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BACTERIAL SPORES

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Post Graduate (M.D)Department Of Microbiology Karnataka Institute Of Medical Sciences HubliDr. MOHAMMED ASHRAF ALI s N

Definition, historyList of medically important spore forming bacteriaSpore formation/ SporulationStructureClassification based on shape and positionProperties Resistance and destructionGermination Demonstration of sporesUses of spores

Endospores were reported by numerous scientistsPerty 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 factorExposure to suboptimal temperature

primarily described them as refractile bodies

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Spore-forming bacteriaBacillus: Bacillus anthracis, Bacillus subtilis,Bacillus cereus, Bacillus clausii,Bacillus halodenitrificansClostridium: 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

- 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 toxin

SporolactobacillusSporolactobacillus 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 genus5

StagesStage IPre-sporulation phase DNA assembles as an axial filamentStage II Spore septum formationStage III Engulfment of the foresporeStage IV Cortex formationStage V Synthesis of spore coats, DIPICOLINIC ACID, calcium uptakeStage VI Spore maturation (coats more thick)Stage VII Lysis of cell and liberation of mature spore

Spore formation

STRUCTURE

5.Exosporiummade of glycoprotein andLipoproteins.

4. Outer Membranes(Spore Coats) keratin-like protein (consists sulphur containing aminoacids with disulphide bonds impermeableprotects spore from chemicals

Cortex thickest spore layer, made of modified peptidoglycansusceptible to lysozyme

2.Core Membrane/germ cell wallstructure similar to that in the vegetative cell wall

Spore protoplast/Core contains DNA,RNA proteinDIPICOLINIC ACIDDivalent ions (calcium)

Core Core Membrane Cortex Spore Coats Exosporium

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SHAPESAND POSITION

ShapesSpherical, oval or elongated in shapeMay be narrower or broader(bulge) than parent cell

sporeBacterial cell

sporeBacterial cell

Bulged Ex- Clostridium sp. Not bulgedEx- Bacillus sp.

PositionTerminal (located at one of the poles)Sub terminal or sub central(between center & one of the poles)Equatorial (central)

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Bacteria can be classified based on the shape and position of spores.

TERMINALSUBTERMINALCENTRAL OVALClostridium tetani( drumstick appearance)Clostridium welchii,Clostridium septicumClostridium sporogenesBacillus anthracisSPHERICALClostridium tertium--------------

spore

spore

sporespore

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)

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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

PROPERTYVEGETATIVE CELLSENDOSPORES Surface coatsmurein cell wall polymerThick cortex with spore coats Microscopic appearanceNon-refractileRefractile Calcium dipicolinic acidAbsentPresent in core Cytoplasmic water activityHighVery low Metabolic activityPresentAbsent RESISTANCEHeat resistanceLowHighResistance to chemicalsLowHighRadiation resistanceLowHigh SENSITIVESensitivity to lysozymeSensitiveResistantSensitivity to dyes & stainingSensitiveResistant

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

(cont.)

Extremely resistant due tolow water content (removal by osmosis)thick wall and impermeability of spore coathigh content of DIPICOLINIC ACIDability to concentrate calcium

Resists boiling for prolonged periods. Can survivedrying, radiation, and many damaging chemicals.Capacity to germinate many years after formation, may be centuries or ages.

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Sporostatic Sporicidal

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. outgrowthGERMINATION

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

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Endospore activationActivation- breaks the dormancy in spores, most spore properties retainedReversible Activators(triggering agents)Heating at 60C for more than 1 hourDecreasing the pHTreatment with mercaptoethanolConformational change in spore macromoleculesNo morphological changes

Germination rate- function of ageing22

Germination:Germination of activated spores occurs when exposedto certain substances(nutrients)aminoacidssugarsnucleosidesIrreversibleRemoval of cortex (enzyme -N acetyl glucosaminidase)- renders it susceptible to chemical agentsLoss of refractilityIn vitro germinationincubated at 37C, 5% CO2, FBS (fetal bovine serum)Dulbeccos modified Eagles medium(DMEM)+10% FBS Roswell Park Memorial Institute(RPMI)1640 medium +10%FBS,

When incubated at 37C and under 5% CO2 in the presence of FBS(fetal bovine serum)23

(b) development of phase dark formsc) Germination completes with full conversion to phase dark cellsmature, phase-bright (REFRACTILE) spores

OutgrowthThe 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 uptakeMolecular biosynthetic process startsRNA synthesis firstProteinDNA synthesisCell wall synthesis coincides with swelling

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.25

DEMONSTRATION OF SPORES

Stained preparationGram stainZN method (modified)Dorners method Malachite green stainSchaeffer and fulton methodAshby modificationMicroscopy: Unstained preparationUsing Phase contrast microscopeMorphology best observedlarge, refractile, oval or spherical bodies within mother cellsStained preparation

Gram stainReveals deep blue bacilli, colorless spore within the bacilli

Modified Ziehl-Neelsen technique0.25% sulphuric acid used as decolorizerSpores- pinkBacteria - blue

Dorners methodFirst spore staining described (1922)

Differential staining methodPrimary stain Carbol fuchsin for 5min Decolorizer acid alcohol for 1minCounterstain- Nigrosin 10%

Dorners Method

Nigrosin (negative stain)

red endospores in colorless vegetative cells.

Malachite green staining

Schaeffer-Fulton Stain Procedure1. Make a smear. Air Dry. Heat fix2. Flood the smear with Malachite Green stain3. flooded smear covered with a square of filter paper4. 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)

Schaeffer-Fulton Stain Procedure (continued)6. Drain slide and rinse for 30 seconds with DI water (discard filter paper)7. Put slide on steam rack8. Flood smear with Safranin (counter stain). This stains the vegetative cell. (Leave for 1 minute)9. Drain the slide and rinse with DI water10. Blot Dry11. Use oil immersion objective to view

Schaeffer-fulton MethodSpore: green Vegetative cell: redhttp://www.arches.uga.edu/~howie/MVC-052endoS.JPGhttp://homepages.wmich.edu/~rossbach/bios312/LabProcedures/endospore.jpg

Uses of sporesSterilization controlBacillus stearothermophilusNanobiotechnologysubstrate for delivery of biomolecules vaccine vehicles sporessource for understanding unknown self-assembling molecules.

Anthrax bioterrorismConcentrated anthrax spores were used for bioterrorism in the 2001, in U.S.Spores mailed in the form of powderled 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.

ReferencesTopley & Wilsons principles of bacteriology, virology and immunity, 8th edition. Vol-1Mackie & McCartney Practical Medical Microbiology, 14th editionA. D. RUSSELL Bacterial Spores and Chemical Sporicidal Agents Clinical microbiology reviews, ASM, Apr. 1990, p. 99-119.en.wikipedia.org/wiki/2001_anthrax_attacksEmerging Applications of Bacterial Spores in Nanobiotechnology, Journal of Nanobiotechnology http://www.jnanobiotechnology.com/content/1/1/6

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