Introduction to Microbiology

MICROBIOLOGY
Presented by-
Jayesh Soni
Associate professor
Venkteshwar college of nursing
Udaipur

Microbiology
Microbiology -is the specialized area of biology, in which we are study the
microorganisms
Microbiology is a subject which deals with microbes and their related concepts
It is the science that studies very small living things, Usually requires a
magnification tool – the microscope.
Among the many specialized fields of microbiology-
• Bacteriology- the study of bacteria,
• Virology- the study of Viruses,
• Mycology- the study of fungi
• Parasitology- the study of parasites
• Immunology- the study of immune system,
• Microbial Ecology- The relationship between microorganisms and their
environment
• Environmental Microbiology,
• Food Microbiology
• Forensic Microbiology

Microorganism
Living things which individually are too small to be seen
with the naked eye.
All of the following may be considered microorganisms:
– bacteria
(eu-bacteria, archae-bacteria)
– fungi (yeasts, molds)
– protozoa
– microscopic algae
– viruses
– various parasitic worms

Viruses
Bacteriophage Avian Flu

Helminth
Tapeworm Ascaris round worm

Development/Historical
Prospective of
Microbiology

Antoni Van Leeuwenhoek
(1673) –
Probably the first person
to observe living cells with
a simple microscope,
amateur scientist, ground
his own lenses and
described- what we know
today as bacteria – rod
shaped , spiral shaped.

Edward Jenner
He credited with first vaccine – in
epidemics of smallpox during the late
1700’s
He observed that milk maids didn’t get
the disease,
Cattle had a similar disease – cowpox,
Milk maids had cow pox lesions, but not
small pox, he purposefully took
scrapings from cowpox blister and
scraped a 8 year old volunteer. With the
material – child got mild illness but not
small pox,
Vaccination comes from Latin word
“vacca” meaning cow. Jenner laid the
foundation for Pasteur’s later work with
other vaccinations.

Known as Father of Bacteriology

Robert Koch –
Developed Koch’s postulates – important technique for determining
the actual microbial cause agent of a disease
1. He discovered the tuberculosis bug (tubercle bacillus,
Mycobacterium tuberculosis)
2. He discovered the cause of anthrax (Bacillus anthracis)
from blood of dead cattle, cultured bacteria in pure culture,
injected bacteria in live cattle and they died, then again cultured
the bacteria in pure culture. This led to the establishment of a
procedure for determining microbial cause of disease.

Koch’s postulates
Koch’s and Pasteur’s work helped
establish the “Germ Theory of
Disease” –
That microorganisms cause
disease (in people, animals, and
even plants)

Louis Pasteur’s experiment
He devised the
ingenious curved
necked flasks that
prevented
contaminated air from
reaching boiled beef
broth – the broth
remained
uncontaminated even
though exposed to
the air

Cont..
1. He developed process that we call Pasteurization –
he heated wine to kill contaminating microbes – cured
sick wine (today we heat treatment to kill pathogens in
milk also)
2. He proved that fermentation was caused by a
microbe – yeast
3. He developed vaccines for rabies and anthrax.
Vaccines led to immunity to diseases that routinely
killed many people, used to help people long before
they understood how they even worked (science of
Immunology)
4. He began the revolution in science that led to the
Golden Age of Microbiology (from 1857-1914)

Iwanowski 1892 –
Discovered that plant disease can be caused by
small organisms that were so small they passed through
filters ,
Tobacco mosaic virus (TMV) was later identified as the
cause - beginning of virology

• Syphilis spirochete Paul Ehrlich

Lister
• Antisepsis in surgery

Microbiology – Chapter 1
Alexander Fleming - Scottish physician and bacteriologist - 1928
Observed mold growing on a bacteria culture, there was a ring of clearing
around the mold where the bacteria didn’t grow, the mold was later
found to be a Penicillium species and the naturally secreted chemical was
called penicillin, an antibiotic
1. Antibiotics are natural agents
2. Synthetic drugs are chemicals produced in labs (sulfas)
3. Problems with them - toxicity, resistance, allergic reactions
4. Fleming’s work - shelved until early WWII, sulfas were failing,
needed penicillin to cure battle field wounds
5. Now have thousands of antibiotics and synthetics (and a significant
problem – resistance)
Alexander Fleming
Scottish physician and bacteriologist - 1928

MicroScope
Unit of Measurement

Microbiology
There are 5 kingdom scheme still widely used-
• Monera – bacteria (Prokaryotic)
• Protista – Protozoans (Eukaryotic)
• Fungi - yeast, molds, etc. (Eukaryotic)
• Plant – photosynthetic producers (Eukaryotic)
• Animals – heterotrophic consumers
(Eukaryotic)

Domain
Kingdom
Phylum
Class
Order
Family
Genus
Species
Categories of Classification

Archaea are single- celled organisms
that lack a membrane-bound
nucleus. - Prokaryote
The cells of
all eukaryotes
have a
membrane-
bound
nucleus.
kingdoms)

Protista
• It is divided into 2 groups-
Prokaryotes & Eukaryotes

DIFFERENCES BETWEEN PROKARYOTES AND EUKARYOTES
Character Prokaryotes Eukaryotes
Nucleus:
Nuclear Membrane Absent Present
Nucleolus Absent Present
Mitotic Division Absent Present
Cytoplasm
Cytoplasmic Streaming Absent Present
Pinocytosis Absent Present
Lysosomes Absent Present
Golgi Apparatus Absent Present
Endoplasmic Reticulum Absent Present
Chemical Composition
Sterols Absent Present
Muramic Acid Present Absent
Teichoic Acid Present Absent

BACTERIA
• Bacteria are Prokaryotic, unicellular that do not
contain chlorophyll.
• Size of bacteria may range from 0.2-1.5 μm
(micrometer) in diameter and 3-5μm in length

Morphology of Bacteria
the study of the form and structure of organisms and their specific structural features.
• Cocci
• Bacilli
• Vibrio
• Spirilla
• Spirochetes
• Actinomycetes
• Mycoplasma

Cellular Arrangement
In Cocci-
• Diplococci: Cocci
arranged in pairs
• Streptococci:
Arranged in chains
• Staphylococci:
Arranged in grape
like clusters

In Bacilli-
• Coccobacilli: Oval
shaped
• Palisades :
Parallel, attached
at any one end of
the cell
• Streptobacilli: In
chains

Vibrio Spirilla
Actinomycetes Mycoplasma

Structure of the Bacterial cell wall
• Bacterial cell wall provides structural integrity to the cell.
• Peptidoglycan is responsible for the rigidity of the
bacterial cell wall and for the determination of cell shape
• Based on the composition of cell wall & Staining bacteria
are classified into “Gram positive” & “Gram Negative”
• The bacterial cell wall differs from that of all other
organisms by the presence of Peptidoglycan
• Peptidoglycan (Muco-peptide) is composed of alternating
chains of ..N -Acetyl Glucoseamine (NAG) and N-Acetyl
Muramic acid (NAM), which is cross linked by Peptide
chains.

Gram Positive Bacterial Cell wall
• The Gram positive cell wall is characterized by the
presence of a very thick Peptidoglycan layer (20-80
nm thick)
• Cell wall contains 90% Peptidoglycan and 10%
Teichoic acid
• Interwoven in the cell wall of gram-positive are
Teichoic acids and lipo-teichoic acids.
• Teichoic acids composed of polymers of glycerol,
phosphates, and the sugar alcohol- ribitol.
• Teichoic acids constitute for the major surface
antigens.

Gram Negative Bacterial Cell wall
• Gram negative bacteria are more complex than Gram
positive bacteria.
• The cell wall is composed of a single layer of peptidoglycan
surrounded by a membranous structure called the outer
membrane.
• The outer membrane of Gram-negative bacteria invariably
contains a unique component lipo-polysaccharide
(LPS or endotoxin) which is toxic to animals.
• LPS is made up of three different components:- 1) O-antigen
or O-polysaccharide, which represents the outermost part
of the structure , 2) the core polysaccharide, & 3) lipid A

Cont…
• LPS plays a role in the host response to pathogenic
gram negative bacteria. The O-antigen triggers an
immune response in an infected host, causing the
generation of antibodies specific to that part of LPS.
• Lipid A acts as a toxin.
• A large amount of lipid A released into the bloodstream
can trigger endotoxic shock, a body-wide inflammatory
response which can be life-threatening.
• Gram negative cells utilize porins, which are
transmembrane proteins

• The peptidoglycan layers are linked to the outer membrane
by the use of a lipoprotein
• This linkage between the two layers provides additional
structural integrity and strength.
Cont…

Difference between Gram + & Gram - Bacteria

Cytoplasmic Membrane
• Bacterial cytoplasmic membrane is composed of
a phospholipid bilayer with embedded proteins.
• It is a thin layer lining the inner surface of the cell wall.
• Semipermiable membrane controlling the flow of
metabolites
• The basic function of the cytoplasmic membrane is to
protect the cell from its surroundings. It controls the
movement of substances in and out of cells and
organelles.

Cytoplasm
• The cytoplasm or protoplasm of bacterial cells is
gel-like matrix composed of water, enzymes,
nutrients, wastes, and gases and contains cell
structures such as ribosomes, a chromosome, and
plasmids.
• The components of the cytoplasm are responsible
for cell growth, metabolism, elimination of waste
and replication (reproduction) of the cell.

• The components of cytoplasm of bacteria are:
Ribosomes, Mesosomes (Chondroids),
Nucleoids, Plasmids, Cytoplasmic Inclusions,
Spore and Cysts.

• Ribosomes are granular-shaped organelles that
are responsible for reading the instructions or
directions in the long strands of DNA and directing
the production of bacterial proteins.
• Large numbers of ribosomes float freely in the
cytoplasm. When they are needed, the ribosomes
fulfill their purpose by attaching to genetic material.
• Providing a platform for protein synthesis
Ribosomes

Mesosomes (Chondroids)
• Mesosomes are the invaginated
structures (usually 2-4 ) formed by the
localized infoldings of the plasma
membrane.
• The invaginated structures comprise of
vesicles & tubules
• Prominent in GM+ bacteria
• The mesosome increases the surface
area of the cell, aiding the cell in
cellular respiration.

Nucleoids
• Bacterial cells have a large, free-lying, double-stranded
DNA molecule in their cytoplasm.
• This DNA molecule is called bacterial chromosome,
which aggregates to form a visible mass called the
“Nucleoid”
• They are chemically composed of about 60% DNA, 30%
RNA, and 10% protein (mostly RNA polymerase) by
weight.
• It is not surrounded by a nuclear membrane
• The nucleoid is essential for controlling the activity of the
cell and reproduction.

Plasmids
• Plasmids naturally exist in bacterial cells
• A plasmid is a small, circular, double-stranded DNA
molecule that is distinct from a cell's chromosomal DNA
• Plasmids have a wide range of lengths, the size of the
plasmid varies from 1 to over 200 thousand base pairs
• The genes carried in plasmids provide bacteria with
genetic advantages, such as antibiotic resistance.

External structures of Bacteria
• Capsule
• Pili (Fimbriae)
• Flagella

Pili(Fimbriae)
• Fimbriae or pili (singlular: pilus) are hair like filaments
(tiny hollow projections) that extend from the cell
membrane into the external environment. A pilus is
composed of subunits of the protein pilin.
• Found mainly in Gram negative organisms
• Bacteria use adherence fimbriae (pili) to overcome the
body’s defense mechanism and cause disease.
• Pili are small hairs that enable some pathogens to
attach and adhere easily to cell surface particularly
mucous membranes.

Length: up to 2 µm
Types: Two general types of Pili are known:
Sex pili (long conjugation pili) and
Common pili (also called fimbriae).
• Common pili (Adhesins): The attachment of bacteria
to specific receptors on the human cell surface
• Sex pili (conjugation tube): It is a specialized kind of
pili that forms the attachment between male (donor)
and the female (recipient) bacteria
during conjugation and acts as a conduit for the
passage of DNA.

Flagella
• Unbranched, long ,filaments ,made up of protein-
“Flagellin”
• Organs of locomotion,
• Flagella are highly antigenic,
• Shape is a 20-nanometer-thick hollow tube.
• it also often has function as a sensory organelle, being
sensitive to chemicals and temperatures outside the cell.
• ATP isn’t needed because bacterial flagellum can use the
energy of the proton-motive force. This means the
energy is derived from ion gradients – usually hydrogen or
sodium – which lie across cell membranes.

Structure
Each flagellum consists of 3 parts
1. Filament
2. Hook
3. Basal body

Gram-positive organisms have two of these basal body
rings- One in the peptidoglycan layer & One in
the plasma membrane.
Gram-negative organisms have four such rings:
L-ring associates with lipopolysaccharides,
P-ring associates with peptidoglycan layer,
S-ring is directly attached to the plasma membrane
M-ring is embedded in the plasma membrane
The filament ends with a capping protein.

• Monotrichous: A single flagellum at one end
of the organism or the other.
• Lophotrichous: Several flagellum on one end
of the organism or the other.
• Amphitrichous: A single flagellum on both
ends of the organism.
• Peritrichous: Several flagellum attached all
over the organism.
Flagellar Arrangement

Mortality:- it is the ability of cell/organism to
move of it’s own accord by expending energy.
Kinds of Motility:
• Darting motility – motion like a shooting star
type of flagellar movement. (Vibrio Cholerae)
• Tumbling motility- To move forward, the
flagella rotate counterclockwise and the
organism "swims" (Listeria)
• Cork &screw motility- motile by bending &
rotating body movements (Spirochaetes)
• Stately motile- moves in a slow and
steady manner (Clostridium)
• Serpentine motility- moves like a serpent or
snake (Salmonella)

Fixation
Fixation is the process used to kill, adhere
and alter the specimen, so it will accept stains.
Staining technique
Stains and dyes are frequently used in
biology and medicine to highlight structures
in biological tissues for viewing, often with the aid
of different microscopes.

Simple Staining
• The simple stain is a very simple staining procedure
involving single solution of stain. Any basic dye such
as methylene blue, safranin, or crystal violet can be
used to color the bacterial cells.
• The simple stain can be used as a quick and easy way
to determine cell shape, size and arrangements of
bacteria

Gram Staining
Gram staining is used to determine gram status to
classify bacteria broadly. It is based on the
composition of their cell wall.
Gram staining uses crystal violet (methyl violet) to stain
cell walls, iodine as a mordant, & safranin (or Carbol
fuchsin may be use) counterstain to mark all bacteria.
The presence or absence of a cell wall changes the
bacterium's susceptibility to some antibiotics.
Gram-positive bacteria stain dark blue or violet.
Their cell wall is typically rich with peptidoglycan
and lacks the secondary membrane and
lipopolysaccharide layer found in Gram-negative
bacteria.

Ziehl Neelsen stain
• The Ziehl–Neelsen stain also known as the acid-
fast stain.
• It was first described by two German doctors: the
bacteriologist Franz Ziehl and the pathologist
Friedrich Neelsen.
• This method is used for those microorganisms
which are not staining by simple or Gram
staining method, particularly the acid-fast
organisms, mainly genus Mycobacterium, can
only be visualized by acid-fast staining.

Acid fast stain or Ziehl Neelsen stain

General structure of Mycobacterium
Acid-fast bacteria are gram-positive, but in
addition to Peptidoglycan, the outer membrane or
envelope of the acid-fast cell wall of contains large
amounts of glycolipids, especially mycolic acids
that in the genus Mycobacterium, which make up
approximately 60% of the acid-fast cell wall.

Acid fast stain or Ziehl Neelsen
staining procedure
Sample Collection & Preparation :
• Direct Smear: Smear prepared directly from a
patient specimen prior to processing.
• Indirect Smear: Smear prepared from a
processed specimen after centrifugation (is used
to concentrate the material)
Reagents required:
• Primary stain - Carbol fuchsin
• Decolorized agent - Acid alcohol 3% v/v
• Counterstain- Methylene blue (5g/l)

Procedure
• Spread smear over the central area of the slide
using a continuous rotational movement. The
recommended size of the smear is about 20 mm by
10 mm.
• Place slides on dryer with smeared surface
upwards, and air dry for about 30 minutes.
• Heat fix dried smear.
• Cover the smear will Carbol fuchsin stain
• Heat the smear until vapor just begins to rise (i.e.
about 60 degree Celsius), Do not overheat, remain
on the slide for 5 minutes.

Cont…
• Wash off the stain with clean water.
• Cover the smear with 3% v/v acid alcohol for 2-5
minutes or until the smear is sufficiently
decolorized, i.e. pale pink.
• Wash well with clean water
• Cover the stain with Methylene blue stain for 1-2
minutes
• Wash off stain with clean water
• Wipe the back of the slide clean, and Examine the
smear microscopically, using the 100x oil
immersion objective.

…Summary / Result…
ACID-FAST STAIN Cell Color Cell Color
Procedure Reagent
Acid-fast
Bacteria
Nonacid-fast
Bacteria
Primary dye Carbol fuchsin RED RED
Decolorizer Acid-alcohol RED COLORLESS
Counterstain Methylene blue RED BLUE
Acid fast: Bright red to intensive
purple (B), Red, straight or
slightly curved rods, occurring
singly or in small groups, may
appear beaded Non-acid
fast: Blue color (A)

Capsule stain
• Capsule stain is a type of differential stain
which uses acidic and basic dyes to stain
background & bacterial cells respectively so that
presence of capsule is easily visualized.
• Capsulated bacteria have a capsule made up of
polysaccharide layer but some bacteria have
capsule made up of polypeptide, or glycoprotein.

India ink method
• In this method two dyes, crystal violet and India ink
are used.
• The capsule is seen as a clear halo around the
microorganism against the black background. This
method is used for demonstrating Cryptococcus.
• The background will be dark (color of India ink).
• The bacterial cells will be stained purple (bacterial
cells takes crystal violet-basic dyes as they are
negatively charged).
• The capsule (if present) will appear clear against
the dark background (capsule does not take any
stain).

Spore Stain
• The spore stain is a differential stain used to
visualize bacterial endospores.
• By forming spores, bacteria can survive in
hostile conditions.
• Spores are resistant to heat, dessication,
chemicals, and radiation.
• Mainly 2 methods are use in spore stain –
Hot method & Cold method

Hot method
Reagents- Carbol Fuchsin Solution, Methylene Blue Solution
Procedure-
 Flood the slide with strong Carbol-Fuchsin and steam.
 After 5 minutes wash the slide well with water.
 Decolorize with ethanol until all traces of red have been
removed.
 Wipe the Bottom of the slide dry to remove excess
stain.
 Wash thoroughly in water
 Counterstain with methylene blue for 1-2 minutes.
 Wash and drain or blot to dry.
Results- The spores stain red while the bacterial bodies stain
blue

Cold method
Reagents- Malachite Green Solution & Safranin Solution
Procedure-
 Fix the smear by passing the slide through a flame.
 Stain for 10 Minutes with malachite green without
heat
 Rinse with tap water for about 10 seconds.
 Counterstain with aqueous safranin (0.25-0.5%) for
15 seconds.
 Rinse with water and drain or blot dry.
Result- The spores will be green and the rest of the cell
will be red.

Flagellar stain
Two techniques for staining flagella are in use-
1. A wet-mount procedure (Ryu method)
2. Dried-smear preparation (Leifson staining
technique)
A wet-mount technique for staining bacterial
flagella is highly successful when a stable stain and
regular slides and cover slips are used. This
technique is simple for routine use when the
number and arrangement of flagella are critical in
identifying species of motile bacteria.

Procedure-
Grow bacteria for 16-24 hrs on a non-
inhibitory medium eg. Soy agar or blood agar,
touch a loopful of water onto the edge of colony
and motile bacteria swim into it. If motile cells are
not seen, do not proceed with the RYU flagella
stain.
Ryu stain has 2 components.-
Solution I - the mordant-
phenol, tannic acid, and
aluminum potassium sulfate.
Solution II - the stain
crystal violet

Hanging Drop Preparation
• Hanging drop preparation is used in
dark illumination to observe the motility
of bacteria.
• In this method a drop of culture is placed
on a coverslip that is encircled with
petroleum jelly (or any other sticky
material). The coverslip and drop are
then inverted over the well of a
depression slide. The drop hangs from
the coverslip, and the petroleum jelly
forms a seal that prevents evaporation.
This preparation gives good views of
microbial motility.

Materials required:
• Glass slides (glass slide with depression) or
Normal glass slide with adhesive or paraffin
ring
• Paraffin wax
• Loop
• Coverslip
• Microscope
• Bunsen burner
• Young broth culture of motile bacteria

Growth and Nutrition of Bacteria
• The bacterial cell has the same general chemical pattern as
the cells of other organisms.
• The bacterial cell contains water (80% of total weight),
proteins, polysaccharides, lipids, nucleic acids, mucopeptides
and low molecular weight compounds.
• Bacteria can be classified nutritionally based on their energy
requirements and on their ability to synthesize essential
metabolites.
• Bacteria which derive energy from sunlight are called
phototrophs.
• Those who obtain energy from chemical reactions are called
chemotrophs.
• Bacteria that can synthesize all their organic compounds are
called autotrophs.
• They are able to use atmospheric carbon dioxide and nitrogen

Bacterial Cell Division
• Bacterial binary fission is the process that bacteria use to carry
out cell division.
• In this cell division, bacteria reproduce, or add more bacteria
to the population.
Process
1-DNA by replication enzymes begins at a
spot on the chromosome called the origin of
replication.
2-The origin is the first part of the DNA to
be copied. As replication continues, the two
origins move towards opposite ends of the
cell, pulling the rest of the chromosome
along with them. The cell also gets longer,
adding to the separation of the newly
forming chromosomes.

Cont…
3-Replication continues until the
entire chromosome is copied and
the replication enzymes meet at
the far side. Once the new
chromosomes have moved to
opposite cell ends and cleared the
center of the cell, division of the
cytoplasm can take place.
4- The membrane pinches inward
and a septum, or new dividing
wall, forms down the middle of
the cell.
5-Finally, the septum itself splits
down the middle, and the two cells
are released to continue their lives
as individual bacteria.

Bacterial Growth Curve
• In higher organism growth refers as increase in size and
volume of organism but in bacteria growth refers as increase
in number.
• When fresh liquid medium is inoculated with a given number
of bacteria and incubated for sufficient period of time, it gives
a characteristic growth pattern of bacteria.
• If the bacterial population is measured periodically and log of
number of viable bacteria is plotted in a graph against time, it
gives a characteristic growth curve which is known as growth
curve or growth cycle.
The growth curve has following phases:-
1- Lag phase 3- Stationary phase
2- Log phase or exponential phase 4- Death phase or decline phase

1- Lag phase
 When bacteria is inoculated into new fresh media, it
do not divide immediately. Bacteria takes some time to
adjust to the new environment. The time period in
which bacteria is metabolically active but do not
divide is called as lag phase.
 Size of bacteria increase continuously so the bacteria
have largest size at the end of lag phase.
 It is the phase of adjustment necessary for the synthesis
of enzymes and co-enzymes for physiological activities.
 At the end of lag phase, bacteria become fully prepared
for cell division.

2- Log phase or exponential phase
 During this phase bacteria divides continuously at constant
rate and the number of bacteria increase exponentially.
 In this phase all bacteria are in their rapid stage of cell
division and show balanced growth.
 Due to rapid cell division, bacteria have smallest size in this
phase.
 Biochemical and physiological characteristics are
commonly used for identification of bacteria are manifested
during log phase of growth.
 Generation time of bacteria is usually determined during log
phase
 Generation time is shortest during log phase and is strongly
dependent upon growth factors present in the medium.

3- Stationary phase
 The bacteria growth reaches a state during which there
is no net increase in bacterial population. This is called as
stationary phase.
 In this phase a constant bacterial population is
maintained by balance between cell division and cell
death.
 In endospore forming bacteria, sporulation occur as the
bacteria enter stationary phase.

4- Death phase or decline phase
 In this phase, number of bacteria decrease
continuously and exponentially.
 During this phase, total count of bacteria may remain
constant but the viable count decreases.
 It is just inverse of log phase. But the death rate is
slower than growth rate.
 Death phase is brought about by various reasons, such
as depletion of nutrition and accumulation of toxic
wastes.
 Not all bacteria die at same rate, some die faster and
some are more resistant and remain viable for longer
time. Eg. Spore forming bacteria.

Introduction to Microbiology

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