Exploring Bacterial Cell Morphology and Microscopy Techniques

1. MORPHOLOGYOF BACTERIA

2. EUKARYOTIC Parasites, fungi Membrane enclosed organelles Cytoskeleton MICROORGANISMS • PROKARYOTIC • Bacterial cell • Do not contain organelles • Cell wall, peptidoglycan

3. Prokaryote and eukaryote cells PROKARYOTE AND EUKARYOTE

4. SIZE - 0.25-1 µm width 1-3 µm length SHAPE – cocci bacilli coccobacilli fusiform spiral ARRANGEMENT MORPHOLOGY OF BACTERIA

5. SHAPE OF BACTERIA Shapes of bacteria: 1. coccus; 2. bacillus; 3. vibrio; 4. spirillum; 5. spirochete

6. ARRANGEMENT OF BACTERIA Arrangement of curved bacteria: 1. vibrio; 2. spirilla; 3. spirochetes

7. OPTICAL/LIGHT MICROSCOPE MICROSCOPY Principle of bright-field (light) microscopy

8. PHASE CONTRAST MICROSCOPE Improves contrast Makes evident structures within cells ‘Phase differences’ converted to differences in intensity of light – producing light and dark contrast in image MICROSCOPY

9. FLUORESCENT MICROSCOPE Light of high intensity source excites a fluorescent agent, which in turn emits a low energy light of longer wavelength that produces the image MICROSCOPY

10. Fluorescent microscopy FLUORESCENT MICROSCOPE

11. FLUORESCENT MICROSCOPE APPLICATIONS Principles of fluorochroming and immunofluoresence

12. MICROSCOPY DARK GROUND MICROSCOPE Improves contrast Reflected light used instead of transmitted light Dark field condenser Light rays falling on the object are reflected or scattered on to objective lens Object self-luminous against dark background

13. DARK GROUND MICROSCOPE Dark-field microscopy

14. ELECTRON MICROSCOPE Beam of electrons instead of beam of light Electron beam focused by circular electromagnets - analogous to lens in light microscope Object held in the path of electron beam and produces an image which is focused on a screen MICROSCOPY

15. Shadow casting Negative staining – phosphotungstic acid Freeze etching ELECTRON MICROSCOPE APPLICATIONS

16. SIMPLE STAIN – Methylene blue or basic fuchsin NEGATIVE STAINING – India ink or nigrosin IMPREGNATION – Silver impregnation STAINING TECHNIQUES

17. Differential stains GRAM STAIN – Christian Gram (1884) Principle: Gram positive – acidic protoplasm - retain basic primary dye Peptidoglycan of Gram-positive bacteria thick – retain the dye iodine complex High lipid content of Gram-negative bacteria makes them permeable to counterstain (secondary dye) STAINING TECHNIQUES

18. ACID FAST STAIN - Ehrlich Modified – ZIEHL and NEELSEN Principle: High lipid content, variety of fatty acids and lipids Mycolic acid – peculiar to acid fast bacilli Integrity of the cell wall STAINING TECHNIQUES

19. ALBERT STAIN - demonstrate metachromatic granules – C.diphtheriae Neisser’s stain Ponder’s stain STAINING TECHNIQUES

20. BACTERIAL CELL Diagram of an idealised bacterial cell

21. Cell envelope and appendages Cell interior Cell structures function as a complex integrated unit BACTERIAL CELL

22. CELL ENVELOPE Outer membrane (GNB) Cell wall – peptidoglycan Periplasm (GNB) Cytoplasmic membrane BACTERIAL CELL

23. Bilayered Composed of lipopolysaccharide (LPS) Gives GNB - net negative charge Scattered throughout LPS - porins Porins - control passage of nutrients and antibiotics OUTER MEMBRANE

24. Composed of disaccharide-pentapeptide Disaccharide-N-acetyl glucosamine N-acetyl muramic acid Amino acid only linked to N-acetyl muramic acid Polymers-crosslink-via peptide bridges to form peptidoglycan sheets CELL WALL

25. CHEMICAL STUCTURE OF BACTERIAL CELL WALL Chemical structure of bacterial cell wall

26. Notable difference - Gram positive and Gram negative Gram positive-Peptidoglycan thick Teichoic acid Glycerol Ribitol phosphate Mycolic acid CELL WALL

27. CELL WALL Gram-positive call wall

28. CELL WALL Gram-negative cell wall

29. Cannot be seen by light microscope Cannot stain with simple stain Demonstration - Plasmolysis - hypertonic solution bacterial ghost - Micro dissection - Reaction with specific antibody - Differential staining - Electron microscope CELL WALL

30. Found in Gram-negative bacilli Inner surface of outer membrane Contains the peptidoglycan layer Helps secure nutrients Has enzymes that degrade macromolecules and detoxify antibiotics PERIPLASMIC SPACE

31. Present in Gram-positive and Gram-negative bacteria Deepest layer of cell envelope Heavily laced with proteins and enzymes vital to cell metabolism Cytoplasmic membrane functionally similar to eukaryotic cell organelles (Mitochondria, Golgi, Lysosomes) CYTOPLASMIC MEMBRANE

32. Transport of solutes Enzymes involved in cell synthesis Generation of chemical energy Cell motility Chromosomal segregation CYTOPLASMIC MEMBRANE FUNCTIONS

33. Capsule Fimbria-pili Flagella Role in causing infection Help identification in laboratory CELLULAR APPENDAGES

34. Immediately exterior to peptidoglycan Glycocalyx/Slime Protect bacteria from attack of cells of human defense mechanism Facilitates and maintains bacterial colonisation of biological surface Example: teeth, prosthetic heart valve CAPSULE

35. SIGNIFICANCE Virulence - Inhibit phagocytosis - Protect cell from lysozyme Permit adherence - Cell surface Example: implant, catheters Prevents cell from drying Toxicity to host cell Protects cell from bacteriophage CAPSULE

36. Covalently linked to cell wall No net charge - so do not bind to dyes Gram stain – clear halo around bacteria Demonstrated by negative staining - India ink - Nigrosin - Congo red Demonstrated immunologically - Quellung reaction CAPSULE

37. CAPSULE S.pneumoniae capsule seen by India ink staining

38. Serological typing - capsular antigen Detection of antigen - CSF, blood Example: S.pneumoniae - CSF Neisseria meningitidis Vaccine - capsular polysaccharide food antigen CAPSULE APPLICATIONS

39. Short, hair-like structures Proteinaceous-antigenic Protrude through cell wall Two types: common pili-adhesins, sex pili FIMBRIAE/PILI

40. SIGNIFICANCE Act as adhesins - bacteria colonise Receptor for bacteriophage Streptococcus pyogenes - M protein Virulence factor Some fimbriae - agglutinate RBCs FIMBRIAE/PILI

41. Complex structure Composed of protein flagellin Embedded in cell envelope Motility - survival FLAGELLA

42. Non-contractile Single protein subunit - flagellin Anchored to bacterial cytoplasmic membrane by disc-like structure FLAGELLA

43. Three parts: filament, hook, basal body Flagella attached to cell body by hook and basal body Hook and basal body embedded in envelope Basal body -1 set of rings-Gram positive 2 sets of rings-Gram negative FLAGELLA

44. FLAGELLA - STRUCTURE General structure of the flagellum of a Gram-negative bacterium

45. ARRANGEMENT OF FLAGELLA Types of flagellar arrangement: 1. monotrichous; 2. lophotrichous; 3. amphitrichous; 4. amphilophotrichous; 5. peritrichous fl agella

46. Detection of motility-Direct-hanging drop Phase contrast Dark ground Motility media Demonstration of flagella-Flagella stain Electron microscope Immunology FLAGELLA

47. Adverse physical and chemical conditions Nutrients scarce Metabolic and structural change Bacterial spores – endospores Favorable conditions endospore germinates Spores killed – autoclave, formaldehyde SPORE

48. BACTERIAL SPORE Diagrammatic representation of a bacterial spore: 1. germinal groove; 2. outer cortical layer; 3. cortex; 4. internal spore coat; 5. subcoat material; 6. outer spore coat; 7. cytoplasmic membrane; 8. cell wall primordium.

49. TYPES OF BACTERIAL SPORES Types of bacterial spores: 1. central, bulging; 2. subterminal, bulging; 3. terminal, spherical; 4. central, not bulging; 5. subterminal, not bulging; 6. terminal, oval

50. Pleomorphism – defective cell wall synthesis Involution forms – activity of autolytic enzymes L forms – Kleineberger and Nobel - aberrant morphological forms Lister Institute, London PLEOMORPHISM