bacterial spore.ppt

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  • primarily described them as refractile bodies
  • - Found in soils.Bacillus: - aerobic,Gram +ve bacilli - express extra-cellular degradative enzymes - used as a means of biological control of insects. - anaerobic, Gram +ve bacilli - grow in canned foods producing toxinSporolactobacillusSporolactobacillus is a group of anaerobic, rod-shaped, spore forming bacteria that include Sporolactobacillus dextrus, Sporolactobacillus inulinus, Sporolactobacillus laevis, Sporolactobacillus terrae and Sporolactobacillus vineae. Sporolactobacillus are also known as lactic-acid bacteria for they are capable of producing the acid from fructose, sucrose, raffinose, mannose, inulin and sorbitol. Sporolactobacillus are found in the soil and often in chicken feed. According to "Fundamentals of Food Microbiology," the spores formed by Sporolactobacillus are less resistant to heat than those formed by the Bacillus genus
  • When spores sense that the environment is suitable for growth the spore is triggered to germinate. Germination is the process whereby spores differentiate into vegetative cells that grow, reproduce & metabolize.; that is, it is easily killed by heat UV light drying
  • Germination rate- function of ageing
  • When incubated at 37°C and under 5% CO2 in the presence of FBS(fetal bovine serum)
  • shed their coats to allow the young vegetative cells to emerge, elongate, and divide.Of the macromolecular biosynthetic processes occurring after germination, RNA synthesis is the first, followed closely in Bacillus spp. by the onset of protein synthesis, with DNA synthesis occurring some time later. During outgrowth, all types of RNA are synthesized. Cell wall synthesis commences after RNA and protein but before DNA and coincides with swelling of the germinated spore.
  • bacterial spore.ppt

    1. 1. 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
    2. 2. 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
    3. 3. • Endospores were reported by numerous scientists Perty 1852; Pasteur 1869; Koch, 1876 and Cohn, 1872.
    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. Spore formation 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
    6. 6. 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 STRUCTURE
    7. 7. SHAPES AND POSITION
    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) Courtesy- microrao.com/anatomy of
    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 dipiclonate • 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
    14. 14. RESISTANCE • 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.”
    15. 15. RESISTANCE 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 • 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. Endospore activation 2. germination 3. outgrowth
    19. 19. 1. Endospore activation • 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
    23. 23. ACTIVATION GERMINATION OUTGROWTH Morphology No change Swelling followed by elongation Reversibility (back to spore state) reversible Not reversible -- Metabolism Inert Active RNA, protein and DNA synthesis starts Resistance retained Loss of resistance, Susceptible to most of chemical agents Not resistant Phase contrast microscopy Refractile Loss of refractility staining Not readily stained Stained Stained readily with simple aniline dyes
    24. 24. DEMONSTRATION OF SPORES
    25. 25. Microscopy: Unstained preparation – Using Phase contrast microscope – Morphology best observed – large, refractile, oval or spherical bodies within mother cells Stained preparation
    26. 26. Stained preparation •Gram stain •ZN method (modified) •Dorner’s method •Malachite green stain 1. Schaeffer and fulton method 2. Ashby modification
    27. 27. Gram stain Reveals – deep blue bacilli, – colorless spore within the bacilli
    28. 28. Modified Ziehl-Neelsen technique • 0.25% sulphuric acid used as decolorizer • Spores- pink Bacteria - blue
    29. 29. 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%
    30. 30. Dorner’s Method Nigrosin (negative stain) red endospores in colorless vegetative cells.
    31. 31. 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) Malachite green staining
    32. 32. 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
    33. 33. Schaeffer-fulton Method Spore: green Vegetative cell: red http://www.arches.uga.edu/~howie/MVC-052endoS.JPG http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/endospore.jpg
    34. 34. Summary of all staining methods Primary stain Decoloriser Counter stain Dorner’s Carbol fuchsin 5% 10min Acid- alcohol 1min Nigrosin 10% Schaeffer & Fulton's Malachite green 0.5% 10 min Tap water Safranin ASHBY’s modification Malachite green 5% 1min Tap water Dilute carbol fuchsin Or 0.5% safranin Modified ZN Strong carbol fuchsin 5-7min 0.25% H2SO4 Methylene blue
    35. 35. Uses of spores • Sterilization control Bacillus steothermophilus • Nanobiotechnology – substrate for DELIVERY of biomolecules – vaccine vehicles spores – source for understanding unknown self-assembling molecules.
    36. 36. 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.
    37. 37. 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. • en.wikipedia.org/wiki/2001_anthrax_attacks • Emerging Applications of Bacterial Spores in Nanobiotechnology, Journal of Nanobiotechnology http://www.jnanobiotechnology.com/content/1/1/6

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