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Bacterial spores ashraf


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Bacterial spores ashraf

  1. 1. Download link: ashraf/
  2. 2. Definition, history List of medically important spore forming bacteria Spore formation/ Sporulation Structure Classification based on shape and position Properties Resistance and destruction Germination Demonstration of spores Uses of spores
  3. 3. Endospores were reported by numerous scientists Perty 1852; Pasteur 1869; Koch, 1876 and Cohn, 1872. Bacterial spores are dormant forms of bacteria which are thick walled, highly refractile and resistant. • Nutritional deprivement(depletion of N2 or C source or both) is the triggering factor • Exposure to suboptimal temperature
  4. 4. Spore-forming bacteria Bacillus: – Bacillus anthracis, – Bacillus subtilis, – Bacillus cereus, – Bacillus clausii, – Bacillus halodenitrificans Clostridium: – Clostridium botulinum, – Clostridium difficile, – Clostridium perfringens, – Clostridium tetani and Clostridium sordellii. – Clostridium thermocellum (ethanol) – Clostridium acetobutylicum (acetone) – Clostridium diolis (propane-di-ol and to convert fatty acids to yeasts)– Thermophilic actinomycetes: – Thermoactinomyces vulgaris
  5. 5. Stages Stage I Pre-sporulation phase DNA assembles as an axial filament Stage II Spore septum formation Stage III Engulfment of the forespore Stage IV Cortex formation Stage V Synthesis of spore coats, DIPICOLINIC ACID, calcium uptake Stage VI Spore maturation (coats more thick) Stage VII Lysis of cell and liberation of mature spore Spore formation
  6. 6. STRUCTURE 5.Exosporium made of glycoprotein and Lipoproteins. 4. Outer Membranes (Spore Coats) • keratin-like protein (consists sulphur containing aminoacids with disulphide bonds • impermeable • protects spore from chemicals 3. Cortex • thickest spore layer, • made of modified peptidoglycan • susceptible to lysozyme 2.Core Membrane/germ cell wall structure similar to that in the vegetative cell wall 1. Spore protoplast/Core • contains DNA,RNA • protein • DIPICOLINIC ACID • Divalent ions (calcium) 1. Core 2. Core Membrane 3. Cortex 4. Spore Coats 5. Exosporium
  8. 8. Shapes – Spherical, oval or elongated in shape – May be narrower or broader(bulge) than parent cell spore Bacterial cell spore Bacterial cell Bulged Ex- Clostridium sp. Not bulged Ex- Bacillus sp.
  9. 9. Position • Terminal (located at one of the poles) • Sub terminal or sub central(between center & one of the poles) • Equatorial (central)
  10. 10. Bacteria can be classified based on the shape and position of spores. TERMINAL SUBTERMINAL CENTRAL OVAL Clostridium tetani ( drumstick appearance) Clostridium welchii, Clostridium septicum Clostridium sporogenes Bacillus anthracis SPHERICAL Clostridium tertium ------- ------- spore spore spore spore
  11. 11. PROPERTIES • Endospores • Extremely resistant • Non-reproducing mechanism of survival rather than a mechanism of reproduction(unlike fungal spores) • Not stained readily with dyes (spore coats)
  12. 12. • Metabolically inactive (dormant forms) • No DNA replication (transcription) • mRNA not produced (translation) • Spore protein synthesis is defective • DNA of the endospores is protected and stabilized by calcium DIPICOLINATE • Specialized DNA binding proteins saturate it and protects it from heat, radiation, chemicals. • Has DNA repair enzymes
  13. 13. PROPERTY VEGETATIVE CELLS ENDOSPORES Surface coats murein cell wall polymer Thick cortex with spore coats Microscopic appearance Non-refractile Refractile Calcium dipicolinic acid Absent Present in core Cytoplasmic water activity High Very low Metabolic activity Present Absent RESISTANCE Heat resistance Low High Resistance to chemicals Low High Radiation resistance Low High SENSITIVE Sensitivity to lysozyme Sensitive Resistant Sensitivity to dyes & staining Sensitive Resistant
  14. 14. Bacterial spores are the toughest forms of life known. “ They are so resistant to destruction that some scientists have proposed that life arrived on earth when bacterial spores drifting through space fell to earth.” RESISTANCE
  15. 15. (cont….) Extremely resistant due to • low water content (removal by osmosis) • thick wall and impermeability of spore coat • high content of DIPICOLINIC ACID • ability to concentrate calcium
  16. 16. • Resists boiling for prolonged periods. • Can survive drying, radiation, and many damaging chemicals. • Capacity to germinate many years after formation, may be centuries or ages.
  17. 17. Sporostatic Sporicidal
  18. 18. • Germination is the process whereby spores differentiate into vegetative cells that grow, reproduce & metabolize. • ONE spore--ONE vegetative cell. • complex process which happens in 3 stages: 1. Endospores activation 2. germination 3. outgrowth GERMINATION
  19. 19. • Activation- breaks the dormancy in spores, • most spore properties retained • Reversible • Activators(triggering agents) Heating at 60°C for more than 1 hour Decreasing the pH Treatment with mercaptoethanol • Conformational change in spore macromolecules • No morphological changes
  20. 20. 2. Germination: • Germination of activated spores occurs when exposed to certain substances(nutrients) – aminoacids – sugars – nucleosides • Irreversible • Removal of cortex (enzyme Ƀ-N acetyl glucosaminidase)- renders it susceptible to chemical agents • Loss of refractility • In vitro germination incubated at 37°C, 5% CO2, FBS (fetal bovine serum) – Dulbecco’s modified Eagle’s medium(DMEM)+10% FBS – Roswell Park Memorial Institute(RPMI)1640 medium +10%FBS,
  21. 21. (b) development of phase dark forms c) Germination completes with full conversion to phase dark cells (a) mature, phase-bright (REFRACTILE) spores
  22. 22. 3. Outgrowth • The core of the endospores leave the old spore coat to develop into a fully functional vegetative cell. • First visible change is swelling of cell--water uptake • Molecular biosynthetic process starts RNA synthesis– first Protein DNA synthesis • Cell wall synthesis coincides with swelling
  24. 24. Stained preparation •Gram stain •ZN method (modified) •Dorner’s method •Malachite green stain 1. Schaeffer and fulton method 2. Ashby modification Microscopy: Unstained preparation – Using Phase contrast microscope – Morphology best observed – large, refractile, oval or spherical bodies within mother cells Stained preparation
  25. 25. Gram stain Reveals – deep blue bacilli, – colorless spore within the bacilli
  26. 26. Modified Ziehl-Neelsen technique • 0.25% sulphuric acid used as decolorizer • Spores- pink Bacteria - blue
  27. 27. Dorner’s method • First spore staining described (1922) • Differential staining method – Primary stain – Carbol fuchsin for 5min – Decolorizer – acid alcohol for 1min – Counterstain- Nigrosin 10%
  28. 28. Dorner’s Method Nigrosin (negative stain) red endospores in colorless vegetative cells.
  29. 29. Malachite green staining
  30. 30. Schaeffer-Fulton Stain Procedure 1. Make a smear. Air Dry. Heat fix 2. Flood the smear with Malachite Green stain 3. flooded smear covered with a square of filter paper 4. Steam slide for 10 minutes (every minute, add a few more drops of Malachite Green stain) 5. Allow slide to cool (after the 10 min. steam process)
  31. 31. Schaeffer-Fulton Stain Procedure (continued) 6. Drain slide and rinse for 30 seconds with DI water (discard filter paper) 7. Put slide on steam rack 8. Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute) 9. Drain the slide and rinse with DI water 10. Blot Dry 11. Use oil immersion objective to view
  32. 32. Schaeffer-fulton Method Spore: green Vegetative cell: red
  33. 33. Uses of spores • Sterilization control Bacillus stearothermophilus • Nanobiotechnology – substrate for delivery of biomolecules – vaccine vehicles spores – source for understanding unknown self-assembling molecules.
  34. 34. Anthrax bioterrorism • Concentrated anthrax spores were used for bioterrorism in the 2001, in U.S. • Spores mailed in the form of powder • led to 11 cases of cutaneous anthrax and 11 cases of inhalational anthrax, with five deaths in the latter. • Used as agent for biological warfare by germans.
  35. 35. References • Topley & Wilson’s principles of bacteriology, virology and immunity, 8th edition. Vol-1 • Mackie & McCartney Practical Medical Microbiology, 14th edition • A. D. RUSSELL “Bacterial Spores and Chemical Sporicidal Agents” Clinical microbiology reviews, ASM, Apr. 1990, p. 99-119. • • Emerging Applications of Bacterial Spores in Nanobiotechnology, Journal of Nanobiotechnology