1. MORPHOLOGY OF BACTERIA

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

3. PROKARYOTE AND EUKARYOTE
Prokaryote and eukaryote cells

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

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

8. MICROSCOPY 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

9. MICROSCOPY 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

10. FLUORESCENT MICROSCOPE
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Fluorescent microscopy

11. FLUORESCENT MICROSCOPE APPLICATIONS
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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. MICROSCOPY 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

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

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

17. STAINING TECHNIQUES 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)

18. STAINING TECHNIQUES 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

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

20. Diagram of an idealised bacterial cell

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

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

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

24. CELL WALL 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

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

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

27. Gram-positive call wall
CELL WALL
Gram-positive call wall

28. Gram-negative cell wall

29. CELL WALL 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

30. PERIPLASMIC SPACE 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

31. CYTOPLASMIC MEMBRANE
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)

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

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

34. CAPSULE 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

35. CAPSULE 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

36. CAPSULE 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
Nigrosin or Nigrosine?

37. S.pneumoniae capsule seen by India ink staining

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

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

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

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

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

43. FLAGELLA 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

44. General structure of the flagellum of a Gram-negative bacterium
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. FLAGELLA Detection of motility-Direct-hanging drop Phase contrast
Dark ground
Motility media
Demonstration of flagella-Flagella stain
Electron microscope
Immunology

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

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