MORPHOLOGY OF BACTERIA

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morphology of bacteria

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BY Dr.S.KRITHIKAA I Yr MD MICROBIOLOGY MORPHOLOGY OF BACTERIA

CLASSIFICATION OF MICROORGANISMS:

CLASSIFICATION OF MICROORGANISMS MICROORGANISMS PLANTS PROTISTA ANIMALS PROKARYOTES EUKARYOTES Bacteria & blue green fungi, algae, slime algae moulds& protozoa

PROPERTIES OF PROKAYOTIC &EUKARYOTIC CELLS:

PROPERTIES OF PROKAYOTIC &EUKARYOTIC CELLS

STRUCTURE OF PROKARYOTIC & EUKARYOTIC CELLS:

STRUCTURE OF PROKARYOTIC & EUKARYOTIC CELLS

MORPHOLOGY OF A BACTERIAL CELL:

MORPHOLOGY OF A BACTERIAL CELL SIZE SHAPE & ARRANGEMENT ANATOMICAL STRUCTURES - CELL WALL - CELL MEMBRANE - CYTOPLASM - SLIME LAYER & CAPSULE APPENDAGES- FLAGELLA & FIMBRIAE SPORES

SIZE:

SIZE 3-5 *0.2-1.5 µm EPULOPISCIUM FISHELSONII- largest bacterium of size 600* 80 µm DEMONSTRATION- electron microscopy, coulter chamber, counting chamber, epifluroscence technique

SHAPE :

SHAPE COCCI- spherical or oval cells BACILLI- rod shaped COCCOBACILLI-length is same as width SPIROCHAETES- slender flexuous spiral forms VIBRIOS- comma shaped SPIRILLA- rigid spiral forms ACTINOMYCETES- branching filamentous bacteria resembling fungi MYCOPLASMA- round or oval bodies & interlacing filaments (cell wall deficient)

ARRANGEMENT:

ARRANGEMENT STREPTO- chains DIPLO- pairs STAPHYLO- clusters TETRADS- groups of four SARCINA- groups of eight CUNEIFORM- chinese letter pattern

DEMONSTRATION:

DEMONSTRATION Simple staining- crystal violet gentian violet carbol fuschin , saffranin differential staining – grams stain acid fast stain

DIFFERENT SHAPES AND ARRANGEMENT:

DIFFERENT SHAPES AND ARRANGEMENT

ANATOMY :

ANATOMY

CELL WALL:

CELL WALL Tough & rigid structure Made of peptidoglycan – mucopeptide ( murein ) composed of N-acetyl muramic acid & N- acetyl glucosamine alternating in chains cross linked by peptide subunits

GRAM POSITIVE CELL WALL:

GRAM POSITIVE CELL WALL tightly cross linked peptides- d- alanine & d- glutamic acid Thickness- 18-80 nm & constitutes 40-80% of the dry weight Techoic acid- water soluble polymers of glycerol phosphate or ribitol phosphate residues two types – wall techoic acid- ribitol &membrane techoic acid- glycerol functions- cell wall stability, association of wall with membrane, adherence, reproduction

CONTD…:

CONTD… Polysaccharides- mannose, arabinose , rhamnose , glucuronic acid & mannuronic acid Thick peptidoglycan layer S LAYER- protein or glycoprotein molecules that self assemble on the outer surface of the organism. Protect from stressful environments, inhibit phagocytosis , contribute to virulence

PEPTIDOGLYCAN LAYER OF GRAM POSITIVE CELL WALL:

PEPTIDOGLYCAN LAYER OF GRAM POSITIVE CELL WALL

GRAM NEGATIVE CELL WALL:

GRAM NEGATIVE CELL WALL Thickness- 3-4 nm Loosely crosslinked by d- diaminipimelic acid or lysine Outer membrane- bilayered LPS containing OMPs – porins , OmpC , D, F, PhoE , LamB,Tsx contains 3 regions- I- polysaccharide determining O ag specificity II- core polysaccharide containing 3-deoxy-D- mannulooctulusonate (KDO) & heptose III- glucolipid responsible for endotoxicity

CONTD...:

CONTD... Lipoprotein layer- connects peptidoglycan to outer membrane & stabilizes the outer membrane Periplasmic space- space between inner& outer membranes containing the peptidoglycan layer and gel like solution of proteins & membrane derived oligosaccharides Thin peptidoglycan layer

PORINS:

PORINS

PEPTIDOGLYCAN OF GRAM NEGATIVE CELL WALL:

PEPTIDOGLYCAN OF GRAM NEGATIVE CELL WALL

ACID FAST CELL WALL:

ACID FAST CELL WALL Lipids constitute 60% of the dry weight & are made of mycolic acid Responsible for virulence, cytotoxicity , granuloma formation, adjuvanticity Also contains phosphatidylinositol mannosides & lipoarabinimannan Polysaccharide- arabinogalactans

CONTD…:

CONTD… Trehalose sulpholipid - 2,3,6,6’ tetracylhelose-2 sulphate - prevents phagosome lysozyme fusion fusion following phagocytosis Proteins- act as porins & involved in biosynthesis of cell wall polymers

STRUCTURE OF ACID FAST CELL WALL:

STRUCTURE OF ACID FAST CELL WALL

FUNCTIONS OF CELL WALL:

FUNCTIONS OF CELL WALL Accounts for the shape of the cell Protects the cell against osmotic damage Confers rigidity Cell division Target site for antibiotics, lysozymes , bacteriophages Carries bacterial antigens

DEMONSTRATION OF CELL WALL:

DEMONSTRATION OF CELL WALL Plasmolysis Microdissection Raection with specific antibody Electron microscopy Indirect methods- grams staining & acid fast staining, fluroscent staining for acid fast bacteria

DEMONSTRATION OF CELL WALL:

DEMONSTRATION OF CELL WALL

GRAMS STAINING:

GRAMS STAINING MECHANISM- permiability of bacterial cell wall integrity of cell wall METHOD- to a heat fixed smear add crystal violet-1min grams iodine-1 min acetone-2-3sec saffranin - 1min

CONTD…:

CONTD… MODIFICATIONS- Kopeloff and Beerman’s method for films & sections, Jensons , Preston & Morrel , quick gram, multiple slide Gram positive- violet Gram negative- pink

GRAMS STAINING PROCEDURE:

GRAMS STAINING PROCEDURE

ZIEHL NEELSON METHOD OF ACID FAST STAINING:

ZIEHL NEELSON METHOD OF ACID FAST STAINING PRINCIPLE- mycolic acid, lipds , fatty acids& higher alchols integrity of the cell wall METHOD- to a heat fixed smear add carbol fuschin - 5-7min and apply gentle heat 20% sulphuric acid- 2-3sec methylene blue 1-2min

CONTD…:

CONTD… MODIFICATIONS- cold method, 5%sulphuric acid Bacilli- stain pink

DIFFERENCE BETWEEN GM+VE &GM-VE CELLS:

DIFFERENCE BETWEEN GM+VE &GM-VE CELLS

CELL WALL DEFICIENT FORMS:

CELL WALL DEFICIENT FORMS MYCOPLASMA- stable oval or round forms L- FORMS- observed in streptobacillus monoliformis . Induced by penicillin PROTOPLASTS- gram positive bacteria when placed in hypertonic saline SPHEROPLASTS- gram negative bacteria when subjected to penicillin. Some cell wall material is retained PLEIOMORPHIC & INVOLUTION FORMS- swollen & aberrant forms resulting from ageing

CELL MEMBRANE:

CELL MEMBRANE 5-10nm thick elastic membrane beneath the cell wall separating it from cytoplasm Composed of lipoprotein. Sterols absent except in mycoplasma Permeases - membrane associated carrier proteins FUNCTIONS- selective permiability and transport of solutes electron transport and oxidative phosphorylation excretion bearing the enzymes and carrier molecules for biosynthesis

BACTERIAL CELL MEMBRANE:

BACTERIAL CELL MEMBRANE

CYTOPLASM:

CYTOPLASM Colloid of organic and inorganic solutes in viscous watery solution RIBOSOMES- centres of protein synmthesis . Composed of rRNA of size 10 - 20nm with a sedimentation constant of 70S MESOSOMES- vesicular, convoluted invaginations from plasma membrane. more prominent in GPB principal site of respiratory enzyme site of synthesis of cross wall septa during binary fission

INCLUSIONS:

INCLUSIONS VOLUTIN- ( BABES ERNST GRANULES) highly refractile , strongly basophilic bodies consisting of polymetaphosphate . stained by Albert or Neisser stain present in diphtheria bacilli reserve of energy POLYSACCHARIDE- stained with iodine LIPIDS- stained with sudan black VACUOLES- fluid filled cavities covered by a membrane

NUCLEUS:

NUCLEUS Contains the cell’s genome made of a single molecule of double stranded DNA arranged in the form of a circle . Measure about 1mm

PLASMIDS or EPISOMES:

PLASMIDS or EPISOMES Extranuclear genetic material Transmitted to daughter cells either by binary fission or from one bacterium to another by conjugation Confer properties like toxigenicity and drug resistance

TRANSPOSONS:

TRANSPOSONS Segments of DNA that can move around to different positions in the genome of a single cell. Responsible for mutations Types- ClassII - DNA segment gets transferred ClassI - DNA converts to RNA and gets transferred Bacterial transposons are proteins imparting antibiotic resistance when incorporated into a plasmid

TRANSPOSONS Tn5:

TRANSPOSONS Tn5

DEMONSTRATION OF NUCLEUS:

DEMONSTRATION OF NUCLEUS - acid or ribonuclease hydrolysis and subsequent staining of nuclear material by Feulgen stain specific for DNA appear as oval or elongated bodies

FUNCTIONS OF NUCLEUS:

FUNCTIONS OF NUCLEUS Binary fission conjugation

SLIME LAYER & CAPSULE:

SLIME LAYER & CAPSULE Amorphous viscid bacterial secretion surrounding the cell wall Loose undemarcated secretion – slime layer or glycocalyx Sharply defined structure- capsule

COMPOSITION OF CAPSULE:

COMPOSITION OF CAPSULE Homo or hetero Polysaccharides made of hexose and pentose sugars plus ribitol , glycerol and other sugar alchohols synthesised by the cell membrane with enzymes- glucosyl and fructosyl transferases producing an insoluble glucan matrix Anthrax bacilli- polypeptide

CAPSULAR FEATURES OF VARIOUS ORGANISMS:

CAPSULAR FEATURES OF VARIOUS ORGANISMS leuconostoc & klebsiella – slime layer pneumococcus - capsule meningococcus - microcapsule streptococcus salivarious - capsule & slime layer

FUNCTIONS OF CAPSULE:

FUNCTIONS OF CAPSULE enhances virulence protective covering increases invasiveness adhesion capsular antigen for identification of bacteria

DEMONSTRATION OF CAPSULE:

DEMONSTRATION OF CAPSULE Negative staining- india ink or nigrosin - capsule appears as a clear halo around the cell Positive staining- Welch method- hot crystal violet followed by rinsing with copper sulphate . Cell background appears dark blue and capsule much paler blue Modifications- Anthony’s , Hiss

CONTD…:

CONTD… Manevals method- background- congo red stain- Manevals solution capsule- unstained halo MACROSCOPY- encapsulated- smooth colonies unencapsulated - rough colonies

CONTD…:

CONTD… Serological methods- Quellung test- loopful of pneumococci + antiserum( antipneumococcal rabit sera) observe in oil immersion under phase contrast microscope capsules become refractile & visible , seperated from the coccal bodies by the width of the capsule

DEMONSTRATION OF CAPSULE:

DEMONSTRATION OF CAPSULE

APPENDAGES:

APPENDAGES

FLAGELLA:

FLAGELLA Long, filamentous appendages arising at the cytoplasmic membrane, protruding through the cell wall into the surrounding medium

ARRANGEMENT OF FLAGELLA:

ARRANGEMENT OF FLAGELLA monotrichous - single polar flagella amphitrichous - single flagellum at both the poles lophotrichous - tuft of flagella at one or both the ends peritrichous - flagella all around the cell

FLAGELLAR ARRANGEMENT:

FLAGELLAR ARRANGEMENT

MORPHOLOGY OF FLAGELLA:

MORPHOLOGY OF FLAGELLA SIZE- 5-20µm long, width-13-17nm PARTS- FILAMENT- made of flagellin semirigid , forms a left handed helix and exits the cell HOOK- Acts as a sleeve from which the filament emerges transmits rotatory motion from basal body to filament BASAL BODY- consists of M,S,P,L rings connected by a rod shaped structure in gram positive bacteria only 2 rings are seen

FLAGELLAR STRUCTURE:

FLAGELLAR STRUCTURE

ANTIGENIC PROPERTY:

ANTIGENIC PROPERTY PHASE VARIATION- 2 types of flagella due to expression of genes coding for 2 different flagellin proteins in the same bacteria flagellar antigen- H-antigen Endoflagellum - arises from one pole, wraps around the cell body interior to the cell. Eg :- vibro , spirochaetes

FUNCTIONS:

FUNCTIONS flagellar antigen H used for identification MOTILITY- impart spinning movement driven by the flow of protons into the cell down the gradient produced by the primary proton pump CHEMOTAXIS, AEROTAXIS, PHOTOTAXIS, ELECTRON ACCEPTOR TAXIS- movement of the cell towards the source of attracant by swimming, tumbling and reorienting itself to the attractant

DEMONSTRATION:

DEMONSTRATION DIRECT METHOD- dark field microscopy & electron microscopy Staining- Leifson method- basic fuschin - primary stain, tannic acid- mordant methylene blue- counter stain flagella stains as red to blue black Ryu method- solution I- phenol, tannic acid, saturated aluminium potassium sulphate 12 hydrate crystals

DIRECT METHODS:

DIRECT METHODS

CONTD…:

CONTD… Solution II- saturated solution of crystal violet Mix 10 parts of sol I & 1 part of sol II Stain for 1-5 min. Flagella & cell bodies stain violet IMPREGNATION METHODS- silver

CONTD…:

CONTD… INDIRECT METHODS- Swarming growth of proteus craiges tube method- spreading of bacteria on semi solid agar hanging drop preparation- motilty of the bacterium examined on a wet film under high power mannitol motility medium- fanning wet mount preparation

INDIRECT METHODS:

INDIRECT METHODS

FIMBRIAE OR PILI:

FIMBRIAE OR PILI Hair like appendages protruding from the cell as straight filaments Found in many gram positive and some gram negative bacteria SIZE- 0.1-1.0µm length, 10nm thick. Each cell possesses 100-500 fimbriae

CONTD…:

CONTD… ARRANGEMENT- peritrichous & helically arranged Possess antigenic property Composed of a protein fimbrillin ( pilin ) which form hollow tubes in the cell membrane ADHESINS- minor proteins on the tips of pili responsible for attachment.

TYPES:

TYPES Common pili - six types- I- responsible for adhesion and are mannose sensitive type II- mannose resistant Sex or fertility pili - long pili present in male bacteria of size 18-20 nm & are 1-4 in number. Helps in forming conjugation tubes for transferring genetic material to female cells Col I ( colicin ) pili

MOTILITY:

MOTILITY TWITCHING- motility established by pili . Bacterium moves in the direction of the adhering tip resulting in surface motility. Seen because the pili donot rotate & lack a basal body

FUNCTIONS:

FUNCTIONS adhesion antigenic property inhibiting phagocytosis transfer of genetic material

DEMONSTRATION:

DEMONSTRATION Electron microscopy Haemagglutination - tile test- drop of dense bacillary deposit + red cell suspension on a white tile at 3-5ºC . develops coarse clumping within a few seconds mannose 0.5% inhibits type I fimbrial haemagglutination RBC’s of guinea pigs, fowl, horses & pigs agglutinate strongly, sheep and human blood weakly and ox blood scarcely

DEMONSTRATION OF PILI:

DEMONSTRATION OF PILI

SPORES:

SPORES

CHARACTERISTICS:

CHARACTERISTICS Spherical or oval structures formed within the bacterial cell Represents the resting or dormant phase formed under unfavourable conditions related to depletion of exogenous nutrients Also called as endospores In sporulation each vegetative cell forms only one spore and during subsequent germination each spore gives rise to only one vegetative bacterium Bacillus and clostridia species form spores

MORPHOLOGY:

MORPHOLOGY CORE- it’s the spore protoplast. Contains nucleus, protein synthesizing apparatus, energy generating system based on glycolysis . Vegetative cell enzymes are increased in amount Contains large amounts of calcium dipicolinate responsible for resistance SPORE WALL- innermost layer surrounding the inner spore membrane. Made of peptidoglycan and forms cell wall CORTEX- Thickest layer made of peptidoglycan sensitive to lysozyme . Role in spore germination

CONTD…:

CONTD… COAT- keratin like protein containing many intermolecular disulphide bonds. Impermeable and provides resistance to antibacterial agents EXOSPORIUM- composed of lipids, proteins and carbohydrates. Consists of paracrystalline basal layer and hair like outer region

CROSS SECTION OF A SPORE:

CROSS SECTION OF A SPORE

SPORULATION:

SPORULATION Process by which spores are formed. Involves production of many new structures , enzymes and metabolites along with disappearance of many vegetative cell components -differentiation Spore composition determining genes are activated by association of RNA polymerase core protein with sigma factor Sporulation process takes about 7hrs under laboratory conditions

STAGES OF SPORULATION & GERMINATION:

STAGES OF SPORULATION & GERMINATION

STAGES OF GERMINATION:

STAGES OF GERMINATION ACTIVATION - spore coat gets damaged INITIATION- triggered by L- alanine or adenosine. Autolysin is secreted that degrades the cortex peptidoglycan . Water is taken up releasing calcium dipicolinate and degrades various spore components by hydrolytic enzymes OUTGROWTH- degradation of cortex and outer layers results in emergence of new vegetative cell

SHAPE &POSITON OF SPORES:

SHAPE &POSITON OF SPORES NON BULGING- diameter of the spore is same as or less than the width of bacteria BULGING- diameter is wider than the bacillary body POSITION- central subterminal terminal

DEMONSTRATION:

DEMONSTRATION GRAMS STAIN- spore appears as clear unstained ares within the cell ZIEHL NEELSON METHOD- 0.25% sulphuric acid is used. Stain red &bacilli blue MALACHITE GREEN STAIN-5% aqueous solution of malachite green- 1min saffranin or basic fuschin – 30sec spores- stain green& bacilli red

DEMONSTRATION OF SPORES:

DEMONSTRATION OF SPORES

Slide 82:

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