Microbilogy

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INTRODUCTION
 The first simple forms of life appeared on
earth more then 3 billion years ago.
 Their descendants have changed &
developed into the several million type of
animals , plants & microorganisms are
recognized.
 Microscopic forms of life are present in vast
numbers in nearly every environment like
soil, water, food, air , etc.
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Microbiology means…..
 this is the science
dealing with the study of
microorganisms.
Microbiology is the science of living
organisms that are not directly visible to
necked eye but only under the microscop.
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Historical events…..
 The credit for observation & description of
bacteria goes to a biologist Antony van
leeuwenhoek.
 Scientific development of microbiology was
ushered by Louis Pasture , perfection on
microbiologycal studies occurred by Robert
Koch & Lord Lister introduced the
antiseptic surgery.
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 Louis Pasteur (1822-1895) was trained
chemist. His studies on fermentation of
wine led him to take interest in
microbiology. He is known as the founder or
father of modern microbiology .
 Joseph Lister was a professor of surgery. He
applied Pasteur’s work & introduced
antiseptic technique in surgery for killing
bacteria in wounds & in the air with carbolic
acid. He is known as father of antiseptic
surgery.
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 Robert Koch(1843-1910),German general
practitioner , perfected bacteriological
techniques & introduced methods for
isolation of pure strains of bacteria. He
introduced staining techniques.
 He is known as father of medical
microbiology.
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Branches of microbiology
1. Medical microbiology
2. Industrial microbiology
3. Food microbiology
4. soil microbiology
5. Plant microbiology
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 also we connected with medical microbiology. It
studied under following headings:
a. Parasitology deals with the study of parasites causing
disease in human being.
b. Mycology deals with the study of fungus causing
disease in human being.
c. Immunology is connected with mechanism involved
in the development of resistance by body to
infectious disease.
d. Bacteriology deals with the study of becteria.
e. Genetics is the study of heredity & variations.
f. Virology is the study of viruses.
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Scope of microbiology
 1.Diagnostic
isolation & identification of causative
organism from pathological lesions.
Widal’s test – typhoid fever
2. Prognosis of disease
in widal’s test rising titer signifies active
disease & ineffective treatment . falling titer
means effective treatment & curing of disease.
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 3. Guidance in treatment
by culturing the organisms in
pure form & then performing drug
sensitivity test that can suggest the effective
drug for the treatment of that particular
infection.
4. Source of infection
in sudden outbreak of
infectious disease we can find out the source
of infection.
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 Bacteria are unicellular free living organisms without
chlorophyll having both DNA and RNA.
 They are capable of performing all essential processes
of life, e.g. growth, metabolism and reproduction.
 They have rigid cell wall containing muramic acid.
They were originally classified under plant and animal
kingdom.
 This being unsatisfactory a third kingdom PROTISTA
was proposed by Hackel in 1866 for them.
 Protista is again divided into 2 groups:
A) Prokaryotes
B) Eukaryotes
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 Bacteria and green algae (photosynthetic
and possess chlorophyll which can exhibit
gliding movement like photosynthetic
bacteria) are prokaryotes while fungi, algae,
slime moulds and protozoa are eukaryotes.
 It is worth mentioning to enumerate the
differences between prokaryotic and
eukaryotic cell.
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Bacterial Anatomy
 The outer layer or cell envelope of bacterial cell
consists of two components
 A) A rigid cell wall
 B) underlying cytoplasmic membrane (plasma
membrane)
 The cell wall encloses the protoplasm comprising of
cytoplasm (ribosomes inclusion granules mesosomes
and nuclear body) and single circular chromosome of
DNA.
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Some bacteria in addition may possess
additional structure such as protective
gelatinous covering outside the cell
wall known as capsule.
When it is too thing it is known as
microcapsule
Apart from this some bacteria possess
filamentous appendages flagella and
fimbria which protruded from cell
surface.
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capsule
 It is an outer covering of thick, jelly-like material that
surrounds the bacterial cell.
 It contain: a) 0.2 µm width
b) 98% water
c) 2% solids
 Capsules which are much narrower than true capsule
and cannot be demonstrated by light microscope are
called microcapsules.
E.g. N. meningititidis; H. influenzae.
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Function:
 Capsule serves as protective covering against anti bacterial
substance such as bacteriophage, phagocytes and enzymes.
 it enhance bacterial virulence
 Capsular antigen is hapten in nature and specific for the
bacteria.
Capsulated organisms:
 H. Enfluenza, S. pneumoniae, bacillus anthracis
Demostration:
 The capsule is best seen in pathological specimence like
pus, blood, sputum & exudate.
 In artificial culture medium, the size of capsule is reduced
and finaly inlatter stages of growth the capsule may
disappear due to accumulation of capsule degrating
enzymes or due to carbon or energy starvation.
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Demonstration
 A) negative staining with India ink: in this procedure,
bacterial bodies and space in between are filled with
India ink and capsule is seen as halo around cell.
 B)Special capsule staining technique using copper salt
as moderant.
 C) Serological method: If suspension of capsulated
bacterium is mixed with its specific serum and
examine under microscope, capsule becomes
prominent and appears swollen due to increase in
refractivity (Quellung reaction).
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Cell wall
 Cell wall is a tough & rigid structure surrounding
the bacterium like a shell.
 The cell wall is 10-20nm in thickness & confers
rigidity upon bacteria in addition to giving
protection to the cell against osmotic damage.
 It 20 to 25% of the dry weight of the bacterial cell.
 It is elastic & porous also freely permeable to solute
molecules of less than 10,000 molecular weight.
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 The cell wall takes part in cell division by
forming an ingrowths from the cell wall.
 The mucopeptide component of cell wall
possess target site for antibiotics , lysozymes
& bacteriophages.
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Structure of cell wall
 The chemical structure of cell wall of gram positive
& gram negative bacteria is different.
 The rigid part of the cell wall is peptidoglycan.
 A) peptidoglycan (murein)
it is the principal structural component of
the cell wall . It is present in both gram positive &
negative bacteria.
I ) lipoprotein layer-
it connects the outer membrane
to peptidoglycan.
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 Peptidoglycan structure-
the peptidoglycan polymers
consist of alternating stands of N-acetylmuramic
acid & N-acatylglucosamine .
The N-acetylmuramic acid links the disaccharides
to an oligopeptide chain consisting of four amino
acids.
 Peptidoglycan cross-linking involves the cross-
linking
between the terminal residue of the peptide side
chain with the penultimate residue of an adjoining
side chain.
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B)autolysins- all bacterial cell wall
possess associated enzymes called
autolysins which can hydrolyze their
own cell wall substance .
The capacity of autolysins to dissolve
the peptidoglycan in essential for cell
growth, cell septation, sporulation, &
in transformation.
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Gram-positive cell wall
I ) The peptidoglycan layer of gram positive bacteria is
much as 50% of the dry weight of the cell wall & 10% of
the total cell much thicker(15-25nm) than in gram
negative bacteria(10-15nm) & it is consist of multiple
layers.
 The periplasmic space is absent & the peptidoglycan is
closely associated with the cytoplasmic membrane.
II)Special components: cell wall contain……
significant amount of teichoid & teichuronic
acids as.
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 They are water soluble polymers containing ribotol
or glycerol polymers & maintain the level of
divalent cations outside the cell membrane.
 The teichoic acids constitute major surface
antigens of grampositive bacteria.
III) Other components:
certain gram positive cell walls also
contain antigens such as the polysaccharide &
protein.
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Gram negative cell wall
 This cell wall is a complex structure & thinner than
that of gram-positive cell with a bimolecular layer
of peptidoglycan.
Outer membrane:
it is an additional outer membrane &
much thicker than the single peptidoglycan layer .
It lies above the peptidoglycan layer .
it is made of phospholipid bilayer
proteins
lipopolysaccharide (LPS)
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Bacteria with defective cell wall
 Removal of cell wall result in lyses of bacteria.
 1) gram positive bacteria-
complete removal of cell wall of gram + bacteria
result in the formation of is protoplast . it lyses unless it
is osmotically stabilised . the protoplast constituted by the
cytoplasmic membrane & bacterial contents
2)Gram negative bacteria-
the complexity of cell wall result in resistance
to enzymatic destruction of cell wall . that gram- bacteria
with damaged cell wall become spheroplast.
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Difference between gram pos.& gram
Neg.
No. Contain Gram positive Gram negative
1 Thickness 15-25 10-15
2 Varity of amino acid Few several
3 Aromatic & sulfur
containing amino
acids
Absent Present
4 Lipids Low 2 to 4% High 15 to 20%
5 Teichoid acids Present Absent
6 Periplasmic space Absent Present
7 Result of enzyme
digestion
protoplast spheroplast
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Demonstration
 When intact bacteria are placed in a solution of very high
solute concentrations & osmotic pressure ,protoplast
shrinks thus retracting the cytoplsmic membrane from cell
wall . The process is called plasmolysois & is useful in
demonstrating cell wall.
 Cell wall may also be demonstrated by a special technique
is called micro dissection.
 Reaction with specific antibody is also a way to study cell
wall.
 Electron microscope gives detailed structural information
of even very particles like parts of cell wall.
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Function
 1) protection of internal structure
 2)Gives shape to the cell
 3) confers rigidity & ductility
 4) role in division of bacteria
 5) offers resistance to harmful effect of environment.
 6) contains receptor sites for phages & colicin
 7)Provide attachment to complement
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Cytoplasmic membrane
 Definition:
“thin semipermiable membrane which lies just
beneath the cell wall that is called as cytoplasmic
membrane”
The whole bacterial cytoplasm is bound peripherally
by very thin , elastic and semipermiable cytoplasmic
membrane also known as cell membrane.
It is 5-10nm in width
Electron microscope shows the presence of three layer
constituting a unit membrane structure.
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 Chemically the membrane consist of phospholipid
with small amount of protein.
 DEMONSTRATION:
The separation of membrane from cell wall is
achieved by readily in gram negative bacteria when
they are suspended in medium of high osmotic
tension. such phenomenon is called as
plasmolysis.
Electron microscope.
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Function
 Transport:
(1) Active transport:
it is site of numerous enzymes (oxidase,
polymerase, permease) involved in the active transport
of selective nutrients. It is impermeable to
macromolecules & ionised substances.
(2) Passive transport:
It is act as semipermiable membrane through
inward and outward passage of water and passive
transport of molecule lipid soluble solutes take place
by diffusion.
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(2) Concentration:
it is also concentration sugar, amino acids
and phosphate so that a 300-400 fold gradient
exists across osmotic barrier.
(3) Enzymatic function:
it also contain cytochrome oxidase, enzyme of
tricarboxylic acid cycle and polymerizing enzyme
necessary for synthesis of cell wall.
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Cytoplasm
 The bacterial cytoplasm is suspension of
organic & inorganic solutes in viscous
watery solution.
 It dose not exhibit protoplasmic streaming
(internal mobility) .
 It lacks endoplasmic reticulum or
mitochondria.
 It contains ribosomes, mesosomes,
inclusions,& vacuoles.
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Ribosome
 Ribosome appear as small granules & pack the whole cytoplasm.
 The ribosomal particles become linked up & travel along the
mRNA stand.
 Ribonucleoprotein granules measure 10 to 20nm units in
diameter & their sedimentation coefficient is 70 Svedberg units.
The 70s ribosome is composed of two smaller units of 50s &30s.
 These are strung together on stands of mRNA to from polymers.
 The code of mRNA is translated into peptide sequence at this
place. the ribosomal particles become linked up & travel along
the mRNA stand . it determines the sequence of amino acids
brought to the site on tRNA molecules & build up the
polypeptide.
 FUNCTION-they are site of protein synthesis.
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 Polysomes-
they are group of ribosome's linked together like beads of
chain by messenger RNA .
 Mesosomes- they are vesicular , convoluted , or multilaminated
structure.
 They are prominent in gram-positive bacteria.
 Two types- 1) septal mesosomes
2)lateral mesosomes
1) The septal mesosomes is attached to bacterial chromosome & is
involved in DNA segregation & the formation of cross walls during cell
division. They are also called as chondroids & are visualized under
electron microscop.
2) The lateral mesosomes in lateral site.
Function-
1) They are the sites of respiratory enzymes in bacteria.
2) Coordinate nuclear & cytoplasmic division during binary fission.
3) Responsible for compartmenting DNA at sporulation.
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Nucleus
 It is a long filament of DNA rightly coiled inside the
cytoplasm.
 the bacterial nucleus is not surrounded by nuclear
membrane.
 The nuclear DNA dose not appear to contain some
basic protein .
 Nucleus cannot be demonstrated under direct light
microscope. It appears as oval or elongated body ,
generally one per cell.
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 The genome consist of a single molecule of double
stranded DNA arranged in the form of circle.
 It may open under certain conditions to from long chain
about 1000 µ in length.
 The bacterial chromosome is haploid & replicates by
simple fission .
 Genes are arranged along the length of chromosome in
fixed order.
 Bacteria may sometimes have extra nuclear genetic
material these are called plasmid or episomes.
 Plasmids are not essential for the life of cell. they may
confer certain properties like toxigenecity virulence & drug
resistance.
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Flagella
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Flagella
 Parts-
These are long, sinnous contractile filamentous
appendages known as flagella.
These are organs of locomotion . ex:-Escherichia coli
salmonella, vibrio , pseudomonas, etc.
The number of flagella varies up to 10 to 20 per cells
according to species of bacteria.
These are extremely thin (diameter)12 to 30 nm, helical
shaped structure of uniform diameter throughout their
length . these are 3 to 20 nm long.
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Each flagellum consist of hook & basal body.
It originates in a spherical body located just
inside cell wall.
These are antigenic & are composed of
protein called flagellin which has
properties of fibrous protein , keratin , &
myosin.
The number & arrangement of flagella are
characteristic of each bacteria.
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 Types :-
There are 4 types of flagellar
distribution on bacteria.
Monotrichous- singal polar
flagellum, ex.-cholera vibrios .
Amphitrichous- single flagellum
attached to each end , ex.-
alcaligenes fecales.
Lophotrichous- tufts of flagella
at the end , ex.- pseudomonas ,
spirilla.
Peritrichous- numerous flagella
all over the bacterial body , ex.-
typhoid bacilli.
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Function
It is responsible for bacterial motility.
Motility may be observed microscopically or by
detecting the spreading growth in semi solid agar
medium.
Demonstration –
Dark ground microscopy.
Special staining techniques in which their thickness is
increased by mordanting.
Electron microscop.
Hanging drop preparation.
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Fimbria (Pili)
 Fimbria are filamentous , short , thin , straight , hair like
appendage.
 This is 0.1 to 1.5 µ long & less than 4 to 8 nm thick.
 They are also called as Pili.
 Fimbriae are seen only in some gram negative bacteria.
 Each bacterium may have 100 to 500 Fimbriae on all over
the body of bacteria.
 They project from cell surface as a straight filaments.
 They are best developed in freshly isolated strains & in
liquid culture.
 They are composed of protein known as pillin (molecular
weight 18000 Daltons).
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 Different forms of fimbria –
i)common pili
ii)F (fertility) pili
iii) Col I (colicin)pili
i)common pili-
They are numerous, short in size(1.5µ) & Peritrichous in
distribution. Ex.- red cell of various animal species.
Common pile are of 6 types based on their morphology
number per cell, adhesive properties, & antigenic nature.
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 TYPE:-
 TYPE 1:-It is relatively thick & involved in hem
agglutinating activity.
 TYPE 2:- They are same as type 1 but they are non-
hem agglutinating.
 TYPE 3:- They are thin & are mannose resistance.
They are confer hem agglutinating activity on
parent cell.
 TYPE 4:-They are thinner than type 3 & are
mannose resistance with hem agglutinating
activity on fresh red blood cell.
 Type 5:-They are monopolar which have been
found only in one species of pseudomonas.
 Type 6:-They are very long & few of them are seen
in one species of klebsiella.
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 F pili:-
they are associated with fertility & help in
bacterial conjugation process . They are longer (20µ
length) than common & Col I pili.
 Col I pili:-they are about 2µ in length & associated
with colicin factor I.
 DEMONSTRATION:-
 Electron microscop.
 Hem agglutination.
 Fimbriated bacteria form pellicle in liquid media.
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 FUNCTION:-
a) Organ of adhesion.
b) Hem agglutination.
c) They are antigenic.
d) Agglutination & pellicle formation.
e) Genetic material is transferred from the donor to
recipient cell.
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SR
NO
FLEGELLA FIMBRIAE
1 Size larger & thicker. Smaller & thinner.
2 Arise from the cytoplasm or
cytoplasmic membrane but not
attached to the cell wall.
Attached to the cell wall.
3 Organ of movement. Organ of adhesion &
conjugation.
4 They are never straight. They are always straight.
5 No require for conjugation. Required for conjugation.
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SPORES
“spores are highly resistant dormant
stage of bacteria formed in unfavourable
environmental condition such as starvation and
dessication.”
As the spores are formed within the parent
bacterial cell so they are also called as endospores.
During germination each spore give rise to only
one vegetative bacteria.
Exospores found in fungi(conidia) formed
extracellularly from end of parent cells.
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Spore forming bacteria
A. Gram positive bacilli:
(1) Obliterate aerobic- genus bacillus.
E.g:- B. anthracis, B. subtilis.
(2)Obliterate anaerobic : genus clostridia.
E.g:-C.tetani,C.welchii,C.botulinum
B. Other bacteria:
Gram positive coccus (sporosarcina)
Gram negative bacilli(coxiella burnetii)
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Spore morphology
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Sporogenesis
 spontaneous sporulation occurs in
condition unfavorable condition such
as starvation, dessication, presence of
disinfectants and in extreme
temperature.
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SPOROGENESIS PROCESS
Spore formation is initiated by appearance of clear area in portion
of protoplasm near one end of bacterial cell and that protoplasm
gradually become more opaque that form fore spore
The cell membrane grow inwards and undergoes in folding forming
double layered membrane structure around the core
The inner most layer of spore wall forms spore membrane in future
vegetative bacterium will develop. The spore wall synthesis a thick
covering layer cortex and multilayered thin but tough outer layer
spore coat
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Conti……
Spores of some spesis have additional apparently rather
loose outer covering called exosporium
Spore cortex contains unusual type of peptidoglycan sensetive to
lysosomes the spore cot is made up of keratin which is impervious to
antibacterial chemical agent
Exosporium is a lipoprotein membrane with some carbohydrate
residue. Young spores remain attached to parent cell
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Shape and position
 The young spore remain attached to the parent cell.
The precise position and shape and relative size of
spores remain constant within a particular bacteria.
 Spore may be central , sub terminal or terminal in
position.
 Its diameter may be same or less than the width of the
bacteria.
 It may be oval or spherical.
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Resistance
 Bacterial spores are resistant to ordinary boiling,
heating, and disinfectant. They can withstand
boiling up to 3 hr, dry heat at 150˚c for 1 hr however
they are destroyed by autoclaving at 121˚c for 15-20
min.
 The highly impervious spore coat, low water
content, low metabolic activity and high
concentration of calcium dipicolinate of spore
make resistant to drying and heating.
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Germination
 Definition:
“ The process of conversation of spore in to
vegetative cell under suitable environment is known as
germination”
There are three stages of germination……
 A. activation
 B. initiation
 C. outgrowth
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1. Activation
 The germination of bacterial spore do not
occur even when placed in environment that
favours process.
 Unless first activated by one or another
agent damage the coat of spore such as heat,
abrasion and compound containing free
sulphydryl groups.
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2.Initiation
 The process of initiation is not clear,
however the spore will initiate germination
in favorable condition.
 Different species of bacteria recognizes
different effectors as signalling a rich
medium such as L-alanine for one species &
adenosine for another species.
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3. Out growth
 With the swelling of spore wall and disintegration of
cortex a single germ cell emerge after breaking open
the spore coat.
 The new vegetative cell consist of spore protoplast with
its surrounding wall.
 This is followed by a period of active biosynthesis
producing an outgrowth.
 Outgrowth is denoted as the stage from germination
up to the formation of vegetative cell & prior to first
cell division.
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Demonstration
 By ordinary stain and modified Z-N stain.
 Laboratory use:
for making sterilization
1. B. stearothermophilus-destroyed at a tem.
of 121˚c in 10-20 min.
2. B. subtilis-destroyed at 105˚c in 5 min.
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Microbilogy

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Microbilogy