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  1. 1. Dr. KRITHIKAA SEKAR, MD Assistant Professor, Microbiology, SLMCH
  3. 3.  Unstained Preparation- hanging drop (motility), dark ground microscopy (Spirochates)  Simple staining (single stain)- crystal violet, gentian violet, carbol fuschin, saffranin  Negative staining- bacterial capsules (India Ink)  Impregnation Techniques- flagella (silver impregnation)  Differential staining – Grams stain, Acid fast stain, Albert’s Stain
  4. 4.  COCCI- spherical or oval cells- eg. Staphylococcus  BACILLI- rod shaped- eg. E.coli, Brucella,  COCCOBACILLI-length is same as width, eg.Klebsiella  SPIROCHAETES- slender flexuous spiral forms. Eg. Treponema  VIBRIOS- comma shaped  SPIRILLA- rigid spiral forms  ACTINOMYCETES- branching filamentous bacteria resembling fungi  MYCOPLASMA- round or oval bodies & interlacing filaments (cell wall deficient)  RICKETTSIAE and CHLAMYDIAE- small obligate parasites classified as viruses, but included in bacteria due to presence of cell wall, bacterial enzymes and structural similarities with bacteria
  5. 5.  STREPTO- chains  DIPLO- pairs  STAPHYLO- clusters  TETRADS- groups of four eg. Micrococci  SARCINA- groups of eight  CUNEIFORM- chinese letter pattern eg. Corynebacterium
  6. 6.  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
  7. 7.  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
  8. 8.  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
  9. 9.  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
  10. 10.  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
  11. 11.  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
  12. 12.  Plasmolysis  Microdissection  Reaction with specific antibody  Electron microscopy  Indirect methods- Grams staining & Acid fast staining, fluorescent staining for acid fast bacteria
  13. 13.  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
  14. 14.  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
  15. 15.  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
  16. 16.  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
  17. 17.  Contains the cell’s genome made of a single molecule of double stranded DNA arranged in the form of a circle . Measure about 1mm
  18. 18.  Extranuclear genetic material  Transmitted to daughter cells either by binary fission or from one bacterium to another by conjugation or bacteriophage  Confer properties like toxigenicity and drug resistance
  19. 19.  - acid or ribonuclease hydrolysis and subsequent staining of nuclear material by Feulgen stain specific for DNA appear as oval or elongated bodies  Binary fission and conjugation
  20. 20.  Amorphous viscid bacterial secretion surrounding the cell wall Loose undemarcated secretion – slime layer or glycocalyx Sharply defined structure- capsule
  21. 21.  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
  22. 22. leuconostoc & klebsiella– slime layer pneumococcus- capsule meningococcus- microcapsule streptococcus salivarious- capsule & slime layer
  23. 23. enhances virulence protective covering increases invasiveness adhesion capsular antigen for identification of bacteria
  24. 24.  Negative staining- india ink or nigrosin- capsule appears as a clear halo around the cell  Positive staining- B.anthracis- polychrome methylene blue- McFadyean capsule stain
  25. 25.  Manevals method- background- congo red stain- Manevals solution capsule- unstained halo MACROSCOPY- encapsulated- smooth colonies unencapsulated- rough colonies
  26. 26. Serological methods- Quellung test (Neufeld reaction)- 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
  27. 27.  Long, filamentous appendages arising at the cytoplasmic membrane, protruding through the cell wall into the surrounding medium
  28. 28. monotrichous- single polar flagella- Vibrio amphitrichous- single flagellum at both the poles- A.fecalis lophotrichous- tuft of flagella at one or both the ends- Spirilla peritrichous- flagella all around the cell- S.typhi
  29. 29.  V.cholerae- darting  Listeria- Tumbling  Clostridium- Stately  Mycoplasma- Gliding
  30. 30.  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
  31. 31.  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
  32. 32.  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
  33. 33.  DIRECT METHOD- dark field microscopy & electron microscopy  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
  34. 34.  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
  35. 35.  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.
  36. 36.  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
  37. 37.  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
  38. 38. adhesion antigenic property inhibiting phagocytosis transfer of genetic material
  39. 39.  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
  40. 40.  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
  41. 41.  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
  42. 42.  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
  43. 43.  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
  44. 44.  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
  45. 45. 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
  46. 46.  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
  47. 47.  Indicator for sterilization- G.stearothermophilus is destroyed by autoclaving, hot air oven  Ethylene oxide sterilizer- B.atrophaecus
  48. 48.  https://drive.google.com/file/d/1vq9iEoo6o meomDXatLYrPicFY2cCQwVF/view?usp=sha ring  https://drive.google.com/file/d/1SR8FG6Iu MHuErW5tUffWoI_II- jMZTBw/view?usp=sharing