cell wall synthesis
• Drug prevents formation of peptide cross bridges in
actively growing bacteria resulting in weakened cell
wall and cell lyses.
• Examples of drugs: Penicillin, ampicillin, methicillin,
oxacillin- effective against gram positive
organisms.
Inhibition of Protein synthesis
• Antibiotics combine with either 50s or 30s subunits
of 70s ribosomes where they inhibit peptide bond
formation in growing polypeptide chain of protein.
• Examples of drugs: chloramphenicol,
erythromycin, tetracycline
Injury to cytoplasmic membrane
• Polypeptide antibiotics attach to cytoplasmic
membrane where they change its permeability
resulting in loss of metabolites from microbial.
These antibiotics are toxic to host tissue and
have limited use in chemotherapy
• Example: Polymyxin B.
Inhibition of nucleic acid synthesis
• Antibiotics also toxic to host cells due to
similarities in microbial and host cell DNA
and RNA metabolism. Have limited clinical
use
• Example: Trimethoprim
Inhibition of enzymatic activity
• Drugs bind to enzyme in organism
preventing its activity e.g.. Enzyme involved
in folic acid synthesis in bacteria.
• Example: Sulfonamides
Mechanisms of antibacterial
resistance
During treatment with drugs, bacteria may
acquire resistance towards them. The
following are various mechanisms by which
bacteria can acquire drug resistance:
Accumulation barriers
• Cell wall and membrane provide barriers to
drugs.
• Outer membrane of Gram negative bacteria
provides a formidable barrier for access to
the interior of the cell.
• Some bacteria have energy dependent
efflux mechanisms that pump drugs from
the cell.
Altered target
• Once in the cell; drugs act by binding
and inactivating their targets which is
typically a crucial enzyme or ribosomal
site.
• If the target is altered by substitution of
a single amino acid at a certain
location in a protein, then it alters its
binding affinity to the drug without
affecting its function in the bacterial
cell.
Enzymatic inactivation
• Enzymes produced by resistant bacteria
may inactivate the drug in the cell; in
periplasmic space or outside cell.
• They may act by disrupting the structure of
the drug.
• For example, B-lactamases may break open
the B-lactam ring of Penicillin and inactivate
the drug.
• Some enzymes produced modify the drug by
phosphorylating the amino group on
aminoglycosides (e.g.. Streptomycin,
neomycin, kanamycin, gentamicin)
rendering them ineffective.
Chromosomal resistance
• Develops as a result of mutation on
chromosome.
• Presence of drug serves as selecting
mechanism to suppress susceptible
microorganisms and favor growth of
resistant mutant.
Extrachromosomal resistance
• Plasmids e.g. R-factor carry genes for
resistance to one or several antimicrobial
drugs.
• Plasmids code for enzymes that destroy
antimicrobial drug.
Antiviral drugs
Mode of action of antivirals
Inhibition of cell penetration and uncoating
• Inhibits several early steps in viral replication
including viral uncoating.
• Examples: Amantadine; rimantadine
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Inhibition of reverse transcriptase enzyme
(Nucleoside analogue of Thymidine)
Inhibits the reverse transcriptase enzyme of RNA
containing viruses e.g. HIV
All terminates the elongation of the chain that is
initially synthesized to initiate replication of viral
genome.
Toxicity frequently occurs.
Examples: Lamivudine, Stavudine,
Inhibition of reverse transcriptase
enzyme (Non-Nucleoside analogue)
• Also inhibits reverse transcriptase enzyme in RNA
viruses e.g. HIV
• Relatively non-toxic.
• Examples: Nevirapine; Efavirenz
Inhibition of protease enzyme
• These block the action of viral coded enzyme
protease.
• Protease is responsible for cleaving polypeptides to
produce structural proteins.
• Inhibition of this enzyme leads to blockage of viral
assembly and release.
• Causes hepatotoxicity.
• Examples; Saquinavir, ritonavir, indinavir, nelfinavir
Mechanisms of antiviral resistance
• Resistance is largely due to mutations
in genes that code for production of
enzymes.
Antifungal drugs
• These act against fungi.
Mode of action of antifungals
P.T.O
• Azoles act by inhibiting the action of
cytochrome P450 dependent sterol 14αdemethylase enzyme with resulting in
cooperation of 14-methyl sterol which is
thought to disrupt membrane structure
and function thereby inhibiting fungal
growth.
Examples: Ketoconazole, miconazole,
Itraconazole, clotrimazole
• Polyenes acts by binding to ergosterol
leading to distortion and damage of
normal membrane permeability. Results
in leakage of intracellular constituents
and cell death.
Examples: amphotericin B; Nystatin
Mechanisms of antifungal resistance
• Secretion of some cellular enzymes to
extracellular medium which degrade drug.
• The drug is pumped out by an efflux
pump.
• The drug target is altered so that the drug
cannot bind to the target.
• The target enzyme is overproduced so
that the drug does not inhibit biochemical
reactions completely.
• The cell develops a bypass route for
production of drug target.
Antiparasitic drugs
These drugs act on parasites.
• Mode of action of antiparasitic drugs
• Quinolones block nucleic acid synthesis by
intercalation into double stranded DNA.
• Example: chloroquine
• Quinones blocks pyrimidine biosynthesis and
inhibition of parasites mitochondrial electron
transport chain.
• Example: Atovaquone
• Praziquantel induces loss of intracellular
calcium, titanic muscular contraction and
destruction of the tegument.