INTRODUCTION TO MEDICAL MICROBIOLOGY

Instructor:
Abdella G. (BSc, MSc, PhD Candidate)

December, 2021

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1.1 Historical background
 What is Microbiology?
Defn. Microbiology is a subject which deals with living
organisms that are individually too small to be seen with the
naked eye.

• It considers the microscopic forms of life and deals about:

• their reproduction and physiology
• participation in the process of nature
• helpful and harmful relationship with other living things
• significance in science and industry

NB: These organisms include bacteria, algae, protozoa, fungi, and

virus.
• Prions (“infectious proteins”) are recent addition. 2
History of Medical Microbiology

Hippocratus, Father of Medicine, observed that ill health resulted due to

changes in air, winds, water, climate, food, nature of soil and habits of
people.

Fracastorius (1500 G.C.) proposed that the agents of communicable disease

were living germs, that could be transmitted by direct contact with humans
and animals, and indirectly by objects ; but no proof because of lacking
experimental evidence.

Antony Van Leeuwenhoek (1632-1723 G.C.), observed “animalcules”

using his simple microscope with one lens.
 He was the first who properly described the different shapes of bacteria

 Question raised - where did they originate?

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 Although Leeuwenhoek was not concerned about the
origin of micro-organism

Many other scientists were searching for an explanation

for spontaneous appearance of living things from
decaying meat, stagnating ponds, fermenting grains and
infected wounds

 On the bases of this observation, two major theories were

formulated
1. Theory of Abiogenesis

2. Theory of Biogenesis
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Theory of Abiogenesis deals with the theory of spontaneous
generation; stating that living things originated “spontaneously”
from non-living things.
 Aristotle (384-322 BC): The founder of a theory spontaneous
generation.
He observed spontaneous existence of fishes from dried ponds,
when the pond was filled with rain.

Theory of Biogenesis: - States that life comes from pre existing life

 Francesco Redi (1626-1697): He is the scientist who first tried to

set an experiment to disprove spontaneous generation.

o Utilized jars containing meat. Some were covered, some were

not.
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o Maggots appeared in uncovered jars.

o Conclude that maggots' did not emerge spontaneously but

from the eggs laid on the meat by the fly.

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Louis Pasture (1822- 1895) was the scientist who
disproved the theory of abiogenesis once and for all and
remembered as the Father of Microbiology

• He performed experiment to disprove theory of spontaneous

generation

• He established that fermentation was the result of microbial

activity

• He developed microbiology as a scientific discipline

• He introduced techniques of sterilization , etc

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* In ‘A’ air freely moved through the tube, but dust
particles were trapped in the curved portion of the
flask. And no microbial growth was observed.

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Therefore, Pasteur proved that microorganisms entered to the
broth with the air and micro organisms did not evolve
spontaneously
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 Other contributions of Leus pasture

• Microbial theory of fermentation

• Principles and practices of sterilization and pasteurization

• Control of disease of silk worm

• Development of vaccines against anthrax & rabies

• Discovery of Streptococci

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 Sub divisions of Medical Microbiology

 Bacteriology – which deals with bacteria

 Mycology- which deals with fungi

 Virology –studies about viruses

 Rickettisiology - studies about Rickettisia

 Immunology – concerned with resistance of the body

to infectious diseases

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 Distribution of Microorganisms in nature

• Microorganisms can be found nearly everywhere as normal

inhabitants of the earth (biosphere)
• They exist in soil, water, air, in our food, in our clothing, in/on
our body etc.
– Microorganisms can also survive in most unlikely environment
like in cold air, in hot springs at temperatures of 900C.

– Microorganisms inhabit the surface of living human and animal

bodies and grow abundantly in the mouth and intestinal tract

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Actually only a small percentage microbes are pathogenic- few
are able to cause disease

The others are considered beneficial or harmless

 Some may cause disease only if they accidentally invade the

wrong place at the right time such as when the host immunity is
low. These microbes are considered opportunistic

Most of this Microorganisms that live on the human body with

out causing disease and apparent physiological response comprise
the normal flora

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 The Germ Theory of infectious diseases
• Pasture has also developed the germ theory of diseases, which
states that “a specific disease is caused by a specific type of
microorganism”.
• Robert Koch, in 1876 established an experimental procedure to
prove the germ theory of disease, which states that specific disease
is caused by specific pathogen. The scientific procedure is known
as Koch’s Postulate
• He studied anthrax, a disease of cattle & can be transmitted to
humans
o Koch isolated the organism from infected cattle in pure culture and then
injected a small amount of the pure culture in to healthy animals
o The injected animals become infected and anthrax developed
o The infectious agent was then isolated from this second group of animals
 This sequence of isolation, reinfection and recovery of the
infective agent is called Koch's postulate
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 Koch’s Postulate: - proof of germ theory of disease
• A Micro-organism can be accepted as a causative agent of an
infectious disease only if the following conditions are satisfied

1. The microorganism should be found in every case of the disease

and under conditions, which explain the pathological changes and
chemical features

2. It should be possible to isolate the causative agent in pure culture

from the lesion

3. When such pure culture is inoculated in to appropriate lab

animal, the lesion of the disease should be reproduced

4. It should be possible to re-isolate the bacterium in pure culture

from the lesion produced in the experimental animal

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Fig. Koch’s postulate 16
 Exceptions to Koch’s postulate
• Many healthy people carry pathogens but do not exhibit
symptoms of the disease
• Some microbes are very difficult or impossible to grow in vitro
(in the laboratory) in artificial media. E.g. Treponema pallidum.
• Many pathogens are species specific. Eg. Brucella abortus cause
abortion in animals but not in humans.
• Certain diseases develop only when an opportunistic pathogen
invades immuno-compromised host

Major achievements of Robert Koch

1. Discovery and use of solid medium in bacteriology
2. Discovery of causative agents of tuberculosis and cholera
3. Koch’s postulate
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 1.2 Basic Structures of Cells

Eukaryotic cells
– Eukaryotic cells are more advanced, larger, contain organelles

– All higher species: animals, plants, fungi, protozoa, cells of

plants and animals

– Eukaryotic cells generally contain nucleus within a nuclear

membrane

– They have organelles with numerous discrete membranes

– They divide /multiply/ by a process called mitosis

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Getachew k. 19
 Prokaryotic cells
– Prokaryotic cells are more primitive, small and without
organelles
Example: Bacteria, Rickettsia, Mycoplasma, blue green algae
etc

– Are cells of lower life forms having nuclear materials

/DNA/ which is not enclosed by membrane.
– But the nuclear material is distributed in mass through the
cytoplasm.

– Their genetic material is not organized in to chromosome

– They divide by simple dividing system called binary fission

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 Basic structures of cells
Tab.1 Basic difference of eukaryotic and prokaryotic cells
Characteristic Prokaryotes Eukaryotes

Nucleus No Membrane bounded Present with Nuclear Membrane

Organelles No Membrane bounded Present
DNA structure Single closed loop Naked multiple chromosome with histone
strand with no histone protein protein

Chlorophyll When present dissolved in when present contained in chloroplast

cytoplasm

Ribosome Smaller (70 s) Larger (80 s)

Cell walls Present Present some type e. g. plants
Reproduction usually by fission Mitosis
Examples. Fungi, protozoa, animals,
no evidence of mitosis. human
Example: Bacteria, Rickettsia,
Chlamydia

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 Bacteriology
Basic features of Bacterial Cell

– Typical prokaryotic cell

– Contain both DNA and RNA
– Most grow in artificial media
– Replication is by binary fission
– Contain rigid cell wall

• Bacteria with defective cell walls include: Protoplasts,

Spheroplast, L-forms –Bacterium-and Mycoplasma

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 Function of cell wall

• Provides shape or rigidity to the bacterium and it Protects from

destructive environmental factors

• Provides staining characteristics to the bacterium. The gram stain

divide bacteria in to two classes

• It contains unique and varied surface antigens (Ags).

• The serologic identification of these Ags is a major diagnostic tool’

and cell wall biosynthesis is the site of action of many antibiotics

• Contains receptor sites for phages and action of antibody

• Contains components toxic to host (endotoxins)
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 Types of cell wall

I. Gram positive cell wall of bacteria

– has two layers of Peptidoglycan (PG) cross linked with
teichoic acid
– The PG layers is much thicker than Gram negative bacteria
and is 15 – 50 nm thick
– The PG layer comprises 50 – 90% of the cell wall and 20 –
40% of the cell wall weight
• The large amount of peptidoglycan make gram- positive
bacteria susceptible to the enzyme lysozyme and penicillin.
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•Lysozyme hydrolyzes peptidoglycan by specific cleavage between
N-acetyl muramic acid and the N-acetyl glucosamine of the glycon
strand.

•Penicillin specifically inhibits peptidoglycan synthesis

Teichoic acids and cell wall- associated proteins are the major
surface antigens of the gram- positive cell wall

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Teichoic acid
– These polymers of glycerol phosphate or ribitol
phosphate are located in the outer layer of the gram
positive cell wall

Function of Teichoic acid

– Used to bind (keep) Mg+2 concentration in the cell
wall

– Used to activate autolytic enzyme (enzymes which

are secreted by bacteria usually when it dies)

– To bind bacteriophage in the cell wall

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 II. Gram negative Cell wall of bacteria
• Is some what complex than Gram positive bacterial cell
wall
• Has a thin peptidoglycan layer (3 – 8nm)
• Has high lipid content (lipopolysaccharide) in the
outer cell membrane
• Has periplasmic space

Getachew k. 28
Fig. Gram-Positive (a) and Gram negative (b) cell wall of bacteria
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Outer Membrane
• Contains receptors (sites) for bacteriophage attachment or
bacteriocine (bacteriocine – are antibacterial agents produced by
bacteria)
• It participates in cell division
• Used in transport of materials (either out of or towards the cell)_

• Porins, proteins that form pores in the outer membrane, allow

passage of hydrophilic, low-molecular-weight substances into the
periplasmic space

Lipopolysaccharides (LPS)_is comprised of the lipoid A, the core

polysaccharide, and the O-specific polysaccharide chain
• It is responsible for antigenicity of the outer membrane
Periplasmic space
• Found between outer membrane(PG) and the cell membrane
• Mostly contain enzymes and endotoxins
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 Cytoplasmic membrane (Plasma membrane)
 Can be seen under electron microscope
 It accounts for 30% of the dry weight of bacterial cell
 It is the actual barrier between the interior and exterior of the
bacteria cell.

 Exhibit a well- defined selective permeability, excretion of

enzyme, and biosynthesis of cell wall and other proteins
 The principal energy system (oxidative phosphophorylation) are
located in the cytoplasmic membrane.

 It assists DNA replication / cell division

 Chemically, the plasma membrane consists of proteins and

phospholipids
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 It is 60% protein, 20 – 30% lipid and 10-20% carbohydrate
 Cellular Element Enclosed with in the Cell Envelope
Mesosomes_ can be seen only under electron microscope
• are complex invaginations of cytoplasmic membrane
– Mesosomes are attached to chromosomes and are involved in
DNA segregation during cell division
– It is involved in respiratory enzyme -activity (Site of oxidative
phosphorulation)
Ribosomes
• Cytoplasmic particles, which are the sites of protein synthesis
• It is composed of ribosomal RNA (rRNA) (70%) and proteins
(30%)
• Prokaryotic ribosomes are 70S in size, being composed of 30S and
50S subunits.
– S or Svedberg unit designates the sedimentation coefficient of
the rRNA
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Cytoplasmic inclusions_ are nor permanent or essential structures.
Concerned with the cell metabolism
• The granules in bacteria that represent the accumulated food
reserve. For example polysaccharides, lipid, reserves of PO4,
starch or sulphur
Nuclear material
• The prokaryotic nucleoid is considered primitive nucleus,
• it is not surrounded by a nuclear membrane
• It does not have a definite shape, and has little or no protein
material
• The nucleoid consists of one long double-stranded circular DNA
molecule (chromosome)

• Apart from nucleus, the bacteria may have some extra

chromosomal genetic material in the form of DNA, which is
known as Plasmid
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 Cellular Element External to the Cell Envelope

Flagella
• It is the organ of locomotion in bacterial cell and consists
of filament
• is free on the surface of bacterial cell
• Help in adhesion, invasion & spread of infection (in
pathogenicity)
• It is composed of protein named as flagellins
• The flagellar antigen in motile bacterium
(Enterobacteriaceae ) is named as H (Hauch) antigen
• Together with the O-antigens, they are used to classify
bacteria in serovars
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Flagellar arrangements

1. Atrichous: Bacteria with no flagellum: E.g. All cocci

2. Monotrichous: Bacteria with single polar flagellum:
E.g. [Link]
3. Lophotrichous: Bacteria with bunch of flagella at one pole:
E.g. Pseudomonas flouresense
4. Amphitrichous: Bacteria with flagella at both poles:
E.g. Alcaligenes faecales
5. Peritrichous: Bacteria with flagella all over their surface:
E.g. [Link] , [Link]

The presence of flagella in bacterial cell is detected by:

 Hanging drop preparation

 Swarming phenomenon on surface of plate agar

 Motility in semi solid media

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Fig. Different flagellar arrangements 36
 Pilli (Fimbriae)
 interchangeable terms used to designate short, hair-like
structures (finer filaments) on the surfaces of prokaryotic cells
 are shorter and stiffer than flagella, and slightly smaller in
diameter

 Common pili /fimbriae: Help for attachment of bacteria to

epithelial cell

 Sex pili or F pili: appear to be specifically involved in

bacterial conjugation, i.e. transfer of genetic material (DNA)
from one bacterium to another

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 Capsules
• Outside & immediately in contact with cell wall, gelatinous
material made of complex polysaccharide in most species
• In some species it consists of polypeptide or proteins

• Demonstrated by negative staining in wet films with Indian Ink

• Protects the cell wall against attack by various antibacterial agents

(bacteriophages, colicines, complement, lysozyme) & against
ingestion by the phagocytes

• NB: The cell wall, capsule, flagella, & fimbriae play a

role in the process of infection

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 Bacterial spores

• Under conditions of limited supply of nutrition, vegetative

forms of certain bacteria especially gram-positive bacilli and
actinomycets form highly resistant and dehydrated forms, which
are called endospores

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 CLASSIFICATION OF BACTERIA
1. Taxonomy: Taxonomy is the science of classification,
identification, and nomenclature.
– For classification purposes, organisms are usually organized
into subspecies, species, genera, families & higher orders.

2. Classification
– Classification is the orderly arrangement of bacteria into
groups
– For example, clinical microbiologists are interested in the
serotype, antimicrobial resistance pattern, toxin and
invasiveness factors in Escherichia coli, whereas geneticists
are concerned with specific mutations and plasmids

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3. Identification
– Identification is the practical use of classification criteria to
distinguish certain organisms from others
4. Nomenclature
– Nomenclature (naming) is the means by which the
characteristics of a species are defined and communicated
among microbiologists.
– For example, the organism known as Clostridium perfringens
in the United States is called Clostridium welchii in England.
5. Species
– A bacterial species is a distinct organism with certain
characteristic features or a group of organisms that resemble
one another closely in the most important features of their
organization.
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 Classification below Species Level

 Particularly for epidemiological purposes, clinical

microbiologists must distinguish strains with particular traits
from other strains in the same species

 Serotype, phage type, colicin type, biotype, bioserotype (a group

of strains from the same species with common biochemical and
serologic characteristics that set them apart from other members
of the species), and pathotype

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• Bacteria are classified in to 19 different categories in
Bergey’s manual of determinative bacteriology, 8th
(1974), and the classification is based on

1. Morphology

2. Staining

3. Motility

4. Growth characteristics

5. Nutritional requirement

6. Bio chemical and metabolic activity

7. Pathogenecity

8. Amino acid sequencing of proteins

9. Genetic composition(GC to AT) 43
Fig. Bacterial identification.
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 BACTERIAL METABOLISM AND GROWTH

 Bacterial metabolism involves all the cellular processes required

for the survival and replication of the organism

 It refers to all of the chemical reactions occurring within a cell,

including the production of energy, intermediate products, and end
products

 Most biochemical reactions fall into two categories: Catabolism

and Anabolism

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Catabolism
• The metabolic degradation (breakdown) of organic compounds
that results in the production of energy and smaller molecules
• Catabolic reactions involve the breaking of bonds; whenever
chemical bonds are broken, energy is released

Anabolism
• Refers to those biosynthetic processes that use energy for the
synthesis of protoplasmic materials needed for growth,
maintenance, and other cellular functions
– Anabolic reaction involves the creation of bonds; it takes
energy to create chemical bonds

– Smaller molecules are bonded together to create large

molecules
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 NUTRITION OF BACTERIA

• For their optimal growth bacteria have well defined requirements

of proper nutrients, oxygen, PH and temperature

• All bacteria need some form of the element C, H, O2, S, P, & N

for growth

• Special elements such as K, Ca, Fe, Mn, Mg, Co, Cu, Z, Ur are
needed by certain bacteria

• Some have specific vitamin, and growth factor requirements and

others need organic substances secreted by other microorganisms
during their growth and they are known as fastidious organism

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• From a nutritional, or metabolic, viewpoint physiologic types of
bacteria exist:
– The heterotrophs (or chemoorganotrophs), most
bacteria
– The autotrophs (or chemolithotrophs), and
– The photosynthetic bacteria (or phototrophs)

Heterotrophic Metabolism
 Heterotrophic bacteria, which include all pathogens, obtain energy
from oxidation or fermentation of organic compounds

• Biologic oxidation of these organic compounds by bacteria results in

synthesis of ATP and generation of simpler organic compounds
(precursor molecules) e.g. acid, alcohol, gas, etc….

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• All heterotrophic bacteria require preformed organic compounds
 2. Oxygen requirement
On the basis of this requirement, bacteria have been divided in to:
• Facultative aerobes/anaerobes:- these can grow under both
aerobic and anaerobic conditions, e.g. Enterobacteriaceae

• Obligate aerobes:- these cannot grow unless oxygen is present

in the medium e.g. Pseudomonas

• Microaerophilic:- these organisms can grow under conditions

with low oxygen tension e.g. Helicobacter spp.

• Aero-tolerant anaerobes:– These bacteria oxidize nutrient

substrates without using elemental oxygen. Although, unlike
obligate anaerobes, they can tolerate it. e.g. Saprophytic bacteria

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3. Temperature
• The minimum and maximum temperature at which a micro-organism
can grow is different in the different species of bacteria. This is known
as Temperature tolerance range.
• Based on temperature requirement bacteria are classified as:

– Psychrophilic- are those grow in the range of -5 to 300C with an

optimum of 10-200 C.
 Cause spoilages of food at refrigeration temperature (2-8 ⁰C)

– Mesophilic - are those bacteria, which grow at 20-450C and show

optimum growth at 37oC. And virtually, medically important
bacteria (pathogenic bacteria) belong to this group.

– Thermophilic – are those organisms which prefer high

temperature (50-800C) for their growth and show maximum
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growth at 50- 600C
 4. pH requirement

Based on their pH requirement bacteria can be classified as

– Neutrophilic:- bacteria grow best at neutral pH

(pH=7)
• Most pathogenic micro-organism best grow at neutral pH
(pH=7)
– Acidophilic
• Bacterial grow best at acidic pH (pH<7)
E.g. Lactobacilli, fungi and yeast
– Alkalophilic
• Bacterial grow best at Alkaline pH (pH>7)
E.g. Vibrio cholerea grow at a pH of 8.6
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 5. Salinity (salt concentration)
• In most case bacteria need small amount of salt
concentration to grow

 Halophytes are bacteria which need high concentration

of salt for their growth,7-10% NaCl
E.g. Staphylococcus epidermidis, Vibro vulnificus

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 Bacterial growth
• Whenever adequate nutrition and conducive environment factors
are available a bacterium enlarges and eventually divides by binary
fission to form two daughter cells
• Nuclear division precedes cell division

Fig. Bacterial binary fission 53

Generation time or population doubling time

• The interval of time between two cell division, or the time

required for a bacterium to give rise to two daughter cells
under optimum conditions

• The generation time of bacteria ranges from as little as 20

minutes for [Link] to more than 20 hrs for Mycobacterium
tuberculosis

• The generation time varies not only with the species but also
with the amount of nutrients, the temperature, the pH, and
other environmental factors

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• The growth cycle of bacteria has four major phases:
1. The Lag phase

2. The log phase (exponential phase)

3. The stationary phase

4. The decline phase

• If a small number of bacteria are inoculated into a nutrient medium

and the bacteria are counted at frequent interval, the typical phase
of a standard growth curve can be demonstrated
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The Lag Phase
o This phase is of short duration in which bacteria adapt themselves
to new environment

o This is a period of active macro molecular synthesis like DNA,

RNA, various enzymes and other structural components

o It is the preparation time for division

o No increase in cell number occurs, however, vigorous metabolic

activity occurs
o This can last for a few minutes up to many hours

o The duration of lag phases varies with the species, nature of

culture medium, temperature of incubation etc.
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The Log, Logarithmic, or Exponential Phase

• During this phase, the population can double

• It has limited duration because of:-

– Exhaustion of nutrients
– Accumulation of toxic metabolic end products
– Rise in cell density or number
– Change in pH and
– Decrease in oxygen tension (in case of aerobic organisms)
– Become sensitive for antimicrobial agents (antibiotics)

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• The number of bacteria during log phase growth can be calculated
by the following equation

Nt = N o x 2 t/d
• Nt = is the number of bacteria after time (t),
• No = the initial number of bacteria
• t/d = is the amount of time divided by the doubling time

E.g. Suppose the generation time of Salmonella is 15 minutes.

Calculate the number of bacteria that can result from a bacterium
inoculated in the artificial culture medium for 10hrs.

• The morphology of the bacteria is best developed in this phase

and organisms manifest typical biochemical characters
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Stationary Phase
• Occur when nutrients depletion or toxic products cause slow
growth until the number of new cells produced balances the
number of cells that die resulting in a steady/constant state
• The number of viable cell remain constant
• There is almost a balance between the bacterial reproduction and
bacterial death

The death/decline phase

• Due to severe depletion of nutrients and accumulation of toxic end
products the number of bacteria dying is much more than those
dividing and hence there is gradual decline in the total number of
organism
• There is drastic decline in viable cells
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