Microbilogy

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INTRODUCTIONThe 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 vanleeuwenhoek.

Scientific development of microbiology wasushered by Louis Pasture , perfection onmicrobiologycal studies occurred by RobertKoch & Lord Lister introduced theantiseptic 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 microbiology1. 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 anthracisDemostration: 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-acetylmuramicacid & 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 wallI ) 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 ribotolor 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 justbeneath the cell wall that is called as cytoplasmicmembrane”

The whole bacterial cytoplasm is bound peripherallyby very thin , elastic and semipermiable cytoplasmicmembrane also known as cell membrane.

It is 5-10nm in width

Electron microscope shows the presence of three layerconstituting a unit membrane structure.

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Chemically the membrane consist of phospholipidwith small amount of protein.

DEMONSTRATION:

The separation of membrane from cell wall isachieved by readily in gram negative bacteria whenthey are suspended in medium of high osmotictension. such phenomenon is called asplasmolysis.

Electron microscope.

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Function Transport:

(1) Active transport:

it is site of numerous enzymes (oxidase,polymerase, permease) involved in the active transportof selective nutrients. It is impermeable tomacromolecules & ionised substances.

(2) Passive transport:

It is act as semipermiable membrane throughinward and outward passage of water and passivetransport of molecule lipid soluble solutes take placeby diffusion.

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(2) Concentration:

it is also concentration sugar, amino acidsand phosphate so that a 300-400 fold gradientexists across osmotic barrier.

(3) Enzymatic function:

it also contain cytochrome oxidase, enzyme oftricarboxylic acid cycle and polymerizing enzymenecessary 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 mesosomes1) 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 unfavourableenvironmental condition such as starvation anddessication.”

As the spores are formed within the parentbacterial cell so they are also called as endospores.

During germination each spore give rise to onlyone vegetative bacteria.

Exospores found in fungi(conidia) formedextracellularly from end of parent cells.

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Spore forming bacteriaA. 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 PROCESSSpore formation is initiated by appearance of clear area in portionof protoplasm near one end of bacterial cell and that protoplasmgradually become more opaque that form fore spore

The cell membrane grow inwards and undergoes in folding formingdouble layered membrane structure around the core

The inner most layer of spore wall forms spore membrane in futurevegetative bacterium will develop. The spore wall synthesis a thickcovering layer cortex and multilayered thin but tough outer layerspore coat

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Conti……Spores of some spesis have additional apparently ratherloose outer covering called exosporium

Spore cortex contains unusual type of peptidoglycan sensetive tolysosomes the spore cot is made up of keratin which is impervious toantibacterial chemical agent

Exosporium is a lipoprotein membrane with some carbohydrateresidue. 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 ofspores remain constant within a particular bacteria.

Spore may be central , sub terminal or terminal inposition.

Its diameter may be same or less than the width of thebacteria.

It may be oval or spherical.

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Resistance Bacterial spores are resistant to ordinary boiling,

heating, and disinfectant. They can withstandboiling up to 3 hr, dry heat at 150˚c for 1 hr howeverthey are destroyed by autoclaving at 121˚c for 15-20min.

The highly impervious spore coat, low watercontent, low metabolic activity and highconcentration of calcium dipicolinate of sporemake 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. ActivationThe germination of bacterial spore do not

occur even when placed in environment thatfavours process.

Unless first activated by one or anotheragent damage the coat of spore such as heat,abrasion and compound containing freesulphydryl groups.

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2.InitiationThe process of initiation is not clear,

however the spore will initiate germinationin favorable condition.

Different species of bacteria recognizesdifferent effectors as signalling a richmedium 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 openthe spore coat.

The new vegetative cell consist of spore protoplast withits surrounding wall.

This is followed by a period of active biosynthesisproducing an outgrowth.

Outgrowth is denoted as the stage from germinationup to the formation of vegetative cell & prior to firstcell 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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Education

Microbilogy