Lec 2,3 Micro.pptx microbioooooooooology
- 1. Bacteriolo gy Basics Morphology, Classification, Staining Methods
- 2. Introducti on 2 • Microorganisms – several classes of living beings • Based on the organization of their cellular structures, all living cells can be divided into two groups: eukaryotic and prokaryotic – Eukaryotic cell types - Animals, plants, fungi, protozoans, and algae – Prokaryotic cell types - bacteria & blue green algae
- 3. Schematic of typical animal (eukaryotic) cell, showing subcellular components. 3 (5) rough endoplasmic reticulum (ER) Organelles: (1) nucleolus (2) nucleus (3) ribosome (6) Golgi apparatus (4) vesicle (7) Cytoskeleton (8) smooth ER (11) cytoplasm (9) mitochondria (12) lysosome (10) vacuole (13) centrioles
- 4. Background Information Prokaryotes 4 • Prokaryotes represent two domains, bacteria and archaea. • Archaea live in Earth’s extreme environments. • Bacteria are the most abundant and diversified organisms on Earth.
- 5. Eukaryotes have organelles 5 • Much larger; more complex than prokaryotes • Processes compartmentalized into organelles – Nucleus – Protein synthesis (ribosomes, RER, Golgi) – Mitochondria; chloroplasts – Lysosomes – Plasma membranes have different modifications – Cytoskeleton
- 6. Differences between prokaryotic & eukaryotic cells 6 Character Prokaryotes Eukaryotes Nucleus Nuclear membran e Absent Present Nucleolus Absent Present Chromoso m e One circular One or more paired and linear Cell division Binary fission Mitosis Cytoplasmi c membrane Structure and Compositio n fluid phospholipid bilayer, lacks sterols fluid phospholipid bilayer containing sterols
- 7. Differences between prokaryotic & eukaryotic cells Character Prokaryotes Eukaryotes Cytoplasm Mitochondria Absent Present Lysosomes Absent Present Golgi apparatus Absent Present Endoplasmi c reticulum Absent Present Vacuoles Absent Present Ribosomes 70 S 80 S 7
- 8. Differences between prokaryotic & eukaryotic cells Character Prokaryotes Eukaryotes Cell Wall Present Animals & Protozoans – Absent Plants, Fungi & Algae - Present Composition Peptidoglycan – complex carbohydrate Cellulose or chitin Locomoto r organelle s Flagella Flagella/ Cilia 8
- 9. Prokaryotic Cells 9 • Much smaller (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.
- 10. Size of Bacteria 10 • Unit of measurement in bacteriology is the micron (micrometre, µm) • Bacteria of medical importance –0.2 – 1.5 µm in diameter –3 – 5 µm in length
- 11. 11 Eukaryotic cell Gram - Cell wall (e.g. animal) Nucleoid Rough endoplasmic reticulum Nucleus Cell membrane Cytoplasm Mitochondria Prokaryotic cell Gram + Flagellum Outer membrane Granule Capsule Cell (inner) membrane Ribosomes Cell wall Pili
- 12. Shapes of Bacteria 12 major groups • Cocci – spherical/ oval shaped • Bacilli – rod shaped • Vibrios – comma shaped • Spirilla – rigid spiral forms • Spirochetes – flexible spiral forms • Actinomycetes – branching filamentous bacteria • Mycoplasmas – lack cell wall
- 13. Bacteria Have One of Three Cellular Shapes 13 • Rods (bacilli) • Coccoid-Shaped • Spirilla
- 14. Arrangement of bacteria: Cocci 14 Sarcina – groups of eight Cocci in chain - Streptococci Coccus Cocci in pair – Diplococcus Tetrad – groups of four Cocci in cluster - Staphylococci
- 15. Reproducti on 15 • Prokaryotic cell division is binary fission. – Single DNA molecule that first replicates. – Attaches each copy to a different part of the cell membrane. – Cell begins to pull apart. – Following cytokinesis, there are then two cells of identical genetic composition.
- 18. Anatomy of a Bacterial Cell 18
- 19. Anatomy of A Bacterial Cell 19 • Outer layer – two components: 1. Rigid cell wall 2. Cytoplasmic (Cell/ Plasma) membrane – present beneath cell wall • Cytoplasm – cytoplasmic inclusions, ribosomes, mesosomes and nucleus • Additional structures – plasmid, slime layer, capsule, flagella, fimbriae (pili), spores
- 20. Structure of Bacteria 20 • All cells have 3 main components: – DNA (‘nucleoid”) • genetic instructions – surrounding membrane (“cytoplasmic membrane”) • limits access to the cell’s interior – cytoplasm, between the DNA and the membrane • where all metabolic reactions occur • especially protein synthesis, which occurs on the ribosomes
- 21. 21 • Bacteria also often have these features: – cell wall • resists osmotic pressure – flagella • movement – pili • attachment – capsule • protection and biofilms
- 22. The Cell Envelope 22 Gram Positive Gram Negative
- 23. GRAM POSITIVE 23 Cytoplasm Cytoplasm Lipoteichoic acid Peptidoglycan-teichoic acid Cytoplasmic membrane Inner (cytoplasmic) membrane Outer Membrane Lipopolysaccharide GRAM NEGATIVE Porin Braun lipoprotein
- 24. Gram-positive and gram-negative bacteria
- 25. Difference Between Gram- Negative and Gram- Positive Bacteria Gram-Negative Bacteria Gram-Positive Bacteria More complex cell wall. Simple cell wall. Thin peptidoglycan celll wall layer. Thick peptidoglycan celll wall layer. Outer lipopolysaccharide wall layer. No outer lipopolysaccharide wall layer. Retain safranin. Retain crystal violet/iodine. Appear pink/red. Appear blue/purple. 25
- 26. Cell Envelope • The cell envelope is all the layers from the cell membrane outward, including the cell wall, the periplasmic space, the outer membrane, and the capsule. – All free-living bacteria have a cell wall – periplasmic space and outer membrane are found in Gram- negatives – the capsule is only found in some strains 26
- 27. Structure & Function of Cell Components
- 28. CELL WALL 28 • 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.
- 29. Cell Wall 29 • 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 simpler chemical nature than Gram –ve bacteria. 7. Several antibiotics may interfere with cell wall synthesis e.g. Penicillin, Cephalosporins
- 30. Outer Membrane 30 Gram negative bacteria • Major permeability barrier • Space between inner and outer membrane –Periplasmic space store degradative enzymes • Gram positive bacteria • no Periplasmic space
- 31. Gram positive cell wall 31 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.
- 32. Gram negative cell wall 32 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.
- 33. Cell Wall 33
- 34. Summary of the differences between Gram positive & Gram negative bacteria 34 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
- 35. Cytoplasmic (Plasma) membrane 35 • Thin layer 5-10 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
- 36. Other Cytoplasmic Components 36 as e • Ribosomes – protein synthesis • Mesosomes – 1. Multilaminated structures formed invaginations of plasma membran 2. Principal sites of respiratory enzymes 3. Coordinate nuclear & cytoplasmic division during binary fission 4. More prominent in Gram +ve bacteria • Intracytoplasmic inclusions – reserve of energy & phosphate for cell metabolism e.g. Metachromatic granules in diphtheria bacilli
- 37. Nucle us 37 • No nucleolus • No nuclear membrane • Genome – –single, circular double stranded DNA
- 38. Additional Organelles 1. Plasmid – – Extra nuclear genetic elements consisting of DNA – Transmitted to daughter cells during binary fission – May be transferred from one bacterium to another – Not essential for life of the cell – Confer certain properties e.g. drug resistance, toxicity 38
- 39. Additional Organelles 2. Capsule & Slime layer – – Viscous layer secreted around the cell wall. – Polysaccharide / polypeptide in nature Capsule – sharply defined structure, antigenic in nature • Protects bacteria from lytic enzymes • Inhibits phagocytosis • Stained by negative staining using India Ink • Can be demonstrated by Quellung 39
- 40. Additional Organelles 40 3. Flagella – Long (3 to 12 µm), filamentous surface appendages – Organs of locomotion – Chemically, composed of proteins called flagellins – The number and distribution of flagella on the bacterial surface are characteristic for a given species - hence are useful in identifying and classifying bacteria – Flagella may serve as antigenic determinants (e.g. the H antigens of Gram-negative enteric bacteria) – Presence shown by motility e.g. hanging drop
- 41. Types of flagellar arrangement 41 Polar/ Monotrichous – single flagellum at one pole Lophotrichous – tuft of flagella at one pole Amphitrichous – flagella at both poles Peritrichous – flagella all over Amphilophotrichous – tuft of flagella at both ends
- 42. Cocci do not have flagella 42
- 43. FLAGELLA 43 • Some bacteria are motile • Locomotory organelles- flagella • Taste environment • Respond to food/poison – chemo
- 44. • Flagella – embedded in cell membrane – project as strand – Flagellin (protein) subunits – move cell by propeller like action 44
- 45. Additional Organelles 45 4. Fimbriae/ Pili – – Thin, hairlike appendages on the surface of many Gram- negative bacteria – 10-20µm long, acts as organs of adhesion (attachment) - allowing bacteria to colonize environmental surfaces or cells and resist flushing – Made up of proteins called pilins. – Pili can be of two types – Common pili – short & abundant Sex pili - small number (one to six), very long pili, helps in conjugation (process of transfer of DNA)
- 46. Additional Organelles 46 5. Spores – – Highly resistant resting stages formed during adverse environment (depletion of nutrients) – Formed inside the parent cell, hence called Endospores – Very resistant to heat, radiation and drying and can remain dormant for hundreds of years. – Formed by bacteria like Clostridia, bacillus
- 47. The cycle of spore formation and germination 47 At the beginning of spore formation, a septum forms, separating the nascent spore from the rest of the cell and all of the genetic material of the cell is copied into the newly-forming cell. The spore contents are dehydrated and the protective outer coatings are laid down. Once the spore is matured it is relleased from the cell. On germination, the spore contents rehydrate and a new bacterium emerges and multiplies.
- 48. Shape & position of bacterial spore 48 Oval central Spherical central Oval sub terminal Oval sub terminal Oval terminal Spherical terminal Free spore Non bulging Bulgin g
- 49. Spore s 49 • Some bacteria can form very tough spores, which are metabolically inactive and can survive a long time under very harsh conditions. –Allegedly, some bacterial spores that were embedded in amber or salt deposits for 25 million years have been revived. These experiments are viewed skeptically by many scientists.
- 50. Spore s 50 • Spores can also survive very high or low temperatures and high UV radiation for extended periods. This makes them difficult to kill during sterilization. • Anthrax • Bacillus species including anthracis (anthrax) and cereus (endotoxin causes ~5% of food poisoning) • Clostridium species including tetani (tetanus), perfringens (gangrene), and botulinum (botulism: food poisoning from improperly canned food)
- 51. Pleomorphic & Involution forms 51 • Pleomorphism – 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
- 52. Bacterial Taxonomy 52 • Includes three components: 1.Classification : orderly arrangement 2.Identification of an unknown unit 3.Nomenclature : naming the units
- 53. Bacterial Taxonomy: Classification 53 • Orderly arrangement : • Kingdom – Division – Class – Order – Family – Genus – Species Tribe – Phylogenetic classification – represents a branching tree like arrangement. One characteristic being used for division at each branch or level Molecular or Genetic classification – based on the degree of genetic relatedness of different organisms Intraspecies classification – based on biochemical properties (biotypes), antigenic features (serotypes), bacteriophage susceptibility (phage types)
- 54. Bacterial Taxonomy: Nomenclature 54 • Two kinds of name are given to bacteria – Casual / common name – for local use, varies from country to country e.g. “typhoid bacillus” – Scientific / International Name – same all over world, consists of two words (in Italics) e.g. Salmonella typhi, Staphylococcus aureus
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- 56. Why we should be Stain Bacteria Bacteria have nearly the same refractive index as water, therefore, when they are observed under a microscope they are opaque or nearly invisible to the naked eye. Different types of staining methods are used to make the cells and their internal structures more visible under the light microscope. 56
- 57. What is a Stain • A stain is a substance that adheres to a cell, giving the cell color. • The presence of color gives the cells significant contrast so are much more visible. • Different stains have different affinities for different organisms, or different parts of organisms • They are used to differentiate different types of organisms or to view specific parts of organisms 57
- 58. Differential Stains Differential Stains use two or more stains and allow the cells to be categorized into various groups or types. Both techniques allow the observation of cell morphology, or shape, but differential staining usually provides more information about the characteristics of the cell wall (Thickness). 58
- 59. Gram staining • Named after Hans Christian Gram, differentiates between Gram- positive purple and Gram- negative pink stains and is used to identify certain pathogens. 59
- 60. Structure and Reactivity to Gram Staining. 60
- 62. Gm+ve cocci & Gm-ve bacilli 62