Introduction

Historical background
• 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,
physiology, and participation in the process of nature, helpful and harmful rela-
tionship with other living things, and significance in science and industry

• Sub divisions of microbiology

 Bacteriology
 Mycology
 Parasitology .
 Virology

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• Medical Bacteriology: involves the study of pathogens, the disease
caused by them, and the body’s defenses against disease.
• It is also concerned with epidemiology, transmission of pathogens,
disease prevention measures, and aseptic techniques, treatment of in-
fectious diseases, immunology, and the production of vaccines to pro-
tect against infectious disease.

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 History of microbiology /The Golden Age of Microbiology

• Mankind has always been affected by diseases which were originally believed
to be visitations by the gods and meant to punish evil doers.
• 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.

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 History of microbiology cont….
• Antony Van Leeuwenhoek (1632-1723 G.C.), father of Microbiology,
– observed “animalcules” using simple microscope with one lens.

– He was the first who properly described the different shapes of bacteria.
– Question raised - where did they originate?
• Although Leeuwenhoek was not concerned about the origin of micro-organism;
many other scientists were searching for an explanation for spontaneous ap-
pearance of living things from decaying meat, stagnating ponds, fermenting
grains and infected wounds.
• On the bases of this observation, two major theories were formulated.
– Theory of Abiogenesis
– Theory of Biogenesis

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 Origin of life Theories
• Theory of abiogenesis deals with the theory of spontaneous generation; stating
that living things originated “spontaneously” from non-living things.
• Aristotle (384-322 BC): is the founder of a theory spontaneous generation/ abio-
genesis.
He observed spontaneous existence of fishes from dried ponds, when the pond
was filled with rain.
• Biogenesis: - States that life comes from pre existing life
– Francesco Redi (1626-1697): He is the scientist who first tried to set an ex-
periment to disprove spontaneous generation

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• Francisco Redi
– Performed experiments that disproved theory of Spontaneous
Generation for more complex forms of life (began approx.1668).
– Utilized jars containing meat. Some were covered, some were not.
– Maggots appeared in uncovered jars.
– Results not accepted for microscopic organisms.
– Introduced experimental procedure to disproof spontaneous gener-
ation
– The controversy on spontaneous generation took 200 years.

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John Needham (1749) Performed experiments similar to Redi’s on the origin of
life in microscopic organisms
• Introduced the first culture medium for microbial growth.
• Utilized infusion broth prepared by boiling meat, grain, etc. to extract nutrients.
Turbidity indicated growth.
• Broth put in flasks, some were sealed with corks, and some were not.
• All flasks became cloudy, result different from Redi’s experiment.
• He suggested that life originate spontaneously from nonliving matters
• The spontaneous generation opponents didn’t accept his conclusion, they said
it could be due to entrance organisms from air or flasks, improper seal.

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Lazzaro Spallanzani (1776)
• Repeated Needham’s experiments to disproof spontaneous genera-
tion in microscopic life.
• Boiled broth after placing in flasks.
• Sealed flasks by plugging with solid stopper.
• Results more consistent with Redi’s.
• Occasionally sealed flask  cloudy.
• Not accepted by spontaneous generation supporters, because they
said that heating may have destroyed, degraded “vital force” and air
was not allowed to enter.
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 Louis pasture (1822- 1895) was the scientist who disproved the theory of
abiogenesis once and for all.
Performed experiment to disprove Theory of spontaneous generation.
In his experiment he filtered air through cotton plug.
He placed plug in infusion broth, broth became cloudy - organisms present in the air.
He designed a large curved flask/swan-necked (pasture goose neck flask) and placed a
sterile infusion broths.
Flasks remained sterile unless tilted or neck broken.

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Therefore, Pasteur proved that microorganisms entered to the broth with
the air and micro organisms did not evolve spontaneously

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 Other major contribution of Louis Pasteur.

• Microbial theory of fermentation

• Principles and practices of sterilization and pasteurization

• Control of disease of silk worm

• Development of vaccines against anthrax and rabies
• Discovery of streptococci.

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 How Can Microbes Be Classified?
• Carolus Linnaeus (Swedish) developed taxonomic system for
naming plants and animals and grouping similar organisms to-
gether
• Leeuwenhoek’s microorganisms grouped into six categories as
follows:

Fungi
Protozoa
Algae

Bacteria
Archaea

Small animals

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 Taxonomy: Naming, Classifying, and Identifying Microorganisms

• Microbial nomenclature- naming microorganisms

• Taxonomy- classifying living things

• Identification- discovering and recording the traits of
organisms so they can be named and classified

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 Traditional Whittaker Classification
Five Kingdoms
Prokaryotae (Monera)
Protista
Fungae
Plantae
Animalia
Based on:
Morphology (0.2 um to 10um long )
Metabolism (Biochemical Activity)
Molecular Techniques
Fatty Acid Profiles
Protein Differentiation
DNA Finger Printing

1. Morphology 8. Amino acid sequence of proteins

2. Staining 9. Genetic composition
3. Motility
4. Growth
5. Nutritional requirement
6. Bio chemical and metabolic activity
7. Pathogenicity

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 Assigning Specific Names

• The binomial system of nomenclature

– The generic (genus) name followed by the species
name
– Generic part is capitalized, species is lowercase
– Both are italicized or underlined if italics aren’t
available
– Staphylococcus aureus

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 The Germ Theory of 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 dis-
ease is caused by specific pathogen.
• The scientific procedure is known as 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 dis-

ease 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 le-
sion

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
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 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. Eg. 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 in-
vades imuno-compromised host.

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 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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The Golden Age of Microbiology/1857-1907

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The Golden Age of Microbiology

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 The Modern Age of Microbiology

• What Role Do Microorganisms

Play in the Environment?
– Bioremediation uses living
bacteria, fungi, and algae to
detoxify polluted environments
– Recycling of chemicals such
as carbon, nitrogen, and sulfur

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The Modern Age of Microbiology

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 The Modern Age of Microbiology
• What Are the Basic Chemical Reactions
of Life?
• Biochemistry
– Began with Pasteur’s work on fermentation
and Buchner’s discovery of enzymes in
yeast extract
– Kluyver and van Niel – microbes used as
model systems for biochemical reactions
– Practical applications
• Design of herbicides and pesticides
• Diagnosis of illnesses and monitoring
of patients’ responses to treatment
• Treatment of metabolic diseases
• Drug design

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 Worldwide Infectious Diseases Affecting Health Sciences

• Increasing number of drug resistant

strains including Nosocomial and
Community Acquired microorganisms
– MRSA - Methicillin Resistant Staphy-
lococcus aureus
– VRE- Vancomycin Resistant Entero-
coccus
– VRSA- Vancomycin Resistant
Staphylococcus aureus
– MDR-TB- Multidrug Resistant Tuber-
culosis

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 Worldwide Infectious Diseases Affecting Health Sciences

• Increasing number of emerging diseases (SARS, AIDS, hepatitis C, viral en-

cephalitis)
• Other diseases previously not linked to microorganisms now are (gastric ulcers,
certain cancers, multiple sclerosis)

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 Morphology, growth and nutrition of bacteria

• Eukaryotic and Prokaryotic cells

• Prokaryotic cell (Pro -means primitive) ( Karyote - means nucleus)
 Prokaryotic cells are less complex cells. Which posses naked (unbound) DNA with out
associated basic proteins, they divide by binary fission are bounded by a semi rigid cell
wall.
 The prokaryotic cells include bacteria and cyanobacteria (blue- green algae.)

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• Eukaryotic cells (Eu- means true, (Karyote- means nucleus)
 The Eukaryotic cell is more complex, has membrane bound nu-
cleus, and many organelles.
 Eukaryotic cells include protozoa, fungi, algae, (green, brown
and red algae) and plant and animal cells.

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Bacterial structure

• Basic features of Bacterial Cell

• General property
– Typical prokaryotic cell
– Contain both DNA and RNA
– Most grow in artificial media
– Replication is by binary fission
– Contain rigid cell wall

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 Structure of Bacteria
Bacterial structure is considered at three levels.
1. Cell envelope proper:
Capsule, cell wall and cell membrane
2. Cellular element enclosed with in the cell envelope:
Mesosomes, ribosome, nuclear material, polyamines and cytoplasmic
granules.
3. Cellular element external to the cell envelope (appendages) :
Flagellum, Pilus

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 1. Cell Envelop proper
• a cell envelope consists of a capsule, cell wall and plasma membrane
• Cell wall:
– a non-living secretion of the cell membrane
– Multi layered structure and constitutes 20% of the bacterial dry weight
– Average thickness is 0.15-0.5 m
– Rigidity of the cell wall is due to the presence of PG/ Provide cell support
Chemical Composition of cell wall
• The major component of cell wall is peptidoglycan (PG)
• polymer of disaccharides cross-linked by peptides-
– peptidoglycan layer (murein) is a complex, interwoven network that surrounds the
entire cells and is composed of a single covalently linked macromolecules.
– Found only in bacterial cell walls
– protects the delicate cell protoplast from osmotic lysis
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 Types of cell wall
I. Gram positive cell wall of bacteria
– has two layers (PG) cross linked with teichoic acid
– The PG layers is much thicker than Gram negative bacteria and i.e. 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 peptideglycan make gram- positive bacteria susceptible to the en-
zyme lysozyme and penicillin.
• Lysozyme hydrolyzes peptidoglycan by specific cleavage between N-acetyl muramic acid
and the N-acetyl glucosamine
• Penicillin specifically inhibits peptidoglycan synthesis
• Teichoic acids and cell well- associated protein are the major surface antigens of the
gram- positive cell well

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 Gram positive cell wall …Cont’d
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 thin peptidoglycan layer (3 – 8nm)

• Has high lipid content (lipopolysaccharied) in the outer membrane

• Has periplasmic space.

Outer membrane
• Contains receptors (sites) for bacteriophage attachment or bacteriocine (bacteriocine –
are antibacterial agents produced by bacteria)
• Used in transport of materials (either out of or towards the cell)
Lipopoly saccharides
• It is responsible for antigenicty of the outer membrane
Periplasmic space
• Found between outer membrane and the cell membrane
• Mostly contain enzymes and endotoxins.

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 (a) (b)

Fig. Gram-Positive (a) and Gram negative (b) cell wall of bacteria
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 Capsule and slim layer
• Capsules are often regarded as portion of the cell envelope
• Capsular constituents vary among the different species of prokaryotes.
• Many bacteria have slimy layers, consisting of polysaccharides only; others have proteins
within the polysaccharide capsule.
• Both serve to enable the bacteria to attach to tissues and to resist phagocytic digestion.
• The basic difference between the capsule and the slim is their property of firm attach-
ment to the cell.
• The gels formed by the capsule adhere to the cell whereas the slim can easily be washed
off.
• Capsule can be detected by Indian ink staining in which the capsule stands out as a halo.

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 Capsule … Cont’d

• Some of the important characteristics of capsule include:

– Usually it is weekly antigenic

– Not necessary for viability
– Endows virulence
– important in adhesion

– Protects from phagocytosis

– Capsulated strains are invariably non motile
• The organism in which capsules have been demonstrated include:
Pneumococci, klebsella, Escerchia coli, hemophilus influenza, etc.
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 Cytoplasmic membrane (Plasma membrane)
• It is the actual barrier between the interior and exterior of the bacteria cell.
• The cytoplasmic membrane exhibits a well- defined selective permeability, ex-
cretion of enzyme, and biosynthesis of cell well and other proteins
• The bacterial transport system and the principal energy system (oxidative phos-
phophorylation) are located in the cytoplasmic membrane.
• It accounts for 30% of the dry weight of bacterial cell

• Chemically, the plasma membrane consists of proteins and phospholipids.

• It is 60% protein, 20 – 30% lipid and 10-20% carbohydrate

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 Cytoplasmic membrane … Cont’d
Function of Cell Membrane
• Regulates the transport of nutrients and waste products into and out of the cell. (It is se-
lectively permeable)
• Assists DNA replication
• Captures energy in ATP (i.e it is site of oxidative phosphorulation )

Cytoplasm

• Viscous watery solution of soft gel, containing a variety organic and inorganic solutes
and numerous small granule called ribosomes

• The cytoplasm consists of water, enzymes oxygen, waste products, essential nutrients,
proteins, carbohydrates, lipids and a complex mixture of all the materials required by the
cell for its metabolic functions
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 2. Cellular Element Enclosed with in the Cell Envelope

Ribosomes :
• Cytoplasmic particles, which are the sites of protein synthesis
• It is composed of ribosomal RNA (rRNA) (70%) and proteins (30%)
• It constitutes 90% of the RNA and 40% of the total protein.

• The ribosomes of procaryotes are smaller than cytoplasmic ribosomes of

eucaryotes.
• Procaryotic 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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 Nuclear material

• is concentrated in the cytoplasm as a nucleoid consisting one long double-

stranded circular DNA molecule (chromosome).
• The chromosome serves as the control center of the bacterial cell, carries
the genetic information needed for producing several thousand enzymes
and other proteins.
• The prokaryotic nucleoid is considered primitive nucleus

• Apart from nucleus, the bacteria may have some extra chromosomal ge-
netic material in the form of DNA, which is known as Plasmid.

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 3. Cellular Element External to the Cell Envelope

Flagellum
• It is the organ of locomotion in bacterial cell and consists of filament
• is free on the surface of bacterial cell
• Size: 3-20m in length and 0.01-0.013m in diameter.
• It is composed of protein named as flagellin
• The flagellar antigen in motile bacterium is named as H (Hauch) antigen

-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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 Flagellum … Cont’d

Flagellar arrangements
1. Atrichous: Bacteria with no flagellum: Eg. All cocci
2. Monotrichous: Bacteria with single polar flagellum: Eg. V.cholara
3. Lophotrichous: Bacteria with bunch of flagella at one pole: Eg. Pseudomonas
flouresense
4. Amphitrichous: Bacteria with flagella at both poles: Eg. Alcaligenes fae-
cales
5. Peritrichous: Bacteria with flagella all over their surface: Eg. S.typhi

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Fig. ? Different flagellar arrangements

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• Flagella provides swimming movement for most motile procaryotic cells.
• Spirochates moves by using a flagellum like structure called the axial fil-
ament, which wraps around the spiral shaped cell to produce an undu-
lating motion.
• Non-motile (non flagellated) bacteria can move by blinding, flexing, and
spinning

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 Pilli and Fimbriae
• Interchangeable terms used to designate short, hair-like structures (finer
filaments) on the surfaces of prokaryotic cells.

• are extruding from the cytoplasmic membrane

• are shorter and stiffer than flagella, and slightly smaller in diameter.

• they are composed of protein called pilin arranged in helical strand

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Two functional types of pili may occur independently or together on some cell.
I. Common pili
– almost always called fimbriae
– Help for attachment of bacteria to epithelial cell
– They considered as virulence factor in some species of bacteria, because
they allow pathogens to attach to (colonize) tissues and to resist attack by
phagocytic white blood cells.
II. Sex pili or F pili
– occur less commonly
– appear to be specifically involved in bacterial conjugation, i.e transfer of
genetic material (DNA) from one bacterium to another.
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 Bacterial spores

• Under conditions of limited supply of nutrition, vegetative forms of certain bacte-

ria especially gram-positive bacilli and actinomycets form highly resistant and
dehydrated forms, which are called endospores.
• endospores are capable of survival under adverse conditions such as heat, dry-
ing, freezing, radiation, and actions of toxic chemicals.
• Spores are usually smooth walled and ovoid, in some species it is spherical.
• It does not take up ordinary stains

• It looks like areas of high refractivity under light microscope

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