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DIRE DAWA UNIVERSITY

COLLEGE OF MEDICINE AND HEALTH


SCIENCES

Medical Microbiology
for BSc Midwifery Students

Instructor: Robel M (MSc in Medical Microbiology)

1
Objectives
At the end of the session, students will be able to:
State the Definition and History of microbiology
Describe the scopes of microbiology
Describe the germ theory of disease
Describe distribution of microorganisms
Explain purpose of studying microorganisms
2
Definitions and scope of microbiology
Micro (too small), Bio (life) & Logy (study)
Microbiology is the branch of biology that deals with
microorganisms
Study of organisms too small to be clearly seen by unaided
eye
It deals with form, structure, reproduction/growth,
metabolism, genetics, transmission, pathogenesis,
identification, treatment and prevention

3
Definitions and scope of
A microorganism or microbe comprises either single
cell (unicellular), cell clusters (multicellular) or no cell
at all (acellular)

Groups:
Bacteria
Virus
Fungi, and
Protozoa & Helminthes
4
Before the discovery of microorganisms, many
felt the world filled with invisible spirits which
would explain things we couldn't understand
Need to know the history of microbiology..

5
History of Microbiology

Important Early Microbiologists:


It was Anton van Leeuwenhoek, a Dutch cloth merchant and
amateur lens grinder, who first made and use lenses to
observe living microorganisms
Introduce simple microscope
First microbiologist
The lenses Leeuwenhoek made were of excellent quality; some
gave magnifications up to 300X
Leeuwenhoek saw bacteria, protozoa, yeasts and described all
the microbial forms we now know, except for viruses
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Some branches of microbiology

Medical microbiology
is both a branch of medicine and microbiology which
deals with the study of microorganisms including
bacteria, viruses, and fungi which are of medical
importance
It includes the study of microbial pathogenesis and
epidemiology, and is related to the study of disease
pathology and immunology
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Pharmaceutical microbiology:
The study of microorganisms that are related to the
production of antibiotics, enzymes, vitamins, vaccines,
and other pharmaceutical products and that cause
pharmaceutical contamination and spoil
Industrial microbiology:
The exploitation of microbes for use in industrial
processes
Examples include industrial fermentation and waste
water treatment
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Theories Of Origin Of Life
Where do microorganisms lives or come from?
In ancient times, there were two contradictory
theories of origin of life
1. Theory of abiogenesis
versus
2. Theory of biogenesis

9
Pasteur's Experimental Equipment
Experiment performed by Pasteur clearly overturned the
theory of spontaneous generation at the microscopic level
He boiled a meat broth in a flask that had a long neck that
curved downward, then upward, like a goose neck
The bend in the neck prevented contaminating particles from
reaching the broth, while still allowing the free diffusion of air
The fact that the flask allowed for the passage of air was a design
breakthrough that finally addressed the critics of Spallanzani
Pasteur's flask remained free of bacterial growth for as long
as the flask remained upright
Pasteur demonstrated that microorganisms are present in air but not
created by air
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Pasteurs swan-necked flasks: remained upright

17
Pasteur's Experimental Equipment.
To show where the contaminating elements were
located, he tipped the flask enough for the broth to
sweep out the bend in the goose neck; the broth would
then quickly become clouded with bacterial growth
Pasteur demonstrated that microorganisms are present
in air but not created by air
This was critical for refutation of the concept of
spontaneous generation and for the development of
germ theory of diseases

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Pasteurs swan-necked flasks: tilted

19
Germ theory of disease
The germ theory of disease proposed that
microorganisms are the cause of many diseases
Although highly controversial when first proposed, germ
theory was validated in the late 19th century
It is now a fundamental part of modern medicine and
clinical microbiology, leading to such important
innovations as antibiotics and hygienic practice

21
Robert Koch
Developed Postulates which are a sequence of
experimental steps for directly relating a specific
microbe to a specific disease
Koch's postulates are four criteria designed to establish
a causal relationship between a causative microbe and a
disease
Koch applied the postulates to establish the etiology of
anthrax and tuberculosis, but they have application to
others

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Koch's postulates

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Robert Koch (1843-1910)
Father of Bacteriology
Established the relationship
between B. anthracis and anthrax

25
Exceptional to Kochs postulates
1. Many healthy people carry pathogens and dont exhibit
symptoms of the disease. E.g. HIV
2. Some microbes are very difficult or impossible to grow in
the laboratory e.g M. leprae, T. pallidum
3. All disease is not caused by microorganisms. e.g. scurvy
4. Some diseases may be caused by mixed infection with
more than one microbe
5. There may be ethical objections to infect subjects with
infectious agents

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Distribution of microorganisms

Found every where/ cosmopolitan


Found in the air we breath, water we drink, on or
in our body, soil and others

What characteristics make them found every where?

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Characteristics which make them found every where:
I. They exist in different forms (spore, cysts)
II. They are small in size
III. Easy adaptation to physical and chemical factors
IV. They have rapid reproduction
V. Easy transmission
VI. They have diverse metabolism (have different
enzyme)

Why we study about microorganisms??


30
Why study about microorganisms??
1. Beneficial to man
Microbiology as a BASIC Science
Bacteria and yeast are useful in studying molecular biology,
biochemistry and genetics
reproduce rapidly
are genetically and biochemically simpler than
higher order organisms
working with bacteria and yeast for understanding
life processes has no ethical ramifications

31
Using microbes bacteria and fungi
Bacterial growth is used to make milk into yoghurt
Fungi can also be used to make food
A meat substituting protein can be produced using
fungi
Yeast is a type of fungus and carries out respiration
Respiration of this microbe can be used in different
ways in baking bread and in brewing

33
Using microbes yeast
The aerobic respiration of yeast is used to make bread rise
Yeast uses the sugar in bread dough to carry out aerobic
respiration

glucose carbon energy


oxygen dioxide water

34
Using microbes yeast
The anaerobic respiration of yeast is used to make beer
and wine
In this case, the yeast respires without oxygen and
produces alcohol (ethanol)
This process is known as fermentation
Yeast converts the sugar into alcohol by anaerobic respiration

Glucose carbon
dioxide ethanol energy

35
Why we study about microorganisms??
2. Enemy of human kind - Pathogenic
Responsible for the loss of many lives
Spoilage of foods
Economic loss (infect both animals and plants)

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Objectives
At the end of the session, the students will be able to:
Define chain of infection
List components of chain of infection
Describe source of microbes/infectious agents

37
Chain of Infection
Series of events that are necessary for the transmission
of infectious diseases

Infectious
Agent
Reservoir

Resident
Risk Factors

Exit

Entry

Transmission

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Source of Microbes/infectious agents
There needs to be someone or something carrying the
microbes that cause the infection
1. Exogenous: from outside the body
Example: some bacteria is carried to the resident via
hands of healthcare worker (HCW)
2. Endogenous: from inside or on the body
e.g. Normal flora
Definition - frequently found on or within the body
of healthy persons
They can cause disease under the right conditions
(endogenous infection/disease)
40
Reservoir
A Place where microbe grows and reproduces
1. Humans: Residents own microbial flora
other sources = healthcare workers, family, visitors

2. Animals: pets

3. Environment: food, beverages, soil, healthcare


equipment

41
Mode of Exit Microbe leaves the Reservoir
There needs to be a way for the microbes to get out of
the infected person or source
Respiratory tract: Cough, sneeze, talking
Gastrointestinal tract: vomitus, feces
Skin, mucous membranes
Genitourinary tract: Urine, semen, vaginal secretions
Blood: from a cut through the skin or contaminated
needle
Artificial openings, e.g. tracheostomy or feeding tubes

42
Transmission How are microbes spread?
The spreading of microbes and disease is known as transmission

A. Direct Transmission-
immediate transfer of the agent from a reservoir to a
susceptible host by direct contact or droplet spread
Example:
Touching
Kissing
Sexual intercourse
Blood transfusion
Trans-placental
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Transmission by contact
Many microbes can be exchanged from one person to another by
direct or indirect contact
Direct contact by hand;
Sexual contact
Indirect contact, e.g. by walking on a
wet floor already contaminated by
someone else who has athletes foot;

44
Transmission through the placenta
If the mother develops the HIV/AIDS
infection, it can be passed on to the unborn
child through the placenta

Transmission via breastfeeding


If a child is being breastfed, he or she can
also pick up microbes from the mother via the
mothers milk

45
B. Indirect Transmission-
an agent is carried from reservoir to a susceptible
host by suspended air particles or by animate (vector-
mosquitoes, fleas, ticks...) or inanimate (vehicle-food, water,
biologic products, fomites) intermediaries.
Example:
I. Vehicle-borne: food, water, towels, ...
II. Vector-borne: insect animals, ...
III. Airborne: dust, droplets
IV. Parenteral injections
46
Transmission by air
A cough or a sneeze can release
millions of microbes into the air
which can then infect somebody
else

Transmission by water
Dirty water can transmit many
diseases, e.g. cholera, which
can be transmitted by drinking

47
Transmission by animals
An animal can carry a microbe from one place to
another, e.g. a mosquito which spreads the malaria
parasite

48
Mode of Entry
Infectious agent enters the new host
Respiratory tract
Breathing contaminated air droplets
Gastrointestinal tract
Eating, drinking, hand-to-mouth (fecal-oral route)
Skin, mucous membranes
Non-intact skin
Hand-to-eye and nose
Genitourinary tract
Urinary catheter is present; bacteria move up catheter into
the bladder
Blood: Contaminated needle 49
Objectives
At the end of the session, the students will be able to:
List the basic classification systems of organisms
Mention the three domains system
Compare the features of prokaryotic and eukaryotic cells
List methods of classifying and identifying microorganisms

50
Classification Systems
Phylogenetic vs Phenetic
1. Phenetic Classification System:
Groups do not necessarily reflect genetic similarity or
evolutionary relatedness
Instead, groups are based on convenient or observable
characteristics
2. Phylogenetic Classification System:
Groups reflect genetic similarity and evolutionary
relatedness

51
Phylogenetic classification of micro-organisms
1. Prokaryotes

Eubacteria: (True bacteria)


Most abundant of the bacteria found in soil, water and
animal digestive tracts
Archaebacteria: (ancient bacteria),
Live in extreme conditions (temperature, pH etc)
Mostly anaerobic (unable to live in the presence of oxygen)

53
Phylogenetic classification of micro-organisms
2. Eukaryotes
Algae:
Live in soil and water,
contains chlorophyll for photosynthesis,
has a cell wall
Fungi (yeast, molds)
Lack chlorophyll and obtains energy from organic
compounds in soil and water,
Has a cell wall
Protozoa:
Colorless, ingests other organisms or organic particles
Lacks a cell wall, 54
Prokaryotes Lack Nucleus and Membrane-Bound Organelles

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Eukaryotes cell

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Eukaryote Prokaryote

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Comparison features of prokaryotic and eukaryotic cells
Characteristic
Prokaryotes Eukaryotes
Typical
bacteria protozoan, fungi, plants, animals
organisms
Type of nucleoid region;
real nucleus with double membrane
nucleus no real nucleus
linear molecules (chromosomes) with
DNA circular (usually)
histone proteins
RNA-
coupled in RNA-synthesis inside the nucleus
/protein-
cytoplasm protein synthesis in cytoplasm
synthesis
Ribosomes 50s+30s=70s 60s+40s=80s

58
Comparison of features.
Characteristic Prokaryotes Eukaryotes

Cytoplasmatic Very few highly structured by endomembranes and


structure structures a cytoskeleton

one to several thousand (though some lack


Mitochondria None
mitochondria)
Chloroplasts None in algae and plants
usually single single cells, higher multicellular
Organization
cells organisms with specialized cells

Binary fission Mitosis


Cell division
(simple division) Meiosis

59
Viruses
Viruses are acellular organisms
do not fit the three domain system
The three domains are:
1. Bacteria/eubacteria
2. Archea/Archeabacteria
3. eukarya

61
Methods of Classifying and Identifying Microorganisms

Identifying:
Determining what species it is
for research or disease treatment
Classifying:
To discover which group the species is most closely related
to (evolutionary relatedness)
how similar (closely related) or
how different (distantly related)

62
Methods of classifying/identifying .
1. Morphological Characteristics
Size, and shape
Presence of endospores,
Presence of flagella, capsules, pili/fimbriae
2. Differential Staining
Gram stain: divides bacteria into two large groups (into
Gram positive or gram negative)
Acid-fast stain: usually used to identify Mycobacterium
3. Biochemical Tests
Fermentation of carbohydrates
Fermentation of end products
Hydrolysis reactions
63
Methods of classifying/identifying.
4. Serological Testing
Reaction of bacteria to antibodies that are specific for
that organisms antigens
i.e. Agglutination tests: if antibody is specific for a certain
bacterium, it will cause agglutination (clumping) of the
organisms
5. Fatty Acid Profile analysis
Separate and identify fatty acids and compare to the
list (profile) of fatty acids that the suspected
bacterium can synthesize
64
Methods of classifying/identifying.
6. Phage Typing
Bacteriophages are specific to the bacterial species
that they infect (similar idea to antigens/antibodies)
If a phage that is specific for a bacterium is placed on
agar with bacterial growth, it will destroy cells where
is it placed creating a plaque
7. Molecular techniques
E.g: DNA Base Composition, Ribosomal RNA
Sequencing etc
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Objectives
At the end of the session, the students will be able to:
Describe nature of bacteria
List structure of prokaryotic/bacterial cells
Describe functions of bacterial cells
Explain nomenclature and taxonomy of bacteria
List methods of bacterial classification
Describe nutritional and atmospheric requirements for
bacteria
Define bacterial growth
List and explain phases of bacterial growth
67
Nature of Bacteria
What are bacteria ?
Small microorganisms with a simple form of cellular
organization described as prokaryotic cells
Do not contain a membrane bound nucleus
Their hereditary material is suspended in a portion of
cytoplasm called Nucleoid or Nuclear region
They are also devoid of mitochondria and other
membrane bound organelles

69
Structure and Function of Prokaryotic Cells
Prokaryotic cells have three architectural regions
1. appendages (proteins attached to the cell surface) in the
form of flagella and Pili
2. a cell envelope consisting of
capsule,
cell wall and
plasma membrane
1. a cytoplasmic region that contains
cell genome (DNA) and
ribosomes and
various sorts of inclusions
71
Bacterial Structure

72
Bacterial Pili
thin, hair-like appendages
Short protein fibers
Protrude from surface of cell
used for attachment
to other cells
to host cell or tissue for
pathogens
Conjugation pili
attachment between cells
for exchange of genetic
material

74
Function in adhesion to other cells and surfaces

75
Joins bacterial cells for DNA transfer (conjugation)

Gene transfer from a donor to a


recipient by direct physical contact
between cells
sex pili attach male to female
bacteria during conjugation
Results in one way transfer of DNA
from donor (male) cell to recipient
(female) cell through conjugative
(sex) pilus

76
Bacterial Flagella
Much longer than cell
It is thread like appendages
Organ of locomotion
Provide motility
made of protein subunit
called flagellin
Flagellins are immunogenic
(H-antigen)

77
Flagellar arrangements
Different species of bacteria have different numbers
and arrangements of flagella
Useful in identifying and classifying bacteria
A. Monotrichous single flagellum
at one end
B. Lophotrichous small bunches
arising from one end of cell
C. Amphitrichous flagella at both
ends of cell
D. Peritrichous flagella dispersed
over surface of cell 78
Importance of Flagella
Are not essential for survival of the cell
Attachment to surfaces (epithelial tissue)
Motility MB videos\Bacterial Flagellar Motility System-1.mp4
as a virulence factor
Diagnostic value and Identification
antigenic nature
Are highly antigenic (H antigens), and some of the
immune responses to infection are directed against
these proteins
arrangement and number

80
Glycocalyx (Capsule and Slime Layer)
Coating of bacteria, external to the cell wall
Made of sugars and/or proteins
2 types
1. capsule - highly organized, tightly attached
2. slime layer - loosely organized and attached,
non uniform in density and thickness

81
Functions
Attachment to other bacteria or host tissues
inhibits killing by WBCs and detergents
Receptor
Some produce biofilm that protects from
antibiotics and host defense ( tooth plaques, S.
mutans)
Capsule and slime layer -unnecessary for
growth but need for survival
important determinants of virulence

82
Cell wall
Thick and relatively rigid layer
Components are unique to bacteria
Contain strong activator of immune response
Structural components and their function used for classification
Crucial for growth and division
Supports and protect the fragile CM and the contents it encloses
Compounds that interfere with synthesis are lethal and include
many antibiotics

83
Cell wall (Gets special attention)
1. They are an essential structure for viability
2. Composed of unique components found nowhere else in nature
3. It is one of the most important sites for attack by antibiotics
4. They provide ligands for adherence and receptor sites for drugs or
viruses
5. They cause symptoms of disease
6. They provide for immunological distinction and immunological
variation among strains of bacteria e.g. LPS, M protein, TA,
84
Rigidity of cell wall is due to the presence of peptidoglycan
Peptidoglycan is
Back bone of the cell wall (gives rigidity)
Made of rope like linear polysaccharides cross links
by peptides

85
Peptidoglycan made of
1. Two amino sugars:
a. N-acetyl glucose amine
b. N-acetyl muramic acid
2. Peptides
L-alanine
D-alanine
D-glutamic acid
L-ysine or diaminopimelic acid (DAP)

86
Peptidoglycan..

Figure . Assembly of the peptidoglycan


87
Cell wall of Gram positive bacteria
Thick peptidoglycan
Peptidoglycan contains: TA, LTA, M-proteins, C-
polysaccharide
LTA anchored to the cell membrane
TA and LTA antigenic (used for classification)
- used as attachment organ (virulent factor)
- Initiate host response

88
Cell wall of Gram positive bacteria

89
Cell wall of gram negative
Structurally more complex
Contain thin peptidoglycan
Contain outer membrane
No TA/LTA
Contain periplasmic space containing:-
variety of hydrolytic enzyme which are used for
metabolism/virulence factors (phosphatase, Beta
lactamase, lipase, protease, collagenase etc.)
Transport proteins, binding proteins
91
Cell wall of gram negative .
Outer membrane contains
Lipopolysacchrides (LPS)- Endotoxin
Powerful stimulator of immune response
activate B cells, macrophages, dendric cells and
cause release of high amount of cytokines(IL-1,IL-2,
TNF-) which cause fever-shock
Structural proteins, receptors

92
Gram negative cell wall
Lipopolysaccharide

Porin proteins

Outer membrane
Periplasmic space
Peptidoglycan
Cytoplasmic membrane

Cytoplasm

93
Lipopolysaccharide = endotoxin
LPS
Commonly called endotoxin
Made of Lipid A, Core polysaccharide and O antigen
Lipid A is the toxic part and essential for bacterial
viability
The O antigens result in a large number of antigenic
variants useful in bacterial typing

94
Lipopolysaccharide = endotoxin

O-antigen

Core poly-
saccharide

Lipid A is
anchored in
outer mem-
brane (OM)

95
Cytoplasmic membrane
Lipid bilayer
Semipermeable barrier
No sterols??
Contains:
Structural proteins
Transport proteins
Electron transport
system (respiration)
Enzymes
Ion pumps
Flagellum anchor
site of DNA synthesis
96
The acid fast cell wall
Resemble Gram positive cell wall
Acid-fast bacteria have a cell wall with a relatively
impermeable cell wall containing a waxy lipid called mycolic
acid
Difficult to stain
Mycolic acids confer resistance to
Desiccation
Most antibiotics
Phagocytosis
Contributes to pathogenicity
e.g. Complex cell wall structure of Mycobacteria
97
Complex cell wall structure of Mycobacteria 98
Chromosome
Single, circular, double-stranded DNA molecule that
contains all the genetic information required by a cell

DNA is aggregated in a dense area called the nucleoid

99
Ribosomes
Made of 60% ribosomal RNA & 40% protein
consist of 2 subunits: large & small 70S
Prokaryotic differ from eukaryotic ribosomes in
size & number of proteins
Site of protein synthesis
Target of some drugs

100
Plasmids
small circular, double-stranded DNA
free or integrated into the chromosome
duplicated and passed on to offspring
not essential to bacterial growth & metabolism
may encode genes for antibiotic resistance, enzymes &
toxins
used in genetic engineering- readily manipulated &
transferred from cell to cell

102
Spore
Multilayered structure
Made by some member of
gram positive bacteria
Made in response of harsh
environmental condition
(depletion of nutrients)
withstand extreme heat,
drying, freezing, radiation &
chemicals
Not a means of reproduction
but for survival
103
Summary

107
Taxonomy
Systematic science of classification of organisms
The goal of showing relationships among
organisms
Also provides a means of identifying organisms

108
Taxonomic Hierarchy
Taxa (taxon) taxonomic category designed to show
degrees of similarities among organisms
1. Kingdom
Groups based on similarity
2. Phylum (pl: Phyla)
Higher taxa very general 3. Class
Lower taxa more restricted 4. Order
5. Family
Species single unique
6. Genus (pl: Genera)
organism group 7. Species (pl: Species
109 )
Three major areas of Taxonomy
1. Nomenclature naming of organisms

2. Classification the process of ordering of


organisms in group based on common properties

3. Identification - of unknown organisms

111
Nomenclature
Every recognized organism is given a two-part scientific
name
This system is called "binomial nomenclature
The scientific name of each species is made up of a
generic name and a specific name
The initial letter of genus is capitalized and can also be
abbreviated; species is not capitalized and abbreviated
Whole name is in italic or underlined
E.g. Klebsiella pneumoniae, or
K. pneumoniae, or
Klebsiella pneumoniae
112
Bacterial morphology
Bacteria are differentiated into major categories
based on microscopic observation of their
morphological features
1. Shape
2. Size
3. Arrangement and
4. Staining Characteristics
Most bacteria range in size from 0.2- 1.2m in width
and 0.4 -14m in length

116
Microscopic morphology and staining reactions
THE GRAM STAIN:
A staining method used to classify different species of
bacteria
It is one of the differential stains
Used for the identification of pathogens in clinical
Specimens and cultures by their gram reactions and
morphology
Differences in Gram reaction due to difference in cell
wall structure
3D-VIDEOS 4 MB\Gram Stain - ok.mp4
117
What is the Difference between Gram positive
and gram negative bacteria?
Gram positive and gram negative refers to how a
bacterium reacts to a gram stain
Gram-positive bacteria are classified as bacteria that
retain a crystal violet dye(primary stain) during the
Gram stain process
Gram-positive bacteria will appear violet under a
microscope, whereas Gram-negative bacteria will
appear red or pink
The difference in classification is largely based on a
difference in the bacteria's cell wall structure 119
Characteristics generally present in Gram-positive bacteria:
A very thick cell wall (peptidoglycan), resist decolonization by
acid alcohol
Teichoic acids are present, which serve to act as chelating agents,
and also for certain types of adherence
Characteristics displayed by Gram-negative bacteria
Cell walls only contain a few layers of peptidoglycan (which is
present in much higher levels in Gram-positive bacteria), easily
decolorized by acid-alcohol
Cells are surrounded by an outer membrane of lipopolysaccharide
outside the peptidoglycan layer
No teichoic acids are present

120
Ziehl- Neelson Staining Reactions (AFB Staining)
Used for diagnosis of Mycobacterium species
Mycobacterium, unlike other bacteria, doesnt stain by
Gram-technique
This is due to the difference in the cell composition of
Mycobacterium species from other bacteria
3D-VIDEOS 4 MB\Ziehl Neelsen (Acid Fast) staining-
1.mp4

122
AFB Staining
There are two methods in ZN:
Ziehl-Neelson methods I and II
The two methods differ in concentration of decolorizer
1. ZN-methods I: use 3% HCl, and used for MTB and M. ulcerans
staining
2. ZN-method II: used for staining of M. leprae
The differences between the acid fastnesses of Mycobacterium
species: MTB and MBU are strongly acid-fast and MBL is weakly
acid fast

126
ZN Stained Smear

128
Reporting: Grading of ZN Smears
WHO scale ZN 1000x, AFB count
Negative 0 AFB / 100 HPF
Scanty 1-9 AFB / 100 HPF
1+ 10-99 AFB / 100 HPF
2+ 1-10 AFB / 1 HPF on average
3+ >10 AFB / 1 HPF on average

129
Bacterial shapes and arrangement
Morphologically bacteria can resemble:
1. Cocci (Singular: Coccus)
2. Bacilli (Singular: Bacillus)
3. Vibrios (Singular: Vibrio)
4. Spirilla (Singular: Spirillum)
5. Spirochaetes (Singular: spirochaete)

130
Morphological features of bacteria

131
1. Cocci:
are round or oval bacteria
may form pairs, chains, or irregular groups (grape like)
cocci in pairs (diplococci), e.g. meningococci and
gonococci
cocci in chains (streptococci), e.g. Streptococcus pyogenes
cocci in irregular groups (staphylococci), e.g.
Staphylococcus aureus
Gram reaction: Staphylococci and streptococci are Gram
positive, where as diplococci can be Gram positive or Gram
negative
132
2. Rods (bacilli):
are stick-like bacteria with rounded, tapered (fusiform),
square, or swollen ends
they may:
form chains, e.g. Streptobacillus species
form branching chains, e.g. Lactobacilli
mass together, e.g. Mycobacterium leprae
remain attached at various angles resembling
Chinese letters, e.g. Corynebacterium diphtheriae
short rods with rounded ends are often called coccobacilli

133
3. Vibrios:
small slightly curved rods
e.g. Vibrio cholerae.
Gram reaction: Vibrios are Gram negative

135
4. Spirilla:
are small, regularly coiled, rigid organisms
Spirilla are motile with groups of flagella at both ends
An example of a spirillum is Spirillum minus
Gram reaction: Spirilla are Gram negative

136
5. Spirochaetes:
are flexible, coiled, motile organisms.
They progress by rapid body movements
Most are not easily stained by the Gram method

137
NUTRITION, GROWTH AND MULTIPLICATION
OF BACTERIA
Nutritional Requirements
Like other living things, bacteria need food and water
Different bacteria have different requirements for certain
substances such as vitamins and amino acids, but all
bacteria require carbon dioxide; most grow better in
neutral or slightly alkaline solutions; all need phosphates
Bacteria require water; drying will cause their death
Some bacteria are AUTOTROPHS self feeding based on
inorganic compounds (CO2)
Others are HETEROTROPHS- dependent on organic
compounds
140
Physical Requirements
Temperature
Effect of temperature on proteins
Effect of temperature on lipid-containing membranes
of cells and organelles
If too low, membranes become rigid and fragile
If too high, membranes become too fluid and
cannot contain the cell or organelle

143
Classification based on temperature
1. Psychrophiles from -5 to 15/20C
2. Mesophiles 25-45C
3. Thermophiles 45-75C
4. Hyperthermophiles 75C up to 110C

[INSERT FIGURE 6.5]

144
Optimum temperature is required for growth

[INSERT FIGURE 6.4]

145
Based on Oxygen requirements
1. Obligate aerobes undergo aerobic respiration
2. Microaerophiles aerobes that require oxygen levels from
5-10% and have a limited ability to detoxify hydrogen
peroxide and superoxide radicals
3. Obligate anaerobes do not use aerobic metabolism
4. Aerotolerant anaerobes do not use aerobic metabolism but
have some enzymes that detoxify oxygens poisonous forms
5. Facultative anaerobes can maintain life via fermentation
or anaerobic respiration or by aerobic respiration

146
Oxygen requirements

[INSERT FIGURE 6.3]

147
Based on pH
Organisms are sensitive to changes in acidity because H+
and OH- interfere with H bonding in proteins and nucleic
acids
1. Neutrophiles: are bacteria and protozoa that grow
best in a narrow range around neutral pH (6.5-7.5)
2. Acidophiles: are bacteria and fungi that grow best in
acidic habitats
3. Alkalophiles: grow best in alkali habitats

148
BACTERIAL GROWTH
Usually refers to increase in population size
Most bacteria reproduce by a relatively simple asexual
process called binary fission each cell increases in size
and divides into two cells
During this process there is an orderly increase in cellular
structures and components, replication and
segregation of the bacterial DNA, and formation of a
septum or cross wall which divides the cell into two
progeny cells

150
Reproduction in bacteria
Binary fission is a common reproduction in bacteria

151
If the environment is optimum
the two daughter cells may divide into four
1, 2, 4, 8, 16, 32, 64... or
20,21, 22 , 23, 24, 25, 2n, exponential growth
Mathematically expressed as: N=No x 2n,
(Number at any time = Number at time zero
times 2 to the nth power),
where "n" = number of generations

logN=logNo+nlog2logN-logNo=nlog2
n=logN-logNo/log2=logN-logNo/0.301
n=3.3(logN-logNo) 152
Where,
n=number of generation
No= number of bacteria at the beginning of a
time interval
N=number of population after a period of
exponential growth or number of bacteria at
the end of the time interval

153
Generation Time (G)
the time needed for a cell population to double in number
Varies among bacterial species
If the conditions are optimal, E. coli and S. aureus will have
a generation time approximately 20 min. or less
Many bacteria have generation times of 1 3 hours.
Mycobacterium species, because of their physical make-up,
can have a generation time up to 10 days
G= t/n
=t/3.3(logN-logNo)
Where t = time interval in hours or minutes
154
Example
1. What is the generation time of a bacterial
population that increases from 10,000 cells to
10,000,000 cells in four hours of growth?

Ans = 0.4hr (24 minutes)

155
Bacterial growth phases
Four phases
1. Lag phase
2. Log (exponential) phase
3. Stationary phase
4. Death or decline phase

157
Growth Curve

Stationary

COLONY
FORMING Death
Log
UNITS

Lag

TIME

158
Phases of growth

1. Lag phase
cells are adapting to their new niche, usually a broth

Enzymes are synthesized to utilize broth nutrients

The bacterial cells are metabolically active

Cell reproduction is not immediate

159
2. Log/exponential phase
necessary chemicals are synthesized for metabolism
rapid replication of genetic material
There is logarithmic growth (dividing at greatest rate)
Metabolic activity has peaked
new cells are young, delicate and immature
Sensitive to antibiotics and UV light
The cells can be Gram stained the best during this phase

160
3. Stationary phase
The growth rate slows
This is a phase of equilibrium, because the number of
cells being replicated equals the number of cells dying
Nutrients are depleted
Waste products are building up being toxic
Metabolic activity is greatly reduced
the starved cells become harder to kill and more resistant
bacterial pathogens become more virulent when starved
161
4. Death/decline phase

All nutrients are totally depleted

A tremendous buildup of metabolic waste products

Cells are dying faster than they are being replicated

Gram stain reaction is Gram variable

Total death will occur if this culture is not transferred


to a new broth tube (continuous culture)
162
3. Microbial genetics

Mutation
Mechanisms Of Gene Transfer In Bacteria 163
Genetic Information in Microbes
Genetic information in bacteria and many
viruses is encoded in DNA, but some viruses use
RNA.
Replication of the genome is essential for
inheritance of genetically determined traits.
Gene expression usually involves transcription
of DNA into messenger RNA and translation of
mRNA into protein
164
Genome Organization
The bacterial chromosome is a circular molecule
of DNA that functions as a self-replicating genetic
element (replicon).
Extrachromosomal genetic elements such as
plasmids and bacteriophages are nonessential
replicons which often determine resistance to
antimicrobial agents, production of virulence
factors, or other functions.
The chromosome replicates semiconservatively;
each DNA strand serves as template for synthesis
of its complementary strand
165
Gene Expression
Genetic information encoded in DNA is expressed by synthesis
of specific RNAs and proteins, and information flows from DNA
to RNA to protein.
The DNA-directed synthesis of RNA is called transcription.
Because the strands of double-helical DNA are antiparallel and
complementary, only one of the two DNA strands can serve as
template for synthesis of a specific mRNA molecule.
Messenger RNAs (mRNAs) transmit information from DNA, and
each mRNA in bacteria functions as the template for synthesis
of one or more specific proteins.
The process by which the nucleotide sequence of mRNA
molecule determines the primary amino acid sequence of a
protein is called translation.
166
Ribosomes, complexes of ribosomal RNAs
(rRNAs) and several ribosomal proteins, translate
each mRNA into the corresponding polypeptide
sequence with the aid of
transfer RNAs (tRNAs),
amino-acyl tRNA synthesases,
initiation factors and elongation factors.
All of these components of the apparatus for
protein synthesis function in the production of
many different proteins.
A gene is a DNA sequence that encodes a protein,
rRNA, or tRNA molecule (gene product). 167
Exchange of Genetic Information
Genetic exchanges among bacteria occur by several
mechanisms.
In transformation, the recipient bacterium takes up
extracellular donor DNA. In transduction, donor DNA
packaged in a bacteriophage infects the recipient
bacterium. In conjugation, the donor bacterium
transfers DNA to the recipient by mating.
Recombination is the rearrangement of donor and
recipient genomes to form new, hybrid genomes.
Transposons are mobile DNA segments that move
from place to place within or between genomes.
170
Mechanisms of gene transfer in bacteria
1. Vertical Gene Transfer When genes are passed
from an organism to its offspring
2. Horizontal Gene Transfer Occurs between
bacteria
In Horizontal Gene Transfer:
There are two types of cells
1. Donor: transfers DNA to recipient
2. Recipient: receives the DNA

171
172
Horizontal Gene Transfer in Bacteria
Three fundamentally distinct mechanisms
1.Transformation- a cell takes up isolated DNA molecules
from the medium surrounding it
Acquisition of new genetic marker by incorporation of
exogenous or foreign DNA
2. Conjugation- involves the direct transfer of DNA from
one cell to another
3. Transduction- the gene transfer is mediated by bacterial
viruses (bacteriophages)
173
Transformation

174
Conjugation
Transfer of DNA by contact of two bacterial cells
Can transfer plasmid or chromosome

175
177
4. Antimicrobial agents

181
The word antimicrobial was derived from the Greek words
anti (against), mikros (little) and bios (life) and refers to
all agents that act against microbial organisms
This is not synonymous with antibiotics, a similar term derived
from the Greek word anti (against) and biotikos (concerning life)

By strict definition, the word antibiotic refers to substances


produced by microorganisms that act against another
microorganism
Thus, antibiotics do not include antimicrobial substances that
are synthetic (sulfonamides and quinolones), or semi-
synthetic (methicillin and amoxicillin), or those which come
from plants (alkaloids) or animals (lysozyme)

182
Antimicrobial agents
In contrast, the term antimicrobials include all agents
that act against all types of microorganisms bacteria
(antibacterial), viruses (antiviral), fungi (antifungal) and
protozoa (antiprotozoal)
Notice that the term antibacterial, being the largest
and most widely known and studied class of
antimicrobials, is often used interchangeably with the
term antimicrobials

183
Modes (mechanism) of action of antimicrobial agents
Five modes of antimicrobial actions:
1. Inhibition of cell wall synthesis
2. Inhibition of protein synthesis
3. Inhibition of nucleic acid synthesis
4. Disruption of cell membrane function
5. Action as anti-metabolites (Folic acid)

185
Action of Specific Agents
Cell wall synthesis is inhibited by -lactams, such as
penicillin and cephalosporin, which inhibit
peptidoglycan polymerization, and by vancomycin,
which combines with cell wall substrates.
Aminoglycosides, tetracycline, chloramphenicol,
erythromycin, and clindamycin all interfere with
ribosome function.
Quinolones bind to a bacterial complex of DNA and
DNA gyrase, blocking DNA replication.
Nitroimidazoles damage DNA.
Rifampin blocks RNA synthesis by binding to
DNA directed RNA polymerase.
186
Polymyxins disrupt the plasma membrane,
causing leakage.
The plasma membrane sterols of fungi are
attacked by polyenes (amphotericin) and
imidazoles.
Sulfonamides and trimethoprim block the
synthesis of the folate needed for DNA
replication
187
Sites of action of different antimicrobial agents. 188
Antimicrobial resistance
What is antimicrobial resistance?
Antimicrobial resistance (AMR) is resistance of a microorganism to
an antimicrobial medicine to which it was previously sensitive
Resistant organisms are able to withstand attack by antimicrobial
medicines, such as antibiotics, antivirals, and antimalarials
So that standard treatments become ineffective and infections
persist and may spread to others
AMR is a consequence of:
1. Misuse of antimicrobial medicines
2. Mutation of the microbes
3. Acquiring of a resistance gene
189
Mechanisms of bacterial drug resistance
1. Drug inactivation or modification: for example,
enzymatic deactivation of penicillin G in some penicillin-
resistant bacteria through the production of -lactamase
2. Alteration of target site: for example, alteration of PBP
the binding target site of penicillin
3. Alteration of metabolic pathway: for example, some
sulfonamide-resistant bacteria do not require para-
aminobenzoic acid (PABA), an important precursor for the
synthesis of folic acid and nucleic acids in bacteria
inhibited by sulfonamides, instead, like mammalian cells,
they turn to using preformed folic acid
4. Reducing drug accumulation: by decreasing drug
permeability and/or increasing active efflux (pumping
out) of the drugs across the cell surface 190
193
5. Sterilization and Disinfection

Objectives
Define sterilization and disinfection
Differentiate between sterilization and disinfection
List methods of sterilization/disinfection
State the principles of sterilization/disinfection methods
Describe the application of sterilization/disinfection in
healthcare settings
195
Sterilization and Disinfection
Introduction
Numerous published articles documented infections
after improper reprocessing of reusable medical
equipments
The effective use of disinfection and sterilization and
procedures is important in preventing healthcare
associated infections

196
Sterilization
Sterilization is defined as the process where all the living
microorganisms, including bacterial spores are killed
Sterilization can be achieved by physical, chemical and
physiochemical means
Chemicals used as sterilizing agents are called
chemisterilants
To be effective, sterilization must be preceded by
meticulous cleaning (mechanical or manual) to remove
all foreign materials from objects prior to undergoing
sterilization
197
Disinfection
Disinfection is the process of elimination of most pathogenic
microorganisms (excluding bacterial spores) on inanimate
objects
Disinfection can be achieved by physical or chemical methods
Chemicals used in disinfection are called disinfectants
Different disinfectants have different target ranges, not all
disinfectants can kill all microorganisms
Some methods of disinfection such as filtration do not kill
bacteria, they separate them out
Sterilization is an absolute condition while disinfection is not
The two are not synonymous
198
Decontamination is the process of removal of
contaminating pathogenic microorganisms from the
articles by a process of sterilization or disinfection
Asepsis is the employment of techniques (such as
usage of gloves, air filters, uv rays etc) to achieve
microbe-free environment
Antisepsis is the use of chemicals (antiseptics) to
make skin or mucus membranes devoid of
pathogenic microorganisms

199
Bacteriostasis is a condition where the multiplication of
the bacteria is inhibited without killing them
Bactericidal is chemical that can kill or inactivate
bacteria
Such chemicals may be called variously depending on
the spectrum of activity, such as bactericidal,
virucidal, fungicidal, microbicidal, sporicidal,
tuberculocidal or germicidal

200
201
Physical Methods
Sunlight
The microbicidal activity of sunlight is mainly due to
the presence of ultra violet rays in it
By killing bacteria suspended in water, sunlight
provides natural method of disinfection of water
bodies such as tanks and lakes
Sunlight is not sporicidal, hence it does not sterilize

202
Heat
Heat is considered to be the most reliable method of
sterilization of articles that can withstand heat
Heat acts by oxidative effects as well as denaturation
and coagulation of proteins
Those articles that cannot withstand high
temperatures can still be sterilized at lower
temperature by prolonging the duration of exposure

203
Factors affecting sterilization by heat are:
Nature of heat: Moist heat is more effective than dry heat
Temperature and time: temperature and time are inversely
proportional. As temperature increases the time taken decreases
Number of microorganisms: More the number of
microorganisms, higher the temperature or longer the duration
Nature of microorganism: Depends on species and strain of
microorganism, sensitivity to heat may vary. Spores are highly
resistant to heat
Type of material: Articles that are heavily contaminated require
higher temperature or prolonged exposure. Certain heat sensitive
articles must be sterilized at lower temperature
Presence of organic material: Organic materials such as protein,
sugars, oils and fats increase the time required 204
Action of heat
Dry heat acts by protein denaturation, oxidative
damage and toxic effects of elevated levels of
electrolytes
The moist heat acts by coagulation and denaturation
of proteins
Moist heat is superior to dry heat in action
Temperature required to kill microbe by dry heat is
more than the moist heat

205
DRY HEAT
Red heat:
Articles such as bacteriological loops, straight wires, tips of
forceps and searing spatulas are sterilized by holding them
in Bunsen flame till they become red hot
Flaming:
This is a method of passing the article over a Bunsen flame,
but not heating it to redness
Articles such as scalpels, mouth of test tubes, flasks, glass
slides and cover slips are passed through the flame a few
times
Even though most vegetative cells are killed, there is no
guarantee that spores too would die on such short
exposure
206
DRY HEAT.
Incineration:
This is a method of destroying contaminated material
by burning them in incinerator
Articles such as soiled dressings; animal carcasses,
pathological material and bedding etc should be
subjected to incineration
This technique results in the loss of the article, hence
is suitable only for those articles that have to be
disposed

207
Hot air oven
This method was introduced by Louis Pasteur
Articles to be sterilized are exposed to high
temperature (160 C) for duration of one hour in an
electrically heated oven
The oven should be fitted with a thermostat control,
temperature indicator, meshed shelves and must have adequate
insulation
Articles sterilized: Metallic instruments (like forceps, scalpels,
scissors), glasswares (such as petri-dishes, pipettes, flasks, all-
glass syringes), swabs, oils, grease, petroleum jelly

208
Hot air oven
Advantages
It is an effective method of sterilization of heat stable
articles
The articles remain dry after sterilization
This is the only method of sterilizing oils and powders
Disadvantages
Since air is poor conductor of heat, hot air has poor
penetration
Cotton wool and paper may get slightly charred
Glasses may become smoky
Takes longer time compared to autoclave

209
Infra red rays
Infrared rays bring about sterilization by generation of
heat
Articles to be sterilized are placed in a moving conveyer
belt and passed through a tunnel that is heated by
infrared radiators to a temperature of 180C
The articles are exposed to that temperature for a
period of 7.5 minutes
Articles sterilized included metallic instruments and
glassware
It requires special equipments and mainly used in
central sterile supply department

210
MOIST HEAT
Moist heat acts by coagulation and denaturation of proteins
At temperature below 100C
Pasteurization:
This process was originally employed by Louis Pasteur
Currently employed in food and dairy industry
There are two methods of pasteurization, the holder method
(heated at 63C for 30 minutes) and flash method (heated at
72C for 15 seconds) followed by quickly cooling to 13C
This method is suitable to destroy most milk borne pathogens
like Salmonella, Mycobacteria, Streptococci, Staphylococci and
Brucella, however Coxiella may survive pasteurization
211
Serum bath
The contaminating bacteria in a serum preparation
can be inactivated by heating in a water bath at 56C
for one hour on several successive days
Proteins in the serum will coagulate at higher
temperature
Only vegetative bacteria are killed and spores survive

212
At temperature 100C
Boiling:
Boiling water (100C) kills most vegetative bacteria and viruses
immediately
Certain bacterial toxins such as Staphylococcal enterotoxin are
also heat resistant
Some bacterial spores are resistant to boiling and survive; hence
this is not a substitute for sterilization
When absolute sterility is not required, certain metal articles and
glasswares can be disinfected by placing them in boiling water for
10-20 minutes
The lid of the boiler must not be opened during the period

213
Steam at 100C
Instead of keeping the articles in boiling water, they are subjected
to free steam at 100C
A steamer is a metal cabinet with perforated trays to hold the
articles and a conical lid
The bottom of steamer is filled with water and heated
Sugar and gelatin in medium may get decomposed on
autoclaving, hence they are exposed to free steaming for 20
minutes for three successive days
This process is known as tyndallisation (after John Tyndall) or
fractional sterilization or intermittent sterilization
The vegetative bacteria are killed in the first exposure and the
spores that germinate by next day are killed in subsequent days
214
At temperature above 100C
Autoclave
Sterilization can be effectively achieved at a temperature
above 100C using an autoclave
Water boils at 100C at atmospheric pressure, but if
pressure is raised, the temperature at which the water
boils also increases
In an autoclave the water is boiled in a closed chamber. As
the pressure rises, the boiling point of water also raises
At a pressure of 15 lbs inside the autoclave, the
temperature is said to be 121C
Exposure of articles to this temperature for 15 minutes
sterilizes them
215
Autoclave..
Advantages of steam: It has more penetrative power
than dry air, it moistens the spores (moisture is
essential for coagulation of proteins)
Advantage: Very effective way of sterilization, quicker
than hot air oven
Disadvantages: Drenching and wetting of articles may
occur, trapped air may reduce the efficacy, takes long
time to cool

216
RADIATION
Two types of radiation are used, ionizing and non-
ionizing
Non-ionizing rays are low energy rays with poor
penetrative power while ionizing rays are high-energy
rays with good penetrative power
Since radiation does not generate heat, it is termed
"cold sterilization"
Fruits and vegetables are irradiated to increase their
shelf life

217
FILTRATION
Filtration does not kill microbes, it separates them out
Membrane filters with pore sizes between 0.2-0.45 m
are commonly used to remove particles from solutions
that can't be autoclaved
It is used to remove microbes from heat labile liquids
such as serum, antibiotic solutions, sugar solutions, urea
solution

218
CHEMICAL METHODS OF DISINFECTION
Disinfectants are those chemicals that destroy
pathogenic bacteria from inanimate surfaces
Some chemical have very narrow spectrum of activity
and some have very wide
Those chemicals that can sterilize are called
chemisterilants
Those chemicals that can be safely applied over skin
and mucus membranes are called antiseptics

219
Classification of disinfectants
1. Based on consistency
a. Liquid (E.g., Alcohols, Phenols)
b. Gaseous (Formaldehyde vapor, Ethylene oxide)
2. Based on spectrum of activity
a. High level
b. Intermediate level
c. Low level
3. Based on mechanism of action
a. Action on membrane (E.g. Alcohol, detergent)
b. Denaturation of cellular proteins (E.g., Alcohol, Phenol)
c. Oxidation of essential sulphydryl groups of enzymes (E.g., H2O2,
Halogens)
d. Alkylation of amino-, carboxyl- and hydroxyl group (E.g., Ethylene Oxide,
Formaldehyde)
e. Damage to nucleic acids (Ethylene Oxide, Formaldehyde)
220
221
ALCOHOLS
Mode of action: Alcohols dehydrate cells, disrupt membranes
and cause coagulation of protein
Examples: Ethyl alcohol, isopropyl alcohol and methyl alcohol
Application: A 70% aqueous solution is more effective at
killing microbes than absolute alcohols
70% ethyl alcohol is used as antiseptic on skin
Disadvantages: Skin irritant, volatile (evaporates rapidly),
inflammable

222
ALDEHYDES
Mode of action: Acts through alkylation of amino-,
carboxyl- or hydroxyl group, and probably damages
nucleic acids
It kills all microorganisms, including spores
Examples: Formaldehyde, Gluteraldehyde
Application: 40% Formaldehyde (formalin) is used for
surface disinfection and fumigation of rooms, chambers,
operation theatres, biological safety cabinets, wards,
sick rooms etc.
It also sterilizes bedding, furniture and books
10% formalin with 0.5% tetraborate sterilizes clean metal
instruments
223
ALDEHYDES
2% gluteraldehyde is used to sterilize thermometers,
cystoscopes, bronchoscopes, centrifuges, anasethetic
equipments etc.
Disadvantages: Vapors are irritating (must be
neutralized by ammonia), has poor penetration,
leaves non-volatile residue, activity is reduced in the
presence of protein
Gluteraldehyde requires alkaline pH and only those
articles that are wettable can be sterilized
224
PHENOL
Mode of action: Act by disruption of membranes, precipitation
of proteins and inactivation of enzymes
Examples: 5% phenol, 1-5% Cresol, 5% Lysol (a saponified
cresol), hexachlorophene, chlorhexidine, chloroxylenol (Dettol)
Applications: Joseph Lister used it to prevent infection of
surgical wounds
Phenols are coal-tar derivatives. They act as disinfectants at
high concentration and as antiseptics at low concentrations
They are bactericidal, fungicidal, mycobactericidal but are
inactive against spores and most viruses
Disadvantages: It is toxic, corrosive and skin irritant
Chlorhexidine is inactivated by anionic soaps. Chloroxylenol is
inactivated by hard water
225
HALOGENS:
Mode of action: They are oxidizing agents and cause
damage by oxidation of essential sulfydryl groups of
enzymes
Chlorine reacts with water to form hypochlorous
acid, which is microbicidal
Examples: Chlorine compounds (chlorine, bleach,
hypochlorite) and iodine compounds (tincture
iodine, iodophores)

226
Applications:
Tincture of iodine (2% iodine in 70% alcohol) is an antiseptic
10% Povidone Iodine is used undiluted in pre and postoperative
skin disinfection
Chlorine gas is used to bleach water. Household bleach can be
used to disinfect floors. Household bleach used in a stock dilution
of 1:10.
In higher concentrations chlorine is used to disinfect swimming
pools
Used at a dilution of 1:10 in decontamination of spillage of
infectious material.
Disadvantages: They are rapidly inactivated in the presence of
organic matter. Iodine is corrosive and staining. Bleach solution is
corrosive and will corrode stainless steel surfaces.
227
HEAVY METALS
Mode of action: Act by precipitation of proteins and oxidation of sulfydryl
groups.
They are bacteriostatic
Examples: Mercuric chloride, silver nitrate, copper sulfate, organic
mercury salts (e.g., mercurochrome, merthiolate)
Applications:
Silver sulphadiazine is used topically to help prevent colonization and
infection of burn tissues
Mercurials are active against viruses at dilution of 1:500 to 1:1000
Merthiolate at a concentration of 1:10000 is used in preservation of
serum
Copper salts are used as a fungicide
Disadvantages: Mercuric chloride is highly toxic, are readily inactivated
228
by organic matter
SURFACE ACTIVE AGENTS
Mode of actions: They have the property of
concentrating at interfaces between lipid containing
membrane of bacterial cell and surrounding aqueous
medium
These compounds have long chain hydrocarbons that are
fat soluble and charged ions that are water-soluble
Since they contain both of these, they concentrate on the
surface of membranes
They disrupt membrane resulting in leakage of cell
constituents.
Examples: These are soaps or detergents

229
Application
They are active against vegetative cells, Mycobacteria
and enveloped viruses
They are widely used as disinfectants at dilution of 1-2%
for domestic use and in hospitals
Disadvantages:
Their activity is reduced by hard water, anionic detergents and
organic matter
Pseudomonas can metabolise cetrimide, using them as a
carbon, nitrogen and energy source

230
HYDROGEN PEROXIDE
Mode of action: It acts on the microorganisms through
its release of nascent oxygen
Hydrogen peroxide produces hydroxyl-free radical that
damages proteins and DNA
Application: It is used at 6% concentration to
decontaminate the instruments, equipments such as
ventilators
3% Hydrogen Peroxide Solution is used for skin
disinfection and deodorising wounds and ulcers
Strong solutions are sporicidal
Disadvantages: Decomposes in light, broken down by
catalase, proteinaceous organic matter drastically
reduces its activity
231
ETHYLENE OXIDE (EO)
Mode of action: It is an alkylating agent
It acts by alkylating sulfydryl-, amino-, carboxyl- and
hydroxyl- groups
Properties: It is a cyclic molecule, which is a colorless
liquid at room temperature. It has a sweet ethereal
odor, readily polymerizes and is flammable

232
Application:
It is a highly effective chemisterilant, capable of killing
spores rapidly
Since it is highly flammable, it is usually combined with CO2
(10% CO2+ 90% EO) or dichlorodifluoromethane
It requires presence of humidity
It has good penetration and is well absorbed by porous
material
It is used to sterilize heat labile articles such as bedding,
textiles, rubber, plastics, syringes, disposable petri dishes,
complex apparatus like heart-lung machine, respiratory
and dental equipments
Disadvantages: It is highly toxic, irritating to eyes, skin,
highly flammable, mutagenic and carcinogenic
233
PHYSIO-CHEMICAL METHOD
Mode of action: A physio-chemical method adopts
both physical and chemical method
Use of steam formaldehyde is a physio-chemical
method of sterilization, which takes into account
action of steam as well as that of formaldehyde

234
Spauldings classification
Spauldings classification provides a simplified
outline of the recommended processing methods for
items of patient care equipment
Three classification of medical equipments
1. Critical items
2. Semi-critical items
3. Non-critical items

235
Spauldings classification

236
Host - pathogen relationship
Interaction between host and pathogen
Competition for superiority
If Host is successful healthy (pathogen removed or exist
benignly)
Pathogen get upper hand Disease
Disease occurs due to imbalance between host resistance and
virulence factors
Under normal condition the host and the parasite exist in balance
Any decrease in host resistance or increase in parasite virulence
result in disease
When host resistance impaired, organisms that are normally present
may cause disease
237
Host parasite relationship

Disease Health

Parasite Host
Virulence resistance

Balance
Health
Disease

238
Normal flora
Bacteria that are consistently associated with animal
are called the normal flora
Resides without causing disease in healthy host
Commonly found in GIT, mouth, skin and URT

Normal flora could be


1. Symbiontes (benefit the host)
2. Commensals ( neither benefit nor harm the host)
3. Opportunists ( waits suitable opportunity to cause
disease nosocomial infection )

239
Significance of the Normal Flora
The normal flora influences the anatomy, physiology, susceptibility to
pathogens, and morbidity of the host.
I. Skin Flora: The varied environment of the skin results in locally dense or
sparse populations, with Gram-positive organisms (e.g., staphylococci,
micrococci, diphtheroids) usually predominating.
II. Oral and Upper Respiratory Tract Flora: A varied microbial flora is found in
the oral cavity, and streptococcal anaerobes inhabit the gingival crevice. The
pharynx can be a point of entry and initial colonization for Neisseria,
Bordetella, Corynebacterium, and Streptococcus spp.
III. Gastrointestinal Tract Flora: Organisms in the stomach are usually transient,
and their populations are kept low (103 to 106/g of contents) by acidity.
IV. Urogenital Flora: The vaginal flora changes with the age of the individual, the
vaginal pH, and hormone levels. Transient organisms (e.g., Candida spp)
frequently cause vaginitis. The distal urethra contains a sparse mixed flora;
these organisms are present in urine specimens (104/ml) unless a clean-
catch, midstream specimen is obtained.
V. Conjunctival Flora: The conjunctiva harbors few or no organisms.
Haemophilus and Staphylococcus are among the genera most often detected.
240
Numbers of bacteria that colonize different parts of the body. 241
Bacterial Pathogenicity
i. Contact: exposure to microbes ( Contamination)
ii. Infection: entrance of pathogen within the body
iii. Invasion: infectious agent enters in host cell/tissue and
spread in the body
iv. Pathogenicity: ability of a pathogen to cause disease
v. Virulence: precise factors used to invade and damage
host tissue
vi. Pathogen: that cause disease in healthy individuals

242
Pathogens do have mechanism to:
Enter, colonize, attach, derive food, and escape from
host defense mechanism
Bacteria cause disease by:
A. destruction of tissues
B. loss of organ function
C. Release toxins
D. Stimulating cytokines (host inflammatory response)
Disease occurrence depends on:
Bacterial strain, inoculums size, host factor
Seriousness of disease:
Type of organ, extent of damage
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246
Manifestations of Infection
The clinical presentation of an infectious disease reflects
the interaction between the host and the microorganism.
This interaction is affected by the host immune status and
microbial virulence factors. Signs and symptoms vary
according to the site and severity of infection.
Diagnosis requires a composite of information, including
history, physical examination, radiographic findings, and
laboratory data.
Microbial Causes of Infection
Infections may be caused by bacteria, viruses, fungi, and
parasites. The pathogen may be exogenous (acquired from
environmental or animal sources or from other persons) or
endogenous (from the normal flora).

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Specimen Selection, Collection, and Processing

Specimens are selected on the basis of signs


and symptoms, should be representative of
the disease process, and should be collected
before administration of antimicrobial agents.
The specimen amount and the rapidity of
transport to the laboratory influence the test
results.

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Microbiologic Examination
Direct Examination and Techniques:
Direct examination of specimens reveals gross pathology.
Microscopy may identify microorganisms. Immunofluorescence,
immunoperoxidase staining, and other immunoassays may detect
specific microbial antigens.
Genetic probes identify genus- or species-specific DNA or RNA
sequences.
Culture:
Isolation of infectious agents frequently requires specialized media.
Nonselective (noninhibitory) media permit the growth of many
microorganisms.
Selective media contain inhibitory substances that permit the
isolation of specific types of microorganisms.

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Microbial Identification:
Colony and cellular morphology may permit preliminary
identification.
Growth characteristics under various conditions, utilization
of carbohydrates and other substrates, enzymatic activity,
immunoassays, and genetic probes are also used.
Serodiagnosis:
A high or rising titer of specific IgG antibodies or the
presence of specific IgM antibodies may suggest or confirm
a diagnosis.
Antimicrobial Susceptibility:
Microorganisms, particularly bacteria, are tested in vitro to
determine whether they are susceptible to antimicrobial
agents.

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General procedure for collecting and processing specimens for
aerobic and/or anaerobic bacterial culture

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Culture and Biochemical Tests
Cultivation is the process of propagating
microorganisms to grow by providing the proper
environmental conditions
Types of Culture Media Used in Microbiology
1. Solid - primarily agar based
2. Fluid
3. Enriched
4. Selective
5. Differential
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A. ENRICHMENT MEDIA
Basic principle is to control the nutrients and culture
conditions in such a way that it suits mainly to a
specific species temperature, air supply, light, pH
When we assume low amount of potential pathogens
being present in the specimen, they have to be
enriched first, to multiply up the low number e.g.
serumbouillon, dextrosebouillon,
choppedmeatbouillon
Promotes the growth of a particular organism by providing it
with the essential nutrients, and rarely contains inhibitory
substances to prevent the growth of normal competitors
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B. SELECTIVE MEDIA
Used for growth of only selected microorganisms.
Selection by Adding antibiotics, prevents the growth of
other cells Lacking amino acids; May contain stains and
color indicators (EMB)
1. Eosin-methylene blue agar (EMB) Contains methylene blue,
toxic to Gram + bacteria, allowing only growth of Gram bacteria
2. MacConkey agar (MAK) For Gram bacteria
3. Buffered charcoal yeast extract agar (BCYE) Selective for
certain Gram -, for example Legionella.
4. Mannitol-salt agar (MSA) Selective for Gram + bacteria
5. Hektoen enteric agar (HE) Shigella, Salmonella
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6. Thiosulfatecitrate bile sucrose (TCBS) Vibrio cholerae
Microbiological Examination Methods contd
C. DIFFERENTIAL MEDIA
Distinguishes one microorganism type from another
growing on the same media on a difference in the colony
appearance Color, shape, growth pattern Dyes in the
medium, pH indicators
1. Eosin-methylene blue agar (EMB) Differential for
lactose and sucrose fermentation
2. MacConkey agar (MCK) Differential for lactose
fermentation
3. Mannitol salt agar (MSA) Differential for mannitol
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fermentation
Gram Negative Rods on MacConkey Agar

Lactose non-fermenter Lactose fermenter


Serratia Escherichia coli
marcescens

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