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morphphysioofbacteriadr-180131090251.pdf
- 1. M ORP H OL OGY & P H YSIOL OGY OF B ACT E RIA Dr.R. Someshwaran, M D., Assistant professor, Department of M icrobiology, K F M S& R
- 2. Objectives of today’sclass T o define the term prokaryote &eukaryote T o understand different shapes of bacteria T o describe bacterial anatomy in detail T o explain about bacterial growth curve T o know the factors affecting bacterial growth 01/31/18 2
- 3. Introduction • L iving organismsclassified under3 kingdoms: 1.Plant kingdom 2.Animal kingdom 3.P rotista • Under Protista- Microorganismsareclassified as i.P ROK ARYOT E S (Bacteria& Blue-green algae) ii.E UK ARYOT E S (Fungi, Protozoa, slimemolds& other algae) 01/31/18 3
- 4. P rokaryotes • B acteria aresingle celled microorganism containing a single circularchromosome • B acteria lack Nuclearmembrane, nucleolus, deoxyribo- nucleoprotein. • M itotic division is absent in bacteria • Cytoplasmic streaming, pinocytosis, M itochondria, L ysosomes, Golgi apparatus, E ndoplasmic reticulum are absent in the bacterial cytoplasm. • Chemical composition (Sterols-, M uramic acid+) • B acteria do not contain chlorophyll; B acteria do not show true branching. E xception: Actinomycetales (H igherbacteria) 01/31/18 4
- 5. P rokaryotic Cells • M uchsmaller(microns) and more simple than eukaryotes • prokaryotes are molecules surrounded by a membrane and cell wall. • they lack a true nucleus and don’t have membrane bound organelles like mitochondria, etc. • large surface-to-volume ratio : nutrients can easily and rapidly reach any part of the cells interior
- 6. Schematic of typicalanimal (eukaryotic) cell, showing subcellular components. Organelles: (1) nucleolus (2) nucleus (3) ribosome (4) vesicle (5) rough endoplasmic reticulum (ER) (6) Golgi apparatus (7) Cytoskeleton (8) smooth ER (9) mitochondria (10) vacuole (11) cytoplasm (12) lysosome (13) centrioles
- 7. Differences between prokaryotic& eukaryotic cells Character Prokaryotes E ukaryotes Nucleus Nuclear membrane Absent Present Nucleolus Absent Present Chromosome One circular One ormore paired and linear Cell division B inary fission M itosis Cytoplasmic membrane Structure and Composition fluid phospholipid bilayer, lacks sterols fluid phospholipid bilayer containing sterols F unction Incapable of endocytosis (phagocytosis and pinocytosis) and exocytosis Capable of endocytosis and exocytosis
- 8. Differences between prokaryotic& eukaryotic cells Character Prokaryotes E ukaryotes Cytoplasm M itochondria Absent P resent L ysosomes Absent P resent Golgi apparatus Absent P resent E ndoplasmic reticulum Absent P resent Vacuoles Absent P resent Ribosomes 70 S 80 S
- 9. Differences between prokaryotic& eukaryotic cells Character Prokaryotes E ukaryotes Cell W all Present Animals &P rotozoans – Absent P lants, F ungi &Algae - P resent Composition Peptidoglycan – complex carbohydrate Cellulose orchitin L ocomotor organelles F lagella F lagella/Cilia
- 10. Size of bacteria •Unit of measurement used in bacteriology is micron or micrometre (µm) • One micron is equal to one thousandth of millimetre • One nanometre (nm) ormillimicron (mµ) is equal to one thousandth of a micron orone millionth of a millimetre • One Angstrom unit (Å) is equal to one tenth of a nanometre 01/31/18 10
- 11. L imit of resolution •L imit of resolution with the unaided human eye is 200 microns (0.2mm) • B acteria are even much smallerand can be visualized only undermagnification. • B acteria of medical importance generally measure 0.2- 1.5µm in diameterand about 3-5µm in length 01/31/18 11
- 12. M icroscopy • L eeuwenhoek –hand ground lenses – F irst observed bacteria • P arts of a microscope: E ye piece (5x/ 10x), Arm, B ase, M echanical stage, L ight source, Condenser, Diaphragm, Objectives (Scanner, L P , H P , OI), Cylindrical tube length, Stage adjustor(H orizontal, vertical), F ine adjustment and coarse adjustment knob. • M agnification power • Resolution power • T otal magnification: Objective x E yepiece • Refractive index of lens (glass), air, water&oil • F ocal length of lens of human eye (Range:16-25cm) 01/31/18 12
- 13. T ypes of M icroscopes a.Optical orlight microscope b. P hase contrast microscope c. F luorescent microscope d. E lectron microscope e. Dark field /Dark Ground microscope: 2 types namely polarisation &interference microscopes 01/31/18 13
- 14. Staining techniques • Simplestains providecolour contrast but impart samecolour to all bacteria. Ex: Methyleneblue, Basic fuchsin. • Negative staining provideuniformly coloured background against which theunstained bacteriastand out in contrast. Ex: Indiaink or Nigrosin. • Impregnation methods for Cellsand structurestoo thin to be seen under ordinary microscopethat may berendered visibleif they arethickened by silver impregnation on thesurface. Used for demonstration of Spirochetesand Bacterial flagella. 01/31/18 14
- 15. Staining techniques –Contd… • Differential stains: Stainsimpart different colour to different bacteriaor itsstructures. Ex: Gram stain & Acid fast stain. 01/31/18 15
- 16. Shape of B acteria •Cocci – spherical/oval shaped majorgroups • B acilli – rod shaped • Vibrios – comma shaped • Spirilla – rigid spiral forms • Spirochetes – flexible spiral forms • Actinomycetes – branching filamentous bacteria • M ycoplasmas – lack cell wall
- 17. Arrangement of bacteria:Cocci Cocci in pair – Diplococcus Sarcina – groups of eight Tetrad – groups of four Cocci in chain - Streptococci Cocci in cluster - Staphylococci Coccus
- 18. Arrangement of bacteria:B acilli
- 19. Othershapes of bacteria Commashaped Spirochetes Spirilla
- 20. Anatomy of aB acterial Cell
- 21. Anatomy of AB acterial Cell • Outer layer – two components: 1. Rigid cell wall 2. Cytoplasmic (Cell/ Plasma) membrane– present beneath cell wall • Cytoplasm – cytoplasmic inclusions, ribosomes, mesosomesand nucleus • Additional structures – plasmid, slimelayer, capsule, flagella, fimbriae(pili), spores
- 22. Structure &F unction of CellComponents
- 23. CE L LW AL L • Outermost layer,encloses cytoplasm 1. Confers shape and rigidity 2. 10 - 25 nm thick 3. Composed of complex polysaccharides (peptidoglycan/mucopeptide) - formed by N acetyl glucosamine (NAG) & N acetyl muramic acid (NAM ) alternating in chains, held by peptide chains.
- 24. • Cell wall– 4. Carries bacterial antigens – important in virulence &immunity 5. Chemical nature of the cell wall helps to divide bacteria into two broad groups – Gram positive &Gram negative 6. Gram +ve bacteria have simplerchemical nature than Gram –ve bacteria. 7. Several antibiotics may interfere with cell wall synthesis e.g. P enicillin, Cephalosporins
- 25. Gram positive cellwall The Gram-positive cell wall is composed of a thick, multilayered peptidoglycan sheath outside of the cytoplasmic membrane. Teichoic acids are linked to and embedded in the peptidoglycan, and lipoteichoic acids extend into the cytoplasmic membrane
- 26. Gram negative cellwall The Gram-negative cell wall is composed of an outer membrane linked to thin, mainly single-layered peptidoglycan by lipoproteins. The peptidoglycan is located within the periplasmic space that is created between the outer and inner membranes. The outer membrane includes porins, which allow the passage of small hydrophilic molecules across the membrane, and lipopolysaccharide molecules that extend into extracellular space.
- 27.
- 28. Differences between Grampositive &Gram negative Property of bacteria Gram Positive Gram Negative Thickness of wall 20-80 nm 10 nm Number of layers in wall 1 2 Peptidoglycan content >50% 10-20% Teichoic acid in wall + - Lipid & lipoprotein content 0-3% 58% Protein content 0% 9% Lipopolysaccharide 0 13% Sensitive to penicillin Yes Less sensitive Digested by lysozyme Yes Weakly
- 29. Cytoplasmic (P lasma) membrane •T hin layer5-10 nm, separates cell wall from cytoplasm • Acts as a semipermeable membrane: controls the inflow and outflow of metabolites • Composed of lipoproteins with small amounts of carbohydrates
- 30. OtherCytoplasmic Components • Ribosomes– protein synthesis • M esosomes – 1. M ultilaminated structures formed as invaginations of plasma membrane 2. P rincipal sites of respiratory enzymes 3. Coordinate nuclear&cytoplasmic division during binary fission 4. M ore prominent in Gram +ve bacteria • Intracytoplasmic inclusions – reserve of energy &phosphate forcell metabolism e.g. M etachromatic granules in diphtheria bacilli
- 31. Nucleus • No nucleolus •No nuclearmembrane • Genome – • single, circulardouble stranded DNA. • H aploid • Divides by binary fission
- 32. Additional Organelles 1. P lasmid– • E xtranucleargenetic elements consisting of DNA • T ransmitted to daughtercells during binary fission • M ay be transferred from one bacterium to another • Not essential forlife of the cell • Confercertain properties e.g. drug resistance, toxicity
- 33. Additional Organelles 2. Capsule&Slime layer– • Viscous layersecreted around the cell wall. • P olysaccharide /polypeptide in nature a) Capsule – sharply defined structure, antigenic in nature • P rotects bacteria from lytic enzymes • Inhibits phagocytosis • Stained by negative staining using India Ink • Can be demonstrated by Quellung reaction (capsule swelling reaction) a) Slime layer– loose undemarcated secretion
- 34. Additional Organelles 3. F lagella– L ong (3 to 12 µm), filamentous surface appendages Organs of locomotion Chemically, composed of proteins called flagellins T he numberand distribution of flagella on the bacterial surface are characteristic fora given species - hence are useful in identifying and classifying bacteria F lagella may serve as antigenic determinants (e.g. the Hantigens of Gram-negative enteric bacteria) P resence shown by motility e.g. hanging drop preparation
- 35. T ypes of flagellararrangement Polar/Monotrichous – single flagellum at one pole Lophotrichous – tuft of flagella at one pole Peritrichous – flagella all over Amphitrichous – flagella at both poles Amphilophotrichous – tuft of flagella at both ends
- 36. Flagella • Long unbranchedsinuous filaments present on motile bacteria • Made of 3 parts • Filament, hook, and basal body • Filament is external and hook basal body complex embedded in the cell envelope • Hook and basal body are antigenically different
- 38. • Detachment offlagella does not affect the viability of cell • 3-20 m long, uniform diameter μ • M ade up of a protein flagellin • F lagella of different genera - similarchemical composition they are antigenically different • F lagellarantibodies help in serodiagnosis
- 39. Flagellar stains –Leifson stain
- 40.
- 41. Flagella and fimbriae(Pili)
- 42. Shape & positionof bacterial spore Oval central Spherical central Oval sub terminal Oval sub terminal Oval terminal Spherical terminal Free spore Non bulging Bulging
- 43. Capsule/ slime layer •B acteria secrete viscid material • organised into capsule - pneumococcus • L oose undemarcated secretion (slime layer) - leuconostac • M icrocapsules – too thin to be seen underlight microscope • B oth capsule and slime layermay be present
- 44. •Slime layer/ capsule • polysaccharideorpolypeptide • mucoid colonies on culture media •Slime layer • little affinity forgram stain •Capsule • stained by negative staining • Capsularmaterial antigenic. • Serological tests with anti capsularserum • Quellung reaction – Neufeld in 1902
- 45. Capsule •contribute to virulenceby inhibiting phagocytosis • lost by mutation or repeated subcultures
- 47.
- 48. • F ine hairlikeappendages • Shorterand thinnerthan flagella projecting from cell membrane • P resent on both motile and non motile bacteria • M ade up of pilin • 8 morphological types known • Classified as common orsex pili • Conferadhesive property to the cell • Disappearfollowing subcultures
- 49. Properties • Agglutinate redblood cells of guinea pigs, horses and pigs strongly • H uman and sheep cells weakly • Detected by hemagglutination • Antigenically similarand may cause cross reaction in serological tests
- 50. Sex pili • L ongerandfewer • F ound on male bacteria • Attach to female bacteria forming hollow conjugation tubes through which genetic material is transferred
- 52. Spores • H ighly resistantresting stages • E ndospores – formed inside cell • Due to deletion of exogenous nutrients /unfavourable environment • Young spores are seen attached to parent cell • Appearas unstained area in gram stain • Special spore stain can be used
- 53. Gram stain –Never comment on spore
- 54.
- 55.
- 56. Types of sporeformation • Spores may bulging or non bulging
- 57. Clostridium tetani – terminalspore B acillus anthracis – central spore
- 58.
- 59. Pleomorphism & involutionforms • Variation in shape and size of bacteria – pleomorphism • Aberrant forms in ageing cultures – involution forms • Due to defective cell wall synthesis
- 60. L forms • Swollencells and aberrant morphological forms observed by Kleinberger Nobel at Lister institute, London • Develop spontaneously or in the presence of agents that interfere with cell wall synthesis • May be stable or unstable • Resemble mycobacteria
- 61.
- 62. Pleomorphism & Involutionforms • P leomorphism – great variation in shape &size of individual cells e.g. Proteus species • Involution forms – swollen &aberrant forms in ageing cultures, especially in the presence of high salt concentration e.g. plague bacillus • Cause – defective cell wall synthesis
- 63. Bacterial Taxonomy • Includesthree components: 1. Classification : orderly arrangement 2. Identification of an unknown unit 3. Nomenclature: naming theunits
- 64. Bacterial Taxonomy: Classification •Orderly arrangement : • Kingdom – Division – Class– Order – Family – Tribe– Genus– Species Phylogenetic classification – representsabranching treelikearrangement. One characteristic being used for division at each branch or level Molecular or Genetic classification – based on thedegreeof genetic relatednessof different organisms Intraspeciesclassification – based on biochemical properties(biotypes), antigenic features(serotypes), bacteriophagesusceptibility (phagetypes)
- 65. Bacterial Taxonomy: Nomenclature •T wo kinds of name are given to bacteria • Casual /common name – forlocal use, varies from country to country e.g. “typhoid bacillus” • Scientific /International Name – same all overworld, consists of two words (in Italics) e.g. Salm onellaty phi, Staphy lococcus aureus
- 66. What is Physiology? •Study of the mechanical, physical and biochemical functions of living organisms.
- 67. Oxygen requirements • Aerobicrespiration is the release of energy from glucose or anotherorganic substrate in the presence of Oxygen. • Obligateaerobes-Pseudomonas • Microaerophilic –Streptococcus
- 68. • Anaerobic respirationisoxidationof m olecules intheabsenceof oxy gentoproduceenergy • Obligateanaerobes - clostridia • Facultativeanaerobes - vibrio • Aerotolerantanaerobes - la ctobacilli • Ferm entation • conversionof carbohy drates intoalcohols oracids underanaerobic conditions. • Eg: lactobacillus
- 69. Carbondioxide – factoraffecting growth • Small amount of CO2 is produced by most bacteria and used endogenously formetabolism • Capnophiles – bacteria which require a higherCO2 tension (5 – 10%) to grow. • eg: B rucellae, meningococcus , gonococcus
- 70. Bacterial vitamins • Growthfactors to be provided exogenously forgrowth. • E ssential • Accessary • H .influenzae requires accessary factors X (haemin) and V (NAD) present in blood to grow.
- 71. Effect of light •Bacteria usually grow well in the dark. • Phototrophs – need light to grow • Pigmented bacteria like cyanobacteria
- 72. Reaction AUTOTROPHS HETEROTROPHS PHOTO CHEMOPHOTO CHEMO ENERGY FIXING Light energy Inorganic compnds Light energy Organic compnds CARBON FIXING CO2 CO2 Organic compnds Organic compnds Cyanobacteria Green sulphur & Purple sulphur bacteria Nitrosomonas Nitrobacter Non Purple sulphur Non green sulphur bacteria Most bacteria All fungi protozoa
- 73. B acterial growth curve (batch culture)
- 74. • L ag phase- adaptation - build necessary enzyme &metabolic intermediates • L og/exponential phase – division by binary fission numberof cells • Stationary phase – cell division stop due to depletion of nutrients & accumulation of toxic products • P hase of decline – population decrease due to cell death
- 75.
- 76. E .coli M .leprae 20 minutes20 days • At extremes we eitherhave a mass progeny (4000 tones) in 24 hours in a continuous culture (or) no replication at all. • In laboratories it is always a batch culture where the nutrients are limited and toxic products accumulate. • GE NE RAT ION T IM E
- 77. Bacterial count • T otalcount – total numberof bacteria in the sample • Direct counting • Coultercounter • Viable count – measures the numberof living organisms • Dilution methods • P lating methods
- 78. Other factors affectingbacteria • T emperature • P H • M echanical stress • Osmosis
- 79.
- 80. Effect of PH onBacteria
- 82. Osmotic effect andmechanical stress • Tolerant to osmotic variations esp Gram positive bacteria • Sudden transfer from concentrated solution to distilled water – plasmoptysis • Mechanical stress like grinding, vigorous shaking with glass beads or exposure ultrasonic vibration – rupture of cell
- 83. Summary • P rokaryote • GPVsGN Difference • P leomorphic bacteria • Lforms • Cell appendages 01/31/18 83
- 84.