University of Gondar
Institute of Biotechnology
Department of Biotechnology
Microbiology Course
By- Mequanente Dagnaw (Assistant professor)
MPH in Epidemiology
BSc in Clinical Nursing
MSc in Biotechnology
BSc in Biotechnology
PGDT in Biology
Email: mequanente@gmail.com/meki2011@yahoo.com
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Learning Methods
•
•
•
•
•
Interactive lecturing styles
Group discussion
Assignment presentation
Video/animation
Practical demonstration
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Learning objectives
 Describe the different fields of microbiology.
 Describe the Historic Backgrounds of Microbiology
 Explain the theories about the origin of life
 Describe the Germ Theory and its contribution
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Introduction
Definition:
 Microbiology is the study of living organisms of
micros copic size or very small organism.
These
microorganisms
includes:-Bacteria,
Fungi,
Certain Algae, and Protozoa, Viruses.
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Introd…
It considers the microscopic forms of life & deals about
their:
 reproduction,
 physiology,
 participation in the process of nature,
 helpful & harmful r/s with other living things,
 Significance in science & industry.
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Introd…
Branches of microbiology
 Some of the main branches of microbiology are:
Medical Microbiology,
Food Microbiology,
Industrial Microbiology,
Agricultural Microbiology,
Soil Microbiology,
Plant Microbiology
Veterinary Microbiology
Environmental Microbiology
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Introd…
 Medical microbiology
 Deals
with
disease
causing
microorganisms
and
their
pathogenesis, laboratory Dx, Rx, prevention and control.
 Medical microbiology includes:
 Bacteriology,
 Parasitology,
 Mycology,
 Virology, and
 Immunology.
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Con…
Why we study microorganisms???
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Con…
Knowledge of Microbes allows humans to :
Prevent and control disease occurrence
Led to aseptic techniques to prevent contamination
in medicine and in microbiology laboratories
Prevent food spoilage which might be due to
Microbial Poisonings
Well understand the natural course of disease
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Are Microorganisms BAD or Good to
Mankind and the Environment ???
 If they are mostly GOOD in what
perspective???
If they are BAD why???
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 Effects of Microorganisms
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 Scope of Microbiology
 Diagnostic
 Isolation & identification of the causative organism from the
biological samples
 Source of infection - in sudden outbreak of diseases
 Prognosis of disease - predicting the likely outcome of a disease
based on the condition of the patient and the usual action of the
disease.
 Guidance in treatment - culture & sensitivity
 We can suggest the effective drug for the treatment of that particular infection
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History of Microbiology
 In ancient civilization disease was believed to be a
punishment sent from God for human’s wrong doing.
 Many philosophers during the early period, also believes
that disease was transmitted by invisible “animals” since
the animal could not be seen, but theory remains just a
theory.
 Hippocrates: father of medicine, observed that ill results
due to change in Air, Nature of soil, Water, Climate, Wind,
Habitat of people and Food.
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History…
Antony van Leeuwenhoek (1632-1723 G.C)
 Observe microscopic organisms in pond water and
debris surrounding the teeth.
 Leeuwenhoek called this organism “animalcules”
meaning little animals from sample taken on human
mouth.
 He was the first to describe different shapes of
bacteria as rod, spherical and spiral.
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Debating issue
• Origin of Life???
• Where Does life comes from???
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Theories about the origin of life
Many scientists were searching for an explanation
for spontaneous appearance of living things from
decaying material, stagnant ponds, infected
wound, fermented grains e.t.c
Based on these observations two major theories
was formulated:
 Abiogenesis
 Biogenesis
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I. Abiogenesis
 Deals with spontaneous generation; stating that living
things originated from non-living things.
 Aristotle (383-322 BC.)–The Founder of theory of
spontaneous generation.
 He observed spontaneous existence of fish from dried ponds,
when the pond was filled with rain.
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II. Biogenesis
 States that life comes
from pre existing life
 Francesco Redi (16261697)
 He was the first scientist
who tried to set an
experiment to disprove
the theory of
spontaneous generation
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 Francisco Redi…
 Performed experiments that disproved
theory of Spontaneous Generation for
more complex forms of life
 Utilized jars containing meat
 Some were covered, some were not
 Maggots appeared in uncovered jars
 Results not accepted for microscopic
organisms
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
Louis pasture (1822- 1895)
Was the scientist who disproved the theory of
abiogenesis once and for all.
Performed experiment to disprove theory of
spontaneous generation
In his experiment he filtered air through
cotton plug
He placed plug in infusion broth, broth
became cloudy - organisms present in the air
He designed a large curved flask/swan-necked
(pasture goose neck flask) and placed a sterile
infusion broths
Flasks remained sterile unless tilted or neck
broken
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 Flask A. was not swan neck contains sterile broth that is directly exposed to the
air, become turbid after 16-24 hour.
A
B
 Flask B. which is swan neck and also contain sterile broth, because of its shape
only air can enter over surface of the broth but dusts were trapped in the curved
portion of the flask.
 The broth in the swan neck flaskMeki
show
D. no turbidity.
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Major contribution of Louis Pasteur
 Microbial theory of fermentation
 Principle and practice of sterilization & pasteurization
 Development of vaccine against anthrax and rabies
 Discovery of streptococci
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Summary
Scientist
Radii
Needham
Materials Condition
Used
Fresh
some
meat
covered and
some not
Pre
heated
broth
Spalanzani broth
heated in
flask
Pasture
broth
heated in
flask
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Result
Conclusion Limitation
maggot
Disprove
not accepted
observed on spontaneous for
unsealed meat generation microscopic
organisms
some sealed Both flasks
support
insufficient
and some not became turbid spontaneous heat, improper
generation seal ,
some tightly
sealed and
some not
swan necked
flask
No turbidity
in sealed
flasks
no turbidity
Disprove
spontaneous
generation
disprove
spontaneous
generation
Air is not
allowed to
enter
no limitation
(accepted
theory)
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The germ theory of disease
 Robert Koch (1843-1910)
 In the late 1800s a German physician named Robert Koch
demonstrated the relation between microorganism and the
infectious disease process.
 Koch studied anthrax which is a disease of cattle that can be
transmitted to human.
 He isolated the organisms from infected animals in pure.
 Then he injected a small amount of the pure culture to a healthy
animal.
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Koch…
 The injected animal developed the disease anthrax.
 The infectious agent was then isolated from the newly
infected animal in pure culture.
 This sequence of isolation, re-infection and recovery
of the infectious agent called Koch’s postulate (proof
of germ theory of a disease).
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Koch’s postulates
 The causative (etiological) agent must be present in all
affected organisms but absent in healthy individuals
 The agent must be capable of being isolated and cultured
in pure form.
 When the cultured agent is introduced to a healthy
organism, the same disease must occur
 The same causative agent must be isolated again from the
affected host.
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Major achievements of Robert Koch
Discovery and use of solid medium in bacteriology.
Discovery of causative agent of tuberculosis and
cholera.
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Exceptions to Koch’s postulate
a) Many healthy people carry pathogens but do not exhibit
symptoms of the disease.
b) Some microbes are very difficult or impossible to grow
in vitro in artificial media. E.g. Treponema pallidum
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Con…
C) Many microorganisms are species specific. E.g.
Brucella abortus cause abortion in animals but not
in humans .
D) Certain diseases develop when an opportunistic
pathogen invades immuno-compromised host.
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1. GENERAL BACTERIOLOGY
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CellGENERAL BACTERIOLOGY
The smallest structural and functional unit
of an organism.
There are two types of cells:
 Eukaryotes
 Prokaryotes
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Eukaryotic Cell
 Eu-true; Karyote-nucleus
 Has true membrane bound nucleus
 Contain multiple chromosomes
 Has a mitotic apparatus
 Has a well defined endoplasmic reticulum
 Has mitochondria
 Example:
Algae, Protozoa & Fungi
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Fig. Eukaryotic cell
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Prokaryotic Cell
 Pro - primitive( before);
Karyote - nucleus
 Posses naked DNA with out associated basic
proteins (histone)
 Divide mitotically by binary fission
 Bounded by a semi rigid cell wall.
 Example:
Bacteria, Cyanobacteria & Archaebacteria
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Prokaryote Vs Eukaryote
Prokaryote
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Eukaryote
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Structure of Bacterial cell
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Differences B/n Eukaryotes & Prokaryotes
Features
Prokaryotic cell
Eukaryotic cell
Nuclear membrane
Absent
Present
Chromosome
Single
Multiple
Nucleolus
Absent
Present
Sexual reproduction
Absent
Present
Cytoplasmic ribosome
70s
80s
Mitochondria
Absent
Present
Endoplasmic reticulum
Absent
Present
Lysosomes
Absent
Present
Micro filaments & tubules
Absent
Present
Site of oxidative phosphorylation
Cell membrane
Mitochondria
Peptydeglycan
Present
Absent
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membrane composition
Phospholipids Meki
& proteins
D.
Sterols
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General Feature of Bacteria
 The smallest free living microorganisms that is visible only
with the aid of microscope.
 Their size ranges from 0.1 to 10µm.
 Represents
the
largest
and
diversified
group
of
microorganisms that can exist as living cells.
 They are able to carry out their own life processes:
Growth
Energy generation
Reproduction independent of other cells
except
Chlamydia and Rickettsia
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Properties of Bacterial cells
 Typical prokaryotic cell
 Contain both DNA & RNA
 Replication is by binary fission
 Most grow in artificial media
 Contain rigid cell wall
 Sensitive to anti microbial agents
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Bacterial Classification and Nomenclature
 Classification
is
one
branch
of
science
called
Taxonomy
 Classification of organisms is based on their similarity
of characteristics.
 The broadest group in classification is kingdom and
bacteria are belongs to the kingdom monora.
 The smallest group is species and related species are
grouped in genera.
 Taxon is a group or category of related organisms
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 Lower level taxa. - e.g.
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Binomial nomenclature
 Organisms
are
named
using
binomial
nomenclature (except Viruses)
 Binomial nomenclature employs the name of the
two level taxa (genes and species).
 Genus name comes before species name
 Genus name always capitalized while species is
not and both should be underlined or italicized
 Genus name some time used alone but not
species
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Important criteria to group bacteria
 A number of criteria have be used to group bacteria
 Energy source: based on this bacteria can be
grouped as Phototrophic, Chemotropic,
 Based on synthesize essential methabolites:

Autotrophic –organic cpds from N&Co2

Heterotrophic-depend on performed organic cpds.
e.g pathogens
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Con..
Nutrient requirements: these are simple or
complex
Ability to grow in/on living tissue:
saprophytes, commensal and parasite
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Bacterial Grouping…
 Optimum temperature requirement:
Psychrophilic,mesophilic& thermophilic.
 Oxygen requirement: aerobic, anaerobic,
microaerophilic and facultative Anaerobes
 PH requirement
acidity, alkality, neutral
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Bacterial classification
 Methods of classification of bacteria are
generally grouped in to:
 Phenotypic
 Analytical
 Genotypic
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Phenotypic classification????
 Based on microscopic and macroscopic observable
characteristics of the microorganism such as:
 Staining properties: G+ve, G–ve and AFB
 Morphology (shape ,size , arrangement)
 Serotype (by using specific antigens of the bacteria
)
 Phage type (susceptibility to infection with virus )
 Macroscopic appearance of colony
 Biochemical markers Meki D.
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Bacterial shapes & arrangements
Phenotypic…
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Phenotypic…
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Analytical classification
 Chromatographic analysis of the following
 Whole protein analysis
 Cell wall Fatty Acid analysis
 Whole cell lipid analysis
 Cellular enzyme analysis
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Genotypic classification
 The most precise method in classifying bacteria
 DNA hybridization
 Plasmid analysis
 Nucleic acid sequence analysis
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Genotypic Classification
• The ideal means of identifying and classifying bacteria
would be to compare each gene
• Sequence in a given strain with the gene sequences for
every known species
• The total DNA of one organism can be compared with
that of any other organism by a method called nucleic
acid hybridization or DNA hybridization
• It is used to measure the number of DNA sequences
that any two organisms have in common and to
estimate the percentage of divergence within DNA
sequences that are related but not identical.
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D.
Structure of Bacterial Cell
 Compared with eukaryotic cells bacteria composed of a
simple base cell structure.
 The structure of bacterial cell is composed of:
 Cell wall
 Cytoplasmic membrane
 Cytoplasm (containing: neucloid, ribosome,
granules, plasmid)
 Cell surface components (capsule, flagella, Pili)
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A. Cell wall
 A thick and rigid layer that lies outside the cytoplasmic membrane
and beneath the capsule.
 So complex than simple cellulose plant cell wall
 The main functions of the cell wall of bacteria are:
 Maintain shape
 Protection
 Give rigidity to the bacterial cell
 Contain components toxic to the host (antigenic characteristics)
 Contain receptor sites for phages
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Cell wall…
 Complex structure made of Peptydeglycan
 Peptydeglycan layer: - Consists of 3 parts
A back bone- composed of N-acetyl glucose amine and
N-acetyl muramic acid
 Tetra-peptide side chain attached to N- acetyl muramic
acid
 A set of identical peptide cross-bridges
 Bacteria can be grouped in to two groups based on the
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differential staining technique
called grams stain.
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Cell wall
• A thick and relatively rigid layer that lies outside the
cytoplasmic membrane and beneath the capsule
• So complex than simple cellulose plant cell wall
• Made of peptydeglycan
• Peptydeglycan layer consists of 3 parts
– A back bone- composed of N-acetyl glucose
amine and N-acetyl muramic acid
– Tetra-peptide side chain attached to N- acetyl
muramic acid
– A set of identical peptide cross-bridges
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NAG and NAM joined as in peptidoglycan
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Structure of peptidoglycan in G-positive bacteria
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Cell wall…
 The difference in staining characteristics is due to the
difference in their cell wall structure.
o Gram positive
o Gram negative
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Cell wall of Gram-positive bacteria
– Composed of relatively very
thick peptydeglycan layer
(about 50-60%)
– The remaining cell wall
layer is a special
polysaccharide called
teichoic acid
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Gram-positive cell wall
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Cell wall of Gram-negative bacteria
 Unlike the gram positive bacteria, the cell wall of gram negative
bacteria is multilayered.
 It consists of:
1.Peptydeglycan
2.Complex outer layer- similar to the plasma membrane, but is less
permeable and composed
3.Lipopolysaccharide- it is a harmful substance classified as
endotoxins.
Endotoxin is responsible for many of the features of diseases
such as fever & shock.
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Gram-negative cell wall
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Gram-negative cell wall…
LPS is composed of 3 distinct units
• Lipid A- toxic effect
• A core polysaccharide of sugars linked to lipid A
• Outer polysaccharide which is called Somatic or O-antigen used
for Identification of many G-ve bacteria.
Lipoprotein (attaches the outer membrane to the pdg.)
Phospholipids
4. Periplasmic space - the region b/n plasma membrane and the outer
membrane
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Comparison of Gm+ve & Gm-ve
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Bacteria with Defective cell wall
– Treatment of bacterial cell with Lysozyme or penicillin
results the formation of cell wall deficient bacteria
– Most Cell Wall deficient bacteria can survive only in
hypertonic medium (high salt conc.)
– Includes the following
•
Protoplast
•
Spheroplast
•
L-forms
•
Natural L-forms
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Protoplast
– lack CW completely
– Derived from Gram
positive bacteria
– Unstable and osmotically
fragile
– Metabolically active
– Unable to reproduce
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Spheroplast
– Partial removal of CW
– Derived from Gram negative
bacteria
– Have damaged cell wall which is
not functional
– Able to change back to their
normal form when the toxic
substances is removed
– Can reproduce in suitable
condition
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L-forms
– Mutant bacteria with out
cell wall
– Bacteria produced in the lab
– Able to reproduce
– Spontaneous or antibiotic
induced formation of Lforms can Cause chronic
infections because such Lforms are resistant to a
treatment
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Natural L-forms
– The genus Mycoplasma lacks
Cell Wall naturally
– They are very small in size
– Grow very slowly
– Grow best in hypertonic media
– Highly irregular in shape & size
(pleomorphic)
– Lacks rigidity
– Not inhibited by Penicillin,
Cycloserine, Bacitracin,
Cephalosporin
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Cytoplasmic membrane (CM)
 Selectively permeable membrane made up of
phospholipids matrix which surrounds the cytoplasmic
membrane.
 Composed of : 60% - protein,
20-30% - lipids,
10-20% - carbohydrate
 It is similar with the eukaryotic membrane except the
sterol which is found in eukaryotic organisms
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Function of bacterial CM
• Transport of molecules
in and out the cell
• Energy generation and
biosynthesis
• Synthesis of precursors
of the Cell Wall
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Cellular components enclosed with in
the cell envelope
Mesosomes
– Invagination of Cell Membrane which is important in
biosynthesis, cell division and energy generation.
Ribosomes
– Are composed 70% RNA and30% Protein
– Are site of protein synthesis (enzyme, toxin)
– The ribosome monomer is 70s unit
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Cellular components…
Cytoplasmic granules
– Serve as storage area of nutrients
Nucleiod
– Area of cytoplasm on which DNA is located
– DNA of bacterial cell is single and circular
– The DNA replicate by attaching with the mesosme
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Cellular components…
Plasmids
–Extra chromosomal, double strand, circular DNA
molecules
–Replicate independently of bacterial chromosomes
–Encode proteins that serve as a virulence factor and drug
resistance
–Transmissible plasmid can be transferred from cell to cell
by conjugation
–Non
contain transfer genes80
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5.2.5. Specialized structure outside the cell wall
5.2.5. 1. Capsule
– Gelatinous material loosely attached to the
exterior of cell wall
– Can be identified microscopically with the aid of
India ink
– Capsule is found in species of bacteria such as
–S. pneumoniae
–B. anthracis
–Bordetella pertusis
–N. meningitides
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Capsule…
• Two types of capsules:
Polysaccharide-polymerization of glucose & fructose
Amino acids - poly glutamic acid
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Features of Capsule
 Usually weakly antigenic
 Not necessary for viability
 Capsulated strains are invariably non-motile
 Visualized by negative staining & capsule staining
 Detected by quellung phenomenon
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Important role of capsule in disease
• Virulence factors (by inhibiting phagocytosis)
• Antigenicity (ability to induce the immune system to
produce antibody)
• Vaccine preparation
• Adherence of the bacteria to human tissues
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Bacterial Capsule
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Specialized structure…
Flagella
– Tread like protein projected out side the cell wall and
important for movement of bacteria.
– Composed of a protein called flagellin
Its presence in bacteria is detected by:
Hanging drop preparation
Swarming phenomenon on surface of plate agar
Motility media
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Flagella
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Flagellar Arrangements
– Based on the arrangement flagella can be classified as:
• Atrichous - bacteria with no flagella
• Monotrichous - bacteria with one flagellum at one end
• Amphitrichous - bacteria with one flagellum at each end
• Lophotrichous - bacteria with a tuft (bunch) of flagella at
one or both end
• Peritrichous- bacteria with flagella over the entire surface
of the body.
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Fig. Different flagellar arrangements
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Specialized structure…
Pili (Fimbriae)
– Hair like protein appendages frequently observed on
Gram negative and few Gram positive bacteria
– Shorter and finer than flagella
– It is not associated with motility
Two types:
Common /attachment / pili - adherence
Sex pili - conjugation
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Function of pilii
• Attach the bacteria with other bacteria or membrane
surface (intestinal lining, RBCs)
• Absorb additional O2 & nutrient
• Provide the site for the attachment of bacterial viruses
• Transfer genetic material (conjugation)  sex Pili
E.g. E.coli and N. gonorhoeae
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Pili
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Difference between flagella and Pili
Character
Flagella
Pili
Large
small
Thickness
+++
+
Origin
CM
CW
Organ of locomotion
+
-
Organ of adhesion
-
+
Required for conjugation
-
+
Size
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D.
Any Questions
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6.Microbial growth and reproduction
6.1. Definition
– Growth is the coordination of physical and chemical
process in the cell that ideally results in cell division.
– Bacteria can grow in a various nutrient containing
preparations called culture media.
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6.2. Factors affecting microbial growth
– External physical and chemical factors can affect
growth.
6.2.1.Chemical factors
– Chemical substances which are necessary for
growth are nutrients which are available in the form
of organic and inorganic substance or
combination of both.
– The chemical components that are universally
required by all living things are:
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Water
– It is important to dissolve the nutrients so that
materials can be easily transported across the
cytoplasmic membrane in to the cell.
Carbon
– The major element in protein, carbohydrate and fat
Nitrogen
– It is also the major element in protein, purine and
pyramidine. Bacteria can get nitrogen from NH3,
NO3, N2 and protein
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Phosphorus
– It is supplied to microbes in the form of in organic
phosphate and important for the synthesis of nucleic
acids (DNA, RNA)
Minerals
– Minerals such as calcium, magnesium potassium and
iron are needed by all bacteria other minerals such as
sodium, zinc cobalt molybdenum copper manganese
are usually required in trace or small amount.
– Based on their requirement for bacterial growth
nutrients can be grouped as
– Macronutrients - C, H, O, N, K, Ca, P, Mg, S
– Micronutriments- Fe, Cl, Cu, Mn, Zn, Mo, B
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6.2.2. Physical factors
6.2.2.1. Oxygen
– Oxygen plays an important role for growth of
microorganisms because it is the final electron
acceptor in respiration.
– Many of the microorganisms contain the enzyme
that can reduce molecular oxygen in to water and
toxic products such as hydrogen peroxide and
super oxide.
– Based on their oxygen requirement micro
organisms are divided into four groups.
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Obligate aerobes
• This group of organisms is totally dependant on
oxygen requirement and they can tolerate high
concentration of oxygen since they contain the
enzyme catalase and supper oxide dismutase
Microaerophils
• They require small concentration of oxygen and
they can’t tolerate large amount of O2
Facultative anaerobes
• They can grow in the presence or absence of
O2 and they can grow best under anaerobic
conditions
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Obligate anaerobes
– This group of bacteria can grow only in the absence
of oxygen
– Oxygen can be lethal for some obligate anaerobes
since they lack the enzyme catalase and supper oxide
dismutase that can reduce the toxic compounded to
non toxic form
– Certain groups of obligate anaerobes are aerotolerant.
They tolerate oxygen simply they lack the enzyme
that reduce molecular oxygen to toxic radicals and
water
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6.2.2.2 Temperature
– Based on temperature requirement for optimal
growth microorganisms have been divided in to
psychrophiles, Mesophiles and Thermopiles
Psychrophiles
– Organisms that have a temperature range of growth
between -10and 20 oC They are found in aquatic and
soil environments of the temperate region and as
well as extremely cold area of the earth.
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Mesophiles
– Organisms that prefer growth at a temperature range of 3037oC. Pathogenic microorganisms are grouped under this
group
Thermopiles
– Organisms that prefer a temperature of growth between 45
and 70 oC They are found in hot sulfur springs and hotter area
of the world.
– They are not pathogenic
– Minimum temperature is the lowest temperature at which the
organism can able to grow; optimum temperature is the
temperature at which the growth is best.
– Maximum temperature is the highest temperature at which
growth is possible and beyond which growth is not possible.
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6.2.2.3. Hydrogen ion concentration
– For each species organisms there is a certain degree of
alkalinity & acidity at which growth is most rapid
– Most bacteria grow optimally in pH range of 7.1-7.4
(Neutrophiles).
– Some bacteria grow best at low PH (acidophiles)
– Some other bacteria grow best at higher PH (alkaloph
iles)
6.2.2.4. Radiation
– Requirement of light is a property of the photosynthetic
bacteria
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6.2.2.5. Osmotic pressure
– The concentration of salt in a medium is an important factor
controlling the growth of organisms
– If the salt concentration out side the cell is higher than the
inside, water will flow from the cytoplasm of the cell to out
side. As a result the cell becomes shrinking and desiccated.
or it may burst if the condition is hypotonic.
– Cell Wall of the bacteria can resist a change in osmotic
pressure however; extreme osmotic pressure can result in
microbial death. Bacteria that can grow at high salt
concentration are referred as osmotolerant.
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6.3. Reproduction (growth)
– When placed on favorable condition population of bacteria
can increase at remarkable rate
– Growth in bacteria is an increase in no or microbial
mass rather than in their size
– Most bacteria reproduce by Binary fission
– Some reproduce by Budding & fragmentation
– In binary fission a bacteria give rise to two identical
daughter cells then each has the potential to divide
again thus cell number increase exponentially.
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– The time interval for a single cell or population of cells to
double is called generation time/ doubling time.
– Generation time vary widely among organisms from 10
minutes – 24 hours.
– For bacteria the generation time is between 1- 3 hour and
24 hrs or more is especially for algae, protozoa and few
bacteria.
– As one bacteria doubles to become two, which then
multiply to become 4 and so on . The number of bacteria
n in any generation can be expressed as:
• 1st generation n = 1*2= 21
• 2nd generation n = 1*2*2=22
• 3rd generation n = 1*2*2*2=23
• Xth generation n= 1*2x= 2x
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6.3.1 Bacterial growth curve
– A population of bacterial population goes through
a number of phases for a time it introduced into a
medium until it ceases growth when this phase are
graphed they produces a typical growth curve.
– The curve contains 4 phase
 Lag phase
 Log phase
 Stationary phase
 Death phase
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A. Lag phase
– A period of adjustment to the new physical and
chemical environment.
– Also called Latent phase.
– Occurs when an organism is transferred in to a fresh
Medium or from a rich culture to a poorer medium.
– Major events:
– There is a lag in cell division and no increase in cell
numbers
– Period of adaptation & acclimatization (adjustment)
– It is the period for Synthesizing DNA, RNA, Structural
molecules and enzymes needed for cell division
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B. Log phase
– A phase next to the lag phase in which the
population of cells is dividing in logarismic or
exponential fashion.
– Also called Exponential phase
– Cell divides at maximum rate
– Cells in this phase are Younger, Smaller,
Physiologically active and More virulent.
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C. Stationary phase
– The population remains constant
– Reasons some cells grow while others die
– Exhaustion of nutrients
– Unacceptable modification of physical factors
– Accumulation of waste products
– Some of the important products are formed at
this stage are Spore, enzymes and Antibiotics .
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D. Death phase
–
–
–
–
–
–
–
Also called Decline phase
There is Progressive death of cells
Factors responsible for the death phase are
Accumulation of waste products
Release of lytic enzymes
Depletion of nutrients
It is Difficult to study the normal shape & structure of
bacteria at this stage.
– Old culture is not recommended for study of bacterial
morphology.
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Fig. Bacterial growth curve
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Microbial Genetics
• Genetics is the study of genes including:
– the structure of genetic materials,
– what information is stored in the genes,
– how the genes are expressed and
– how the genetic information is transferred.
– Genetics is also the study of heredity and
variation.
– Except RNA viruses – all inherited xics are
encoded in DNA.
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Microbial Genetics
• Bacteria have 2 types of DNA that contains their
genes:
– Chromosomal
– Extra chromosomal (Plasmid)
• Since the chromosome of bacteria is haploid =
alteration on the gene is very high
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Genetic variation in Bacteria
• Can occur by:
– Mutation
– Gene-Transfer
• Mutation
– is a stable heritable & irreversible change in the
nucleotide sequences
– lethal mutation
• Mutagens:
– X-rays,
– Uv-light and
– Radioactive substances
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Mutation
• Mutation can be of 3 types
– Base substitution
• Transition – eg GC change to AT
• Transversion- eg GC change to CG
– Deletion
– Insertion
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Gene-Transfer:
•General Features of Gene Transfer in Bacteria
•Unidirectional: Donor to recipient
•Donor does not give an entire chromosome
•Gene transfer can occur between species
There are three types of gene transfer that alter the DNA gene
content of bacteria
A. Transformation
B. Transduction
C. Conjugation
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Transformation
• A process by which a bacterium acquire DNA fragments or
genes from surroundings.
• Usually this occurs in microbial culture.
• Fig. Transformation process in bacteria
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Transduction
• is a method of gene transfer in which a virus (phage) acts as a
vehicle for carrying DNA from a donor bacterium to recipient
bacterium.
Fig.
Transduction of a chromosomal DNA sequence (a) and a plasmid (b). Meki121
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Conjugation
• A process where by DNA is transferred from one bacterium to
another by cell to cell physical contact
• Plasmids are the genetic elements most frequently transferred
by conjugation.
Fig. Transfer/replication process of a conjugative plasmid.
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Bacterial Conjugation
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Identification and Diagnosis of
bacteria
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Major XICs of bacteria used for identification &
classification:
1.Morphology
2.Motility
3.Staining
4.Nutritional requirements
5.Oxygen requirements
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Techniques of bacterial diagnosis
 Microscopic examination
 Culture and sensitivity
 Serology test
 Molecular technique
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Principle and type of microscope
• A microscope is a magnifying instrument.
• The magnified image of the object (specimen) is first
produced by a lens close to the object called the
objective.
• A second lens near the eye called the eyepiece enlarges
the primary image, converting it into one that can enter
the pupil of the eye.
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Types of microscope
1. Standard light microscope- commonly used to
examine both stained and unstained preparations.
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Microscope…
2. Dark field microscope- light scattered bacteria appear brightly
illuminated against a dark background.
•
Used to study spirochete especially, T. Pallidum
3. Phase contraste microscope
•
A special condenser & objectives are used
4. Fluorescence microscope
•
Use UV-light
•
Bacteria or cells stained with auramine and these dyes alter
the wave length & become visible as bright objects against a
dark back ground.
5. Electron microscope
•
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uses a beam of electron and allows resolution of extremely
small objects e.g. 0.001µm
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A. Microscopic examination
I.
Examination of Unstained Preparations
• To determine whether the culture of bacteria that is
motile or to identify different cells and parasites.
– Commonly used unstained preparations are:
Wet mount:
– A Drop of liquid specimens or broth suspension of
culture is directly placed on a slide & covered with a
cover slip.
– Such preparation is used to observe, bacteria, other cells
and motility.
» KOH
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Microsc…
• Hanging drop: is a technique used for checking
whether the organism is motile or not.
• It is done by streaking of cover glass with petroleum
jelly using applicator stick,
• placing bacterial suspension on the cover glass, then
• Invert the well slide over the cover glass, w/c allows it
to adhere to the petroleum jelly
• Turn round the well slide that cover the cover glass
• The drop will be hanging from the cover glass in the
center
• Examine using 10x and 40x for motility
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KOH
– Unstained preparation used to investigate fungal
infections.
– Fungi are eukaryotic organisms that can be
detected in specimens taken from skin, hair or
nails.
– They can be seen by direct microscopy provided
the specimen first softened and cleared with 1020% w/v potassium hydroxide (KOH).
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II Examination of stained preparation
• Staining
– The process of providing artificial Colour for
colorless organisms.
– Staining helps to increase the refractive index of the
organism, therefore it can increase the chance of
observation and identification of microorganisms.
– The difference in morphology of bacteria is
important for identification
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Morphology includes
– Size of bacteria ranges form 0.5 to 40 um
– Shapes of bacteria are Cocci, Bacilli, and spiral
– Arrangements of bacteria can be in pairs , in
chain , in cluster and in tetrads etc
A
B
C
Fig . Different bacterial morphologies A. Cocci, B. Bacilli, C. Spiral shape
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Fig. different bacterial arrangements
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What are stains?
– Stains are salts and depends on the position of the
coloring agent; they can be grouped as basic,
acidic and neutral.
• Basic stains:
– Are stains that contain the coloring agent in the
base part and the acid part is colorless.
– Used to stain acidic components of the organism.
– Some of the basic stains are Crystal violet, basic
fuchsine and methylene blue.
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• Acidic stains:
– Are stains that contain the coloring agent in the
acid part and the base part is colorless.
– Used to stain basic components of the material to
be stained. Ex. eosin.
• Neutral stains:
– Are those stains in which both the acid and base
parts contain the coloring agent
– Some of them are Giemsa stain, Wrights stain etc
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Type of staining techniques
• Simple staining:
– a staining technique only by using a single staining
dye. There are two types of simple staining
technique.
– Positive staining- in this case the Microorganism is
stained E.g. Methylene blue, CF, Crystal violet
– Negative staining- only the background is stained
E.g. India ink preparation
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• Differential staining:
– These are stains like Gram stain, AFS.
– The two commonly used differential sating
techniques in microbiology laboratory are Gram
stain and acid fast stain
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• Gram staining technique
– Most widely used differential stain in bacteriology
levorotatory Developed by Christian Gram’s in 1884.
• Procedure
– Prepare a smear on a slide from the sample
– Air dries the smear and fix with heat or alcohol
– Place the slide on a staining rack
– Flood the smear with crystal violet for 1 minute
– Rinse & cover the smear with Gram’s iodine for 1 minute
– Rinse & cover with acetone alcohol for 30 seconds
– Rinse & Cover with safranin for 1 minute.
– Examine under the microscope first with 40x objective and
then with 100x objective.
– Result: - G+ve ---- Purple ,G-ve ----- Red
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Gram stain…
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Gram stain…
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Gram stains Pictures
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Factors that can affect a Gram stain result
• Gram positives appear negative:
– Bacterial cell wall damage due to antibiotic damage or
excessive heat fixation
– Over decolourisation
– The use of out of date iodine
– Preparation of the slide from an old culture
• Gram negatives appear positive:
– Smear too thick
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Acid fast staining (ziehl- neelsen staining)
– Most commonly used staining method for the
diagnosis of mycobacterium species.
– Unlike most other bacteria, Mycobacterium species do
not stain by Gram stain.
– Once the Mycobacteria are stained with primary stain
they can not be decolorized with acid, so named AFB.
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• Procedure:– Prepare smears of sputum on a clean slide
– Dry in air, fix by passing over the flame
– Cover the smear with carbol fuchsine for 5 minutes
– Heat the slides from underneath with sprit lamp until
vapor rises.
– Rinse off the stain with clean water & cover the with 3%
acid alcohol for 2 minutes or 25% H2SO4
– Rinse & cover with 0.1% methylene blue or malachite
green for 1 or 2 minute(s).
– Examine under the microscope first with 40x objective
and then with 100x objective
– Result: Tissue & other organisms- blue or Green AFBRed bacilli
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AFB
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Microscopic Reporting of AFB
• Reporting system
–1-10 AFB/100feild =+1
–11-100 AFB /100feild =+2
–1-10 AFB /field =+3
–> 10 AFB /field =+4
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Special Staining Methods
1.Spore staining
2.Capsule staining
3.Flagellar staining
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7.1. Culture
– The process of seeding of microorganisms in
artificial medium invitro is referred as culturing.
– The medium that support growth is called culture.
– Culture is important for isolation, identification of
microorganisms and to perform antimicrobial
susceptibility test of the isolated organism.
– Bacteria grow well in vitro on artificial media.
However, different species have different growth
requirements.
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• Common ingredients of culture media
– Water
– mineral salts
– carbohydrates
– Peptone
– meat extract (lamb lacko)
– yeast extract
– Agar- inert carbohydrate derived from sea weed
and it has a Unique property (it melts at 900C and
solidifies at 40 0C)
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Types of culture media
– Basic /Simple / All purpose media
– Enrichment media
– Enriched media
– Selective media
– Differential media
– Transport media
• Form of culture media
– Solid culture media
– Semisolid
– Fluid culture media
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Serological Test
• Serology refers to the measurement of host-derived
antibodies to the pathogen of interest.
• Typically, two classes of antibodies are measured:
IgG and IgM.
• The latter represents acute antibody responses to a
novel antigen, usually peaking within a few weeks of
exposure and generally disappearing by 6 weeks after
infection.
• Positive IgG titers typically persist for many years.
• It is the standard method of diagnosing Treponema
pallidum infection
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Serology…
• Previous T cell encounters can be determined by skin
testing (a measure of delayed-type hypersensitivity).
• The most common method of skin test is:
• The tuberculin skin test, purified protein derivative
(PPD) test, or Mantoux test.
– In this test, 0.1 mL of PPD is injected subcuta
neously and the injected area is examined 48 to 72
hours later for induration.
– A reading of greater than 15 mm is considered
positive in all individuals.
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Uses of Serological Tests in Microbiology
– Used for identification of microorganisms
– Ag-Ab detection
– Used when organisms are not cultured
– For serotyping and species identification of bacteria
– Fluorescent Ab test in Syphils
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Molecular
• Composition of genetic material (DNA) is unique to
each species.
• Thus by determining the base composition &
comparing the cytosine-guanine ratio the degree
similarity can be determined.
• PCR technique
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Control of
Microorganisms
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• There are four different methods of controlling
microorganisms
– Public sanitation measures
– Proper sterilization and disinfection
– Chemotherapeutic gents
– Defense mechanism of the body
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8.1. Public sanitation measures
– Health education
– House hold hygiene
– Adequate and clean water supply
– Proper waste disposal
– Insure safe food preparation techniques
– Chlorination of water
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Definition of terms:
Sterilization:
 Killing or removing all forms of microbial life in a
material or an object.
 involves the removal of vegetative or endospores.
Disinfection:
 Reducing the number of pathogenic mo’s capable
of giving rise to infection.
 Usually involves the removal of Vegetative form
pathogens.
 May not be effective in
spores.
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Disinfectants:
 antimicrobial agents applied on an inanimate
object that destroys harmful organisms except
spores.
Antiseptics:
 chemical compound that can be used on the
surface of living tissue to inhibit bacterial growth.
Degerming: Mechanical removal of most microbes in
a limited area.
Example: Alcohol swab on skin.
 Sanitization: Use of chemical agent on foodhandling equipment to meet public health standards
and minimize chances of disease transmission.
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Example: Hot soap &Meki
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Bacteriostatic Agent: An agent that inhibits the
growth of bacteria
Germicide: An agent that kills certain mo’s.
o
Bactericide: An agent that kills bacteria. Most
do not kill endospores.
o
Viricide: An agent that inactivates viruses.
o
Fungicide: An agent that kills fungi.
o
Sporicide: An agent that kills bacterial
endospores & fungal spores.
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Rate of Microbial Death
 Several factors influence the effectiveness of
antimicrobial treatment.
1. Number of Microbes: The more microbes present, the
more time it takes to eliminate population.
2. Type of Microbes: Endospores are very difficult to
destroy.
3. Environmental influences: Presence of organic
material (blood, feces, saliva) tends to inhibit antimicrobials,
pH etc.
4. Time of Exposure: Chemical antimicrobials and
radiation treatments are more effective at longer times
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Methods of sterilization &
disinfection
• Physical
• Chemical
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1.Physical Methods of Microbial
Control:
A. Heat
• Kills microorganisms by denaturing their
enzymes and other proteins.
• Heat resistance varies widely among microbes.
 There are two forms of heat sterilization
 Dry heat
 Moist heat
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Dry Heat:
Kills by oxidation effects.
 Dry heat sterilization requires higher temperature
and often takes longer than moist heat sterilization.
 The longer time and higher temperature require in a
dry heat sterilization is because heat in water can
ready transfer to cold object than heat in air.
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Incineration:
Effective way to sterilize disposable items (paper
cups, dressings) and biological waste.
Hot air oven:
It is used to sterilize dry glass ware, metal
instrument, test tubes etc using an oven at 160 o C
for at least 2hrs or 170 oC at least for 1 hr.
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Flaming:
 It is a technique of passing an object over a flame
with out allowing it to become red hot.
 Used to disinfect glass slides, mouth of culture test
tubes etc.
Red hot:
 Sterilization of an object by holding them in a flame
till they become red hot
 It is used for sterilizing needles and inoculating
wires
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Moist Heat:
 Sterilization technique by using steam generating
devices
 Kills microorganisms by coagulating their proteins.
 In general, moist heat is much more effective than
dry heat.
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Boiling:
Boiling at 100Oc for 5 minutes can kill all
vegetative forms of microbes
 Boiling is not sporocidal rather it is an effective
means of physical disinfection.
 Not safe to sterilize surgical and dentistry
instruments
 Used to disinfect cups and plates.
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Pasteurization:
 Developed by Louis Pasteur
 A process that uses relatively brief exposure to
moderately high temperature to reduce the number of
viable microorganisms to eliminate human pathogens
 Prolong the shelf life and ensure safety of the food.
 Heating milk at 63 0C for 30 minutes or at 72 0C for 15
seconds kills all pathogenic bacteria likely to be present
in milk.
 M. bovies
 Salmonella
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 Shigella
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 Does not kill heat resistant vegetative mo’s and
spores.
 Used to reduce microbes responsible for spoilage of
beer, milk, wine, juices, etc.
 Classic Method of Pasteurization: Milk was exposed to 65oC
for 30 minutes.
High Temperature Short Time Pasteurization (HTST):
Used today. Milk is exposed to 72oC for 15 seconds.
 Ultra High Temperature Pasteurization (UHT): Milk is
treated at 140oC for 3 seconds and then cooled very quickly in a
vacuum chamber.
Advantage: Milk can be stored at room temperature for
several months.
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Tyndalization:
• It is the process of intermittent steaming at 100 0C
for 30 minutes for three consecutive days.
• First steaming will kill all vegetative forms
• Second steaming then kills the germinated spores.
• Finally the third steaming enables complete
removal of microorganisms.
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Autoclaving:
– The commonly used sterilization method
– Ordinarily performed in a seal chamber called
autoclave
– Heating at 1210C 15 Ib pressure for 15 minute in
an autoclave
– It can destroy any form of life be it spore or
vegetative.
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Factors affecting sterilization by heat
• Nature of the heat (dry or moist)
• Temperature and time
• The type and number of microorganisms
• The developmental stage of the microorganism
• The type of material from which the organism is
to be eradicated
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B. Radiation
• Gamma radiation and x-ray
– They are called ionization radiations
– They have high penetration power
– They kill microorganisms by producing free
radicals that are toxic and used to sterilize:
– Pharmaceutical products such as hormones,
antibodies and enzymes.
– Heat sensitive articles such as surgical suture ,
disposable plastic syringes and catheter
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• UV- radiation
– They can disrupt DNA
– They are powerful germicides that kill mo’s near
or on the surface of clear solution and also on
bench tops.
– Used to sterilize large room
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C. Filtration
 Removal of microbes by passage of a liquid or gas
through a screen like material with small pores.
 Used to sterilize:
 heat sensitive ingredients like blood, serum,
vaccines, enzymes, antibiotics, and some culture
media.
 injection fluid and IV fluids
 used to separate toxin from bacterial cells
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Face masks used to prevent the exchange of mo’s
between people and surrounding environment.
 Microorganisms can be removed from air by
passage through High Efficient Particulate Air filter
(HEPA-filter).
 Used in:
Pharmaceutical preparation room
Operation Theater
Bacteriology laboratory
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Low Temperature
 Refrigeration: Temperatures from 0 to 7oC.
Bacteriostatic effect.
 Freezing: Temperatures below 0oC.
Flash Freezing: Does not kill most microbes.
Slow Freezing: More harmful because ice
crystals disrupt cell structure.
Over a third of vegetative bacteria may survive 1
year.
Most parasites are killed by a few days of freezing.
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Dessication
 is the state of extreme dryness, or the process of
extreme drying.
 A desiccant is a hygroscopic (attracts and holds
water) substance that induces or sustains such a state
in its local vicinity in a moderately sealed container.
 In the absence of water, microbes cannot grow or
reproduce
 Susceptibility to dessication varies widely:
 Neisseria gonnorrhea: Only survives about one hour.
 Mycobacterium tuberculosis: May survive several months.
 Viruses are fairly resistant to dessication.
 Clostridium spp. and Bacillus spp.: May survive decades.
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Osmotic Pressure
 The use of high concentrations of salts and sugars in
foods
 Yeasts and molds: More resistant to high osmotic
pressures.
 Staphylococci spp. that live on skin are fairly
resistant to high osmotic pressure.
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2.Chemical Methods of Microbial
Control
 Chemicals that can destroy microorganisms
are termed as antimicrobial agents
 Chemical substances are used to destroy mo’s
by:
 Coagulation of bacterial population
 Disruption of cell membrane
 Oxidation of bacterial protoplasm
 Affecting the bacterial enzymes
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Types of Disinfectants
1. Halogens
A. Chlorine:
 Kill microorganisms by disrupting membrane and
inactivating enzymes
 When mixed in water forms hypochlorous acid:
Cl2 + H2O ------>H+
+ Cl- + HOCl
Hypochlorous acid
 Used to disinfect drinking water, pools, and sewage.
 Sodium hypochlorite (NaOCl): Is active ingredient of
bleach.
 Hypochlorite solutions such as sodium hypochlorite can be
used
to disinfect room.
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B. Bromine:
 To toxic to be used around people
 Used to disinfect swimming pool
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C. Iodine:
 It is very efficient bactericidal and sporocidal
 Used in:
 Tincture


of iodine(in alcohol solution)
Combines with amino acid tyrosine in proteins and denatures
proteins.
Stains skin and clothes, somewhat irritating.
 Iodophors: in combination with organic molecules
Compounds with iodine that are slow releasing, take
several minutes to act.
 Used to kill microorganisms on the skin
 Used for preoperative skin cleaning and disinfection
 Not effective against bacterial
endospores.
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2. Phenols and Phenolics
 The oldest recognized disinfectant introduced by Joseph
Lister
 Destroy plasma membranes and denature proteins.
 It is toxic and can not be used as antiseptic
 Stable, persist for long times after applied, and remain
active in the presence of organic compounds.
3. Detergents
They do have hydrophobic and hydrophilic ends
 On the basis of their polarity they are divided in to anionic, cationic.
 Detergents disrupt plasma membrane and inactivate bacterial enzymes
 Used as surgical scrub to disinfect floor and walls, and mouth wash.
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4. Alcohols
 They are among the most effective antimicrobials used for
disinfection
 Act by denaturing proteins and disrupting cell membranes.
 Used to mechanically wipe microbes off skin before
injections or blood drawing.
 Used to disinfect, oral thermometers, cabinet surfaces etc.
 Not good for open wounds, because cause proteins to
coagulate.
o
Ethanol: Optimum concentration is 70%. On skin surface 70%
ethanol in water can kill nearly 90% cutaneous bacterial
population within 2 minutes.
o
Isopropanol: Rubbing alcohol. Better disinfectant than ethanol.
Also cheaper and less volatile.
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5. Heavy Metals
 Include copper, selenium, mercury, silver, and zinc.
 Oligodynamic action: Very tiny amounts are
effective.
A. Silver
1% silver nitrate used to protect infants against
gonorrheal eye infections until recently.
B. Mercury
Organic mercury compounds like merthiolate and
mercurochrome are used to disinfect skin
wounds.
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C. Copper
Copper sulfate is used to kill algae in pools and
fish tanks.
D. Selenium
Kills fungi and their spores. Used for fungal
infections.
Also used in dandruff shampoos.
E. Zinc
Zinc chloride is used in mouthwashes.
Zinc oxide is used as antifungal agent in paints.
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6. Aldehydes
 Include some of the most effective antimicrobials.
 Inactivate proteins by forming covalent crosslinks
with several functional groups.
A. Formaldehyde:
 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.
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B. Glutaraldehyde:
 Less irritating and more effective than
formaldehyde.
 A 2% solution of glutaraldehyde (Cidex) is:
Bactericidal, tuberculocidal, and viricidal in 10
minutes.
Sporicidal in 3 to 10 hours.
 Commonly used to disinfect hospital instruments.
 Also used in mortuaries for embalming.
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7. Gaseous Sterilizers
 Chemicals that sterilize in a chamber similar to an
autoclave.
 Denature proteins, by replacing functional groups
with alkyl groups.
Ethylene Oxide:
Kills all microbes and endospores, but requires
exposure of 4 to 18 hours.
Toxic and explosive in pure form.
Most hospitals have ethylene oxide chambers to
sterilize mattresses and large equipment.
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8. Peroxygens (Oxidizing Agents)
 Oxidize cellular components of treated microbes.
 Disrupt membranes and proteins.
A. Hydrogen Peroxide:
 It can kill anaerobic bacteria
 It is not effective against catalase producing
organisms.
H2O2 
catalase
H2O + O2
 Used as an antiseptic.
 Used to disinfect wounds, Not good for open
wounds because quickly broken down by catalase
present in human cells.
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• It is an oxidizing agent
Effective in disinfection of inanimate objects.
Sporicidal at higher temperatures.
Used by food industry and to disinfect contact
lenses.
B. Ozone:
 Strong oxidizing agent (Highly reactive form of
oxygen).
Used along with chlorine to disinfect water.
Helps to neutralize unpleasant tastes and odors.
 More effective killing agent than chlorine, but
expensive.
Made by
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C . Peracetic Acid:
 One of the most effective liquid sporicides available.
 Sterilant :
Kills bacteria and fungi in less than 5 minutes.
Kills endospores and viruses within 30 minutes.
 Used widely in disinfection of food and medical
instruments because it does not leave toxic residues.
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Factors affecting chemical
sterilization
 Concentration of the agent
 Time of exposure
 PH of the medium, Temperature and Nature of
the organism
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Sterility Control and sterility testing
Sterility Control
 A system of assuring the sterilization instrument
and process of sterilization produces a result that is
safe to use.
i. Physical indicator
 Calibration and testing of all the physical
instrument uses to monitor the process.
 Thermocouples
 Pressure gages
 Timers
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ii. The use of biological indicators
 use of standardized bacterial spores which are
prepared in the form of suspension in water or
culture medium, spore dried on paper.
 The test organism is treated with the sterilization
instrument.
iii.Chemical indicators
 Chemical indicator generally under goes melting as
color change which is important to monitor the
efficiency of the sterilizer.
E.g. autoclave tape (paper impregnates with steam sensitive chemical)
 Darkening of ink indicates effective functioning of
the autoclave.
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Sterility testing
 assesses whether a sterilized pharmaceutical and
medical product are free from contaminating
microorganism.
 There are three alternative methods
1. Direct inoculation
 inoculated the test sample directly into nutrient
medium.
 use Medias that support the growth of aerobic and
anaerobic organism.
Thyogloycolate broth  anaerobic organism
Tryptonsoya broth  aerobic organism
 Incubate the inoculated media to optimum
temperature
 Inspect growth of Microorganisms
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2. Membrane filtration
– Any microorganism present is retained on the
surface of the filter (pore size of 0.45nm).
– The filter is subdivided aseptically and portion are
transferred in to a suitable culture media.
– Finally incubated at appropriate temperature and
inspected for growth.
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3. Addition of culture media to the entire
container
 A sensitive method to detect low level of
contamination in intravenous infusion fluids
 A concentrated culture media is added to the fluid
in its original container then incubated and
growth of microbes is inspected.
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2.1. Antimicrobial agents and drug
resistances
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Antibiotic
– Antibiotic are microbial produced substance
or derived from natural source that can
inhibit or kill another microorganisms
– Antibiotic are made in nature by various
microorganisms and inhibit the growth of
other organisms
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Anti-Microbial Agents
• Includes:
–Antibiotics
–Chemical antimicrobials
• Antibiotics- produced by living mo’s
E.g. Polymyxin
Streptomycin
Gentamycin
• Chemical antimicrobial drugs- produced synthetically
E.g. Chloramphenicol
Sulfonamides
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Characteristics of antibiotics:
 Based on their effect on
microorganisms
• Bactericidal –killing agent
• Bacteriostatic-inhibit growth
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Based their spectrum of action
– Broad spectrum-if effective against wide
range of both GM+VE and GM-VE bacteria
– Narrow spectrum–if effective mainly against
gram positive or gram negative bacteria
– Limited spectrum- if effective against a
single organism or disease
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• Clinically important antibiotics should have the
following feature
– Non toxic to the host
– Have wide spectrum
– Non allergic to the host
– Able to reach the infected site of the body
– Chemically stable (longer shelf life)
– Inexpensive and easy to produce
– Water soluble
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• Mechanism of action of antibiotics
Fundamental way that antimicrobials can work
as therapeutic agents of infectious disease
1.Attack bacterial cell wall synthesis, leads to
bacterial lysis.
E.g. Penicillin, cephalosporin, vancomycin
2.Interfere with protein synthesis- this occurs at
the level of translation and bacterial growth
will be arrested.
E.g.Amynoglycoside,tetracycline,
erythromycin and chloramphenicol
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3.Damaging cell membrane and Disorganizing the
structure or inhibiting the function of the cell
membrane leads to lose of cell content and death.
E.g. Polymaxin and Amphoteracin B
4.Interfere with nucleic acid synthesis: Binds with
DNA or RNA to block transcription and this prevent
the growth of cells.
E.g. Quinolone, Rifampicin and Naldixic acid
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5.Inhibition of essential metabolic path ways that
exist in the bacteria but not in the host
• Inhibition of folic acid metabolism
• Inhibition of mycolic acid synthesis
• Inhibition of nucleotide synthesis.
E.g. Sulphanilamide, Isoniazid, thrimetoprim
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Bacterial drug resistance
– Bacterial drug resistance may be come:
– Innate: due to variation in the structure of their
envelope
– Phenotypic (acquired ): resulted from adaptation to
grow within a specific environment.
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• Factors that can contribute towards drug
resistance includes:
– Incorrect diagnosis
– Unnecessary prescriptions
– Improper use of antibiotics by patients
– Impregnation of house hold items and children’s
toys with low level of antibiotics
– The use of antibiotics as live stock food additive
for growth promotion
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– Once resistant gene is generated bacteria can then
transfer genetic information in a horizontal fashion
by acquisition of:
– Conjugative plasmid
– Recombination of foreign DNA in to their
chromosome
– Mutation at different chromosomal loci.
– Genetic exchange is likely to occur in soil, general
environment and at the gut of human and animals.
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Mechanism of resistance
• Antimicrobial drug resistance of an organism can
occur by one or more of the following mechanisms.
– Production of enzymes that can destroy or inactivate
antimicrobial
E.g. Resistance to penicillin
– Altering permeability of bacterial cell membrane
E.g. Resistance to tetracycline, Polymaxin
– Developing an altered structural target to the drug
E.g. Resistance to amino glycosides and
erythromycin
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– Developing an altered metabolic path way that by
passes the reaction inhibited by the drug.
E.g. Resistance to sulphonamides
– Developing an altered enzyme that still perform
metabolic function but much affected by the drug.
E.g. Resistance to trimethoprim
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Drug susceptibility testing
• Susceptibility tests are important to evaluate፡
 Antibiotics which can be used in the treatment of
specific infectious disease.
 know sensitivity of an organism to known
concentration of antibiotics.
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Two methods:
1. Qualitative test
Disc diffusion(most common)
Procedure
i. Standard inoculums quantity of the test organism is
seeded on appropriate agar plate.
ii. Filter paper disc impregnated with known amount of
antibiotics are paced on the seeded agar plate.
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Ctd…
iii. After 24 hrs incubation at 35-37oC the activity of the
antibiotic will be determined by measuring the width
of zone of inhibition around the disc.
iv. Any zone of inhibition should be measured and
compared with the known standard
v. The organism under testing then defined as susceptible,
intermediate and resistant based on the standard.
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• Susceptible: when the growth of the organism is
inhibited at a distance from the disc.
• Intermediate: when the test result is considered to be
equivocal or indeterminate.
• Resistant: when the organism able to grow up to the
edge of the disc.
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2. Quantitative test:
In this method the MIC and MBC of the drug can be
determined.
A. Broth dilution
B. Agar dilution
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a. Broth dilution
Procedure
i. Known concentration of the antibiotic is added to a
series of test tubes or micro titration plate
ii. Standard inoculums quantity of the test organism is
seeded in to the tubes or micro titration pates
containing the antibiotics.
iii. After 24 hours incubation at 35-37oC the MIC of the
antibiotic is determined.
NB: MIC is the lowest concentration of the drug
inhibiting microbial growth.
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iv. The MBC can be determined from broth dilution (MIC)
tests by subculturing to agar plates that do not contain
the test agent.
 The minimum bactericidal concentration (MBC)
is the lowest concentration of an antibacterial
agent required to kill a particular bacterium or the
lowest concentration of the drug in the original
cultures that produce sterile culture.
 The MBC is identified by determining the lowest
concentration of antibacterial agent that reduces
the viability of the initial bacterial inoculum by
≥99.9%.
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b. Agar dilution
Procedure
i. The antibiotics at various concentration is mixed with
molten agar, poured in to Petri dish
ii. Standard inoculum quantity of the test organism is
seeded to each of the agar plate.
iii. After 24 hours incubation at 35-37oc the MIC and
MBC of the antibiotic is determined as broth dilution
technique .
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Limitations of the dilution methods
• Only one organism can be run in a single series
of test tubes
• Contamination is difficult
• expensive
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2.2. Microbial pathogenesis
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Definition:
• The interaction between the host and the
invading pathogen.
–It is bidirectional
–In brief, the host defense mechanism on
one side and the pathogens virulence and
escaping mechanism on the other side.
Host
pathogen
• The host and pathogen factors determine the fate
of the exposed individual.
Exposure
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Microbial pathogenesis
• Pathogens: are organisms capable of causing disease
• Pathogenicity: The ability of a microorganism to cause
disease.
• Pathogenesis: The mechanism and the way that result in
development of disease.
• Virulence: The degree of pathogenicity of a MO.
• Infective dose: Number of infecting MOS required to
produce disease.
• Degree of pathogensity/ virulence of an organism can
be measured:
• The number of organisms that can elicit an infection
or cause death in 50% of the test animal (infectious
dose, ID50 and lethal dose, LD50)
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– Virulence factors
• They are special properties that enhance the
ability of pathogenic microorganism to cause
disease.
• Some of the Virulence factors are:
–Adhesion factors
–Invasiveness
–Growth and survival enhancing factors
–Toxigenicity
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Bacterial virulence mechanisms
• Virulence factors help bacteria to
– invade the host,
– cause disease
– evade host defenses
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• Adhesion factors
– Enhance attachment of microorganisms on the surface of
mammalian cells to establish infection.
– Can be protein or polysaccharides
– Some of adhesive factors are capsule, flagella, Pili
•
Invasiveness
– Ability of microorganism to invade tissue and reproduce
with in the body
– Enzymes of microorganisms enhance invasiveness by
destroying different body tissue and cells.
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• Some of the enzymes that enhance invasiveness are:
– Hyaluronidase breaks down hayluronic acid
(spreading factor)
– Collagenase split collagen
– Coagulase converts fibrinogen to fibrin
– Fibinolysin catalyze the break up of fibrin clot
– Hemolysin lyses red blood cells
– Leukocidin lyses leukocytes
– Lecithinase break down phospholipids collectively
called lecithin.
– Protease, nuclease and lipase break protein, nucleic
acid and lipids respectively
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• Growth and survival enhancing factors
– Enable microorganisms to escape from host defense
mechanism and cause disease
– Capsules protects some bacteria from phagocytosis
– Siderophores enables the organism to grow in iron
deficient area
• Toxigenicity
– The ability of microorganisms to produce biological
poisons called toxins.
– Toxin can be protein (Exotoxin) or polysaccharide
(Endotoxin) in nature
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• Endotoxin
– They are structural components in bacteria which is
released mainly when the cell is lysed.
– Unlike exotoxins they are not secreted in soluble
form by live bacteria
– The prototypical example of endotoxins is
lypopolysaccharide (LPS)
– LPS consists of polysaccharide (sugar) chain and a
lipid moiety (lipid A)
– The polysaccharide is highly variable among
bacteria
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• Exotoxins
– Are protein toxin produced by growing cells.
– Are specific to the microorganism and cause
specific disease because of their mode of action
– Some of the exotoxins are:
• Neurotoxin
• Enterotoxin
• Cytotoxin
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i.
Neurotoxin
• Interferes with the function of the nervous
system
• Blocks the motor neurons not to transmit signals
to the muscles properly
• Botulism and tetanus are fatal disease due to
neurotoxin.
• Botulinum toxin
• Tetanospasmin
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ii. Enterotoxin
• Produced by various enteropathogenic
bacteria, such as
• Salmonella species
• Shigela species (shiga toxin)
• Vibrio cholera(choleragen)
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iii. Cytotoxin
• Toxins that blocks essential cellular metabolism
by destroying enzymes
• They may interfere in transcription or translation
• Hemolysin
• Leukocidin
• Aflatoxines
• Diphtheria toxin
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Major difference between Exotoxin and Endotoxin
Characteristics
Exotoxin
Endotoxin
Chemical composition
Protein
Lipopolysaccharide
Effect of heat
Labile
Stable
Action
Specific
Non specific
Antigenicity
Strong
Weak
Convertibility to toxoid
Yes
No
Produced in
GM+ve and GM –ve
Only gram negative
Toxin production
Secreted by living cells
Cell wall component of
GM- ve
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Components of the infectious process
• Depends up on the type of microorganisms the
source of infection may be
 Humans,
 Animals,
Inanimate objects and plants.
• The infectious process consists of:
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1. The agent
• Etiology of a certain disease ranges from
the smallest virus particle to complex
multicellular organism.
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2. Reservoir
• It is an organism or habitats, in which the
infectious organism normally lives, transform,
develop and multiply.
• A person who does not have apparent clinical
disease, but can be a potential source of infection
to other people are called carrier.
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• Carrier can be classified as:
Incubatory carrier - transmit the disease during the
incubation period.
Convalescent carrier - transmit the disease during
the convalescent period.
Asymptomatic carrier - transmit the disease without
ever showing its symptoms.
Chronic carrier - transmit the disease for a long
period.
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3. Portal of exit
• The way that the agent leaves the reservoir
• All body secretion, discharge, mucus, saliva,
Brest milk, vagina and tissue, cervical discharge,
excretions (faces, urine) and blood.
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4. Mode of transmission
• Mechanism by which the agent are conveyed to a
susceptible host
A. Direct transmission
• Direct contact-direct of the skin ,mucosa of the body to
the infectious agent
• Direct projection- projection of saliva by coughing,
sneezing, talking.
• Transplacental-from mother to fetus
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B. Indirect transmission
• Vehicle borne- transmission through
indirect contact with inanimate object.
• Vector borne- transmission by arthropod to
host
• Biological- if the agent multiplies in the
vector before transmission.
• Mechanical- if the vector carries the
agent on its legs, wings, and proboscis.
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C. Air borne
 occurred by dust or droplets
D. Non vector intermediate host
• Intermediate hosts that are not playing for the
transportation of the agent to human host.
• Aquatic snails in the transmission of
schistosomiasis are good example of non vector
intermediate host.
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5. Portal of entry
• Site where agents can enter in to the body such
as nasal mucosa, respiratory, anal, vaginal
6. Host
• The final link in the infectious process
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Time course of infectious disease
• Pre-patent period- the time interval between
biological onset and first shading of the agent.
• Incubation period- the time interval between
biological onset and clinical onset
• Communicable period- the interval during which the
agent is shed by the host
• Latent period - the interval between recovery and
relapse in clinical disease.
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THANK U!!!
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Basic principle of immunology
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Immunology
• Is the study of the immune defense system to foreign
entities.
Each of us is continuously exposed to mo’s from
food, water and the environment. However, we are
not continuously ill.
 Our immune system protects us against pathogens
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The outcome for exposure to infectious
agent depends on the host factors:
• Nutrition
• Age
• Gender
• Race
• Occupation
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WHAT IS IMMUNITY?
The status or quality of being immune.
Immune
- Free from possibility of acquiring a given infectious
disease.
- Resistant to an infectious disease
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Organs Of Immune System
• Primary Lymphoid Organs
– Bone Marrow and Thymus
– Maturation Site
• Secondary Lymphoid Organs
– Spleen, lymph nodes,
– MALT (mucosal associated lymph tissue)
– GALT (gut associated lymph tissue)
– Trap antigen, APC, Lymphocyte Proliferation
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Defense mechanism of the host
Two types of host defenses (resistance)
1. Nonspecific resistance
a. Effective against wide variety of organisms
b. Due to anatomical and physiological characteristics
of host
2. Specific resistance (immunity)
a.Effective against only for one microbe
b.Must be acquired following exposure to microbe
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The immune system
Overview of the Immune System
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NONSPECIFIC
RESISTANCE
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NONSPECIFIC RESISTANCE:
• They are also called innate or natural defense
mechanisms
• Represents the first line of defense
• Includes:
• Physical
• biochemical
• Cellular and
• Microbial flora
oInflammation
• Fast defense response
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1. Physical barriers
– Physically Block entry of Microorganisms
– Effective means of disease prevention
 Skin
• Tough layer or integument
• Excellent & generally impermeable barrier to invasion
of the tissue by organisms from either the normal flora
of the skin or environment
• Keratin prevents microorganisms from colonizing or
penetrating the skin
• Outer layer of the skin also contains dead cells , which
prevent infection by viruses, which require live cells
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• Cuts and wounds that break the continuity of the skin
expose the body to infection
E.g. Staphylococcus normal flora of the skin and hair
surface causes subcutaneous infection
• Some other organisms enter the circulatory system and
deep body tissue. thus, to avoid entry:
 Area of wound should be cleansed and covered with
gauze
 Care should be taken during surgical procedures to
prevent entry of microorganisms
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 Mucous Membranes
• Lines the surface of the respiratory tract, GIT, and
GUT
• Has two layers (epithelial and connective tissue layer)
• Mucus on the mucus membrane traps mo’s
• The respiratory tract (nose) have mucus membrane &
mucus, cover hairs that filter inhaled air and trap
mo’s
• Wave like motion of cilia drives mo’s upward and out
ward
• Sneezing and coughing remove mo’s from the
respiratory tract
• More penetratable than the skin
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 Fluid Flow
• Movement of fluid washes the surface of various body
tissue
• Tear - contains lysozyme , keeps the surface of the
eyes sterile
• Saliva - washes microorganisms from the oral in to the
stomach
• Urine - sterile body fluid , flashes microorganisms
from the surface of the urinary tract
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2. Biochemical defense
– Chemicals in the fluid, blood and lymph inhibit the
growth or kill potential pathogens.
Acidity
• inhibits or kills microorganisms
• on the outer surface of skin – sebum (rich in lipid)
prevents drying of the skin and hair
• indigenous mo’s break lipids  fatty acids, inhibit the
growth of mo’s on the skin.
• low pH in vagina inhibits growth of microorganisms
• low pH in the stomach–e.g. gastric acid composed of
HCl, enzyme and mucus) inhibit or kill microorganisms
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• Bile and digestive enzymes
• Microorganisms indigenous to the lower intestinal
tract  acidic fermentation products such as Lactic
acid and Acetic acid Prevents growth of mo’s.
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 Lysozyme
• Found in tears and other body fluids
• Degrades the cell wall of bacteria
• Effective against gram positive bacteria
 Iron Binding Proteins
• Transferrin and lactoferrin bind iron, limiting the
growth of pathogens in the blood
• Transferrin is found in serum and lactoferrin in tears ,
semen, bile, breast milk and in mucosal secretions
 Interferon
• Chemical substance produced by virus infected cells
and can able to protect infection of neighboring cells.
• Interferon induces the production of an antiviral
protein that blocks viral replication with in human
cells
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 Complement
• The complement system is an array of approximately 19
plasma protein and at least 9 membrane proteins that
can act both in a specific and non specific ways.
• Hepatocytes, blood monocyte, tissue macrophages and
epithelial cells of the GIT and UT are the main source of
complement proteins.
• Normally they are inactive in serum.
• Antibodies, antigen–antibody complex and surface
component of microbial cells can activate the
complement system
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• Complement proteins work together in an integrated
fashion,
• if one of the protein is activated the other also be
activated to form membrane attack complex (MAC)
• The complement system can be activated :Endotoxin  alternate pathway
Antigen- antibody complex  classical
pathway
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Function of the complement system
• Important mediator of the hummoral immune response
• Acts as chemoatractant factors stimulates migration of
phagocytic cells.
• Involved in opsonization of an antigen (C3b fragment)
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3. Cellular Barrier
• A varity of cells are involved in resistance to infection
and the non specific cellular defense focused on
ingestion and digestion of microorganisms.
i. Phagocytic cells
• Phagocytes are white blood cells involved in the
engulfment and ingestion of foreign cells followed by
distraction.
• Highly effective defense system
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ii.Polymorphonuclear granulocytes
A.neutrophiles
• Also known as Polymorphonuclear neutrophils
• Makes up 90% of the blood granulocytes
• They exhibit chemotaxis and are the first to
reach the site of infection and succumbed
quickly in the battle .
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B. Basophiles
• Makes up 1% of the total white blood cells
• Their granules contain pharmacologically active
substance such a histamine
C. Eosinophiles
• Makes up 3-5 % of the total white blood cells
• Their granule content is important to destroy
protozoa and worms
• Their granules are acidophilic and release to the out
side.
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iii Mononuclear phagocytes
• These are Monocytes
• They are larger than neutrophils and can able to move out
of the blood circulation to tissue and organs
• Depending on their location and histological appearance
they are given more specific names
Kuffer cells in the liver
Microgilial cells in the brain
Macrophages in the tissue
Alveolar Macrophages in the lungs
Langrhans cells in the skin
• They are involved in phagocytosis and antigen presenting
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Mechanism of phagocytosis
• Phagocytic cells respond to in function in a sequence of
activities chemotaxis – target recognition - ingestion –
killing and degradation
i. Chemo taxis – movement of phagocytic cells to the
site of infection in response to chemical stimuli like
anaphylatoxine, leukoterin, and peptide derived from
bacteria
ii.Target recognition – the mechanism of recognizing
the infectious agent by the aid of a receptor on their
surface
iii.Ingestion – internalizing of the infectious agent by
endocytosis
iv.Digestion – lysosomal granules containing the
enzyme acid hydrolase, myeloperoxidase and
protease kill and degrade the foreign particle
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• The mechanism of killing the foreign particle can be
either oxidative or non oxidative:
– Oxidative - based on the conversion of molecular
oxygen to highly active supper oxide molecules like
singlet oxygen, hydrogen peroxide and hydroxyl
radicals
– Non-oxidative - destruction of an engulfed
microorganism by degradative enzyme including
lysozyme, phospholipase, protease and RNAse
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4. Microbial barriers
• Normal microbial flora
• Are organisms found in the body with out causing
disease
• They are found on the skin, GIT, URT, UGT
• They do not allow exogenous pathogens to establish
themselves by:
 Production of antimicrobial substance
 Competition for available nutrients
 reducing oxygen concentration
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Inflammatory response
• Represents a generalized response to infection
• Characterized by redness, swelling, pain and elevated
temperature
• Localize invading Microorganisms and inhibit the
spread of infection
• Increases the flow of blood to the site of infection and
focuses the non specific defense systems on that
localized regions.
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Inflammation is accompanied by:
1. Edema - swelling
Plasma fluids leak from permeable capillaries
Plasma fluids accumulate in tissues
2. Pain
 Plasma fluids in tissues cause  pressure on
nerves
Injury to nerves (wounds)
3. Calor - localized heat
 Increased enzyme activity of phagocytes  heat
 Increased blood flow
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4. Hyperemia - localized redness
Localized heat
Dilation localized capillaries
5. Pus - accumulation of dead PMN’s
(if microbes/foreign material present)
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SPECIFIC HOST
DEFENSES
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Specific Defense Mechanism:
• It also known as adaptive or acquired immune
response
• The specific immune response is characterized by:
Second line of defense
Acquired or learned response, recognizes specific
substances foreign to the body
Occurs after exposure to an antigen
Specific immune response is characterized by
specificity, memory, and diversity (contain a variety of
epitopes and can stimulate the production of antibodies,
specific T cell responses, or both)
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• On the basis of substance introduced to the body in
order to enhance the immune response there are two
form of adaptive immunity.
 Active acquired
 Passive acquired
• Acquired Immunity: Immunity that an organism
develops during lifetime.
– May be acquired naturally or artificially.
• Development of immunity to measles in response
to infection or vaccination.
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Types of Acquired Immunity
I. Naturally Acquired Immunity: Obtained in the
course of daily life.
A. Naturally Acquired Active Immunity:
– Antigens or pathogens enter body naturally.
– Body generates an immune response to antigens.
– Immunity may be lifelong (chickenpox or mumps) or
temporary (influenza or intestinal infections).
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Ctd…
B. Naturally Acquired Passive Immunity:
– Antibodies pass from mother to fetus via placenta
or breast feeding (colostrum). a form of milk
produced by the mammary glands of mammals
(including humans) in late pregnancy
– Immunity is usually short-lived (weeks to months).
– Protection until child’s immune system develops.
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Types of Acquired Immunity (Continued)
II. Artificially Acquired Immunity: Obtained by
receiving
 a vaccine or immune serum.
1. Artificially Acquired Active Immunity:
– Antigens are introduced in vaccines (immunization).
– Body generates an immune response to antigens.
– Immunity can be lifelong (oral polio vaccine) or
temporary (tetanus toxoid).
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Ctd…
2. Artificially Acquired Passive Immunity:
– Preformed antibodies (antiserum) are introduced
into body by injection.
• Snake antivenom injection from horses or
rabbits.
– Immunity is short lived (half life three weeks).
– Host immune system does not respond to antigens.
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Duality of Immune System
I. Hummoral (Antibody-Mediated) Immunity
– Involves production of antibodies against foreign
antigens.
Antigens/ Immunogens
– They are agents capable of inducing the immune
response and can bind specifically to lymphocytes and
antibodies
– The degree of antigencity of a substance is determined
by
• Degree of foreignness
• Molecular weight
• Chemical complexity
All immunogens are antigens but all antigens are not
immunogens
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Ctd…
Antibody
– Are immunoglobulin produced in response to an
antigen
– Antibodies are produced by a subset of
lymphocytes called B cells.
– B cells that are stimulated will actively secrete
antibodies and are called plasma cells.
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Ctd…
– Antibodies are found in extracellular fluids (blood
plasma, lymph, mucus, etc.) and the surface of B
cells.
– Defense against bacteria, bacterial toxins, and
viruses that circulate freely in body fluids, before
they enter cells.
– Also cause certain reactions against transplanted
tissue.
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Ctd…
• Immunoglobulins have a basic pattern of four poly
peptide chains arranged in Y- shaped structure
• Two heavy chain (H-chain), and
• Two light chain (L-chain)
• The arm of the Y represents the aminoterminal (NH2)
while the tail of Y represents the carboxyl terminal
(COOH)
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Structures of AB
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Ctd…
• When immunoglobulin is treated with an enzyme it will
be broken in to two fragments:
– Fab (fragment antigen binding)- the amino terminal
of both the heavy and light chain which is important
in binding antigens
– Fc ( fragment crystalizable)- the carboxyl terminal of
the heavy chain which is important in binding specific
receptors on neutrophiles and complements.
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Ctd…
• Based on the difference in their high molecular weight
polypeptide chain there are five immunoglobulin
classes such as IgE, IgG, IgD, IgA, and IgM
-IgG- for opsonization, complement activation
-IgE- Play role in immunity against helminthes
-IgM-is made early in the course of an infection
-IgA-is important in protecting surface tissue
-Most abundant in epithelial and secretion (milk)
-IgD- B-cell antigen receptors
-IgG- is the most commonly found
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Ctd…
IgG is the most common and play a role in:
 Antibody dependent cell mediated cytotoxicity
• Fab portion binds with target cell ( mo’s or tumor
cell)
• Fc portion binds with specific receptors for Fc on
NK cells
• IgG focuses the killer cells on their target and
destroy the target cell
• This does not involve the complement mediated
killing nor phagocytosis
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II. Cell Mediated Immunity
– Involves specialized set of lymphocytes called T
cells that recognize foreign antigens on the surface
of cells, organisms, or tissues.
– The T-lymphocytes are arising in the bone marrow
and mature in the thymus. In the thymus they
acquire MHC restriction and TCR.
Two major population of T- cells:
• Helper T cells
• Cytotoxic T cells
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Ctd…
– The T-helper cells expresses CD4+ receptors and
can bind with MHC class II bearing cells
– Activated T- helper cells differentiate in to effectors
cells and produce cytokine.
– The cytokine activates B-cells and T- cytotoxic cells
– Based on the nature of the cytokine produced Thelper cells become T-helper I( for hummoral) and
T-helper II ( for cell mediated )
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Source: Abbas – Kuby. Immunology 2007
5th
ed).
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Ctd…
– The cytotoxic T-cells expresses CD8+ receptor
and they can bind to MHC class I bearing cells
– Activated T- cytotoxic cells differentiate into
cytotoxic T-lymphocyte and they can act against
• Altered self cells
• Virus infected cells
• Tumor cells
• Cells of foreign tissue graft
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Ctd…
– Cytotoxic cells release perforins which can
perforate the cytoplasmic membrane which leads
to alteration of the permeability of the membrane
and lysis of cells
– Other subset of T- cell are suppressor T-cell and
delayed type hypersensitivity cells
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Ctd…
– T cells regulate proliferation and activity of other
cells of the immune system: B cells, macrophages,
neutrophils, etc.
– Defense against:
• Bacteria and viruses that are inside host cells
and are inaccessible to antibodies.
• Fungi, protozoa, and helminthes
• Cancer cells
• Transplanted tissue
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Hypersensitivity reactions
• Immunity other than protection produces damage and
fatal result.
• Antigens that cause hypersensitivity or allergic
reaction are called allergens.
• Hypersensitivity is an exaggerated/ inappropriate
immune response that leads to the death of the host.
• Depending on the type of mediator of allergic
reactions there are four types of hypersensitivity
reactions.
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1.Type I hypersensitivity ( anaphylactic
hypersensitivity)
– Also called immediate hypersensitivity since it occurs
with in 5 to 30 minutes after exposure to an antigen.
– When IgE binds with FC receptor on mast cells and
basophiles become degranulated, histamine and
prostaglandin will be released
– Some of the examples of type I hypersensitivity
reactions are:
– Rhinitis (hay fever)
– Asthma
– Food allergies
– Drug allergies
– Insect bite
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2. Type II hypersensitivity
– It is mediated by an antibody directed towards an
antigen present on the surface of cells
– Three different antibody dependant mechanisms
involved in this type of reaction:
• Complement dependant cell cytotoxicity
• Antibody dependant cell mediated cytotoxicity
• Antibody mediated cellular dysfunction (eg.
myasthenia gravis) is a chronic autoimmune neuromuscular
disease characterized by varying degrees of weakness of the skeletal
(voluntary) muscles of the body
– The common type of reactions are blood transfusion
reaction, Rh incompatibility, drug induced hemolytic
anemia and auto immune hemolytic anemia
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3. Type III hypersensitivity
–
Known as immune complex hypersensitivity since it is mediated
by immune complex deposit.
–
Antigen antibody complex form micropricipitate which can block
small blood vessels and inflame tissue.
–
The immunological response to remove the complex may lead to
destruction of cells.
–
Autoimmune disease like rheumatoid arthritis and lupus
erythematus are good example of type III hyper sensitivity
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4. Type IV hypersensitivity
– It is mediated by specifically sensitized Tlymphocyte
– T-cells release soluble mediators some of which
attract and activate monocyte and macrophage.
– The antigen eliciting this type of response may be
foreign tissue, intracellular antigens (virus,
mycobacteria, and fungi) and chemical substance.
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– The most widely applied of the delayed type
hypersensitivity is the tuberculin test.
Example, Purified protein derivative when injected
induces indurated inflammatory reaction.
– It takes more than 12 hours to appear ,usually 48-72
hours after infecting with the antigen.
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Auto immune disease
• Sometimes our immune system can destroy self cells by
producing autoantibody.
• The disease is referred as autoimmune disease.
• The production of autoantibody may be due to the
following conditions:
• Alteration of surface antigens
• Cross reacting antigens
• Exposure of hidden antigen because of surgery and
injury
• Viral infection
• There are two forms of autoimmune disease both of which
are mediated by antibody, complement, cellular immunity
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A. Organ specific autoimmune disease
 The targets for autoantibody in this case are specific organs
 Some of the organ specific autoimmune diseases are:
• Auto immune Hemolytic anemia- destruction of the
red blood cells
• Myasthenia gravis - destruction of acetylcholine
receptors
• Grave’s disease -hyperthyroidism due to stimulation
by autoantibody
• Type one diabetes mellitus - destruction of beta islet
cells of the pancreas.
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B. Systemic autoimmune disease
 Circulating autoantibodies form soluble complex and
deposited in different parts of the body.
This initiates immunologic response that leads to tissue
damage
 Some of the systemic autoimmune diseases are:
• Systemic lupus erythematus - destruction of
double stranded DNA
• Rheumatoid arthritis - destruction of bone and
cartilage at the joints
• Multiple sclerosis - destruction of the central
nervous system
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Principles of Immunoprophylaxis and
therapy
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Immunoprophylaxis
– Is the method of conferring immunity to prevent
infection.
Small pox had be eradicated by the use of
immunization or vaccination.
– If large number of individuals had be immunized,
Herd immunity is achieved, and transmission of
communicable disease is interrupted
– Innate and acquired immunities are the major
mechanism for responding infectious agent
– Acquired immunity, in addition to being a natural
response to infections can be artificial, passive or
active
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– Natural acquired immunity- due to exposure to
antigen/ contact to antigen unintentionally
– Artificial acquired immunity- arises when antigen
or antibody are introduced by artificial means, by
using a vaccine or antiserum respectively
– Active immunity- individual produces antibodies
as the result of the infections (natural) or by
injecting vaccines
– Passive immunity- antibodies are injected either in
the form of antiserum or immunoglobulin that
were obtained from animals or other humans,
artificially acquired passive immunity
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Vaccines
• Vaccines are preparation of antigen suspensions when
injected can stimulate the immune system to produce
memory cells.
Type and composition of vaccines :
Toxoids
• Inactive form of a toxin produced in the laboratory by
denaturing toxin of microorganisms
• Exotoxins of tetanus and diphtheria can be converted
in to non toxogenic form and can be used as vaccines.
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•
•
•
•
•
Whole cell killed vaccines
vaccines consist of suspension of inactivated intact
microorganisms
E.g. Whooping cough, Typhoid fever, Plague
vaccines -may produce serious side effects
Attenuated vaccines
living microorganisms that lack the ability to cause
disease through laboratory processing and can still
multiply in the host are used
the process of weakening the live organisms is
known as attenuation
Induce higher and long lasting levels of immunity
than do non living organisms.
Attenuation process commonly involves adapting
microorganisms to conditions they do not face in the
host. E.g. growing poliovirus in monkey tissue
culture.
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Purified antigens
• Intact microorganisms have various antigens and
purifying the antigen gives an effective vaccine.
• This is the sub-unit vaccine
• Capsular polysaccharide vaccines of streptococcus
pneumonia and Haemophilus influenza are good
example of sub cellular vaccine.
Recombinant vaccine
• The segment of the gene that codes for the specific
antigen is inserted in to bacteria, yeast, or animal
cells- large quantity of antigens can be produced.
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Immunotherapy
• Antisera developed in animals or gamma globulins
obtained from humans are used to provide temporary,
immediate protection  passive immunotherapy
• Animal antisera in vivo have short half life  cause
anaphylactic reactions on the second exposure.
• Human gamma globulins  few side effects
• Not eliminated as rapidly as animal antisera.
• Hence attention is given for immunotherapy because
of its ability in treating and preventing infections in the
immunocompromised individuals.
• Interferon, interleukins, haematopoietic cytokines and
monoclonal antibodies are now available for
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prevention and treatmentMekiofD. infections.
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Assignment
1.What are nosocomial infections and write the factors
important in nosocomial infections to occur?
2.Discus the prevention and control of nosocomial
infections
3.List and describe types of culture medias
4.Discus how gene is transferred b/n bacteria
5. Protect susceptible population
–Active immunization
–Passive immunization
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