Spore staining

Spore staining

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  SPORE STAINING PRESENTED BY KABERINATH ROLL NO- 17PBT206
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  WHAT IS SPORE? Highly resistant, dormant structures (i.e. no metabolic activity) formed in response to adverse environmental conditions.  Help in the survival of the organisms during adverse environmental conditions; do not have a role in reproduction.  Spore formation (sporulation) occurs when nutrients, such as sources of carbon and nitrogen are depleted.  Are resistant to heat, dehydration, radiation and chemicals.  Structurally and chemically more complex than the vegetative cell.  The shape and the position of spores vary in different species and can be useful for classification and identification purposes. 2
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  Spores may be: Central or equatorial, giving the bacillus a spindle shape (eg. Clostridium bifermentans)  Sub-terminal, the bacillus appearing Club shaped (eg. Clostridium perfringens)  Oval and terminal, resembling a tennis racket (eg. Clostridium tertium)  Spherical and terminal, giving a drumstick appearance (Clostridium tetani)  Mature endospores are released from the vegetative cell to become free endospores.  When the free endospores are placed in an environment that supports growth, the endospores will revert back to a vegetative cell in a process called germination.  endospore formation is not a reproductive process but a process of differentiation that provides the bacteria with a mechanism for survival. 3
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  THE ENDOSPORE STAIN A differential staining technique is used to distinguish between the vegetative cells and the endospores.  The procedure was designed by Alice B. Schaeffer and MacDonald Fulton, during the 1930s.(aka Wirtz-Conklin method)  The identification of spores is very important for the clinical microbiologist who is analysing a patient's body fluid or tissue since there are not that many spore forming genera.  As a spore forms inside of the vegetative cell, the spore wall chemically changes and thicken. This sporulation process changes the spore’s stainability, making it increasingly resistant to the staining dyes, and so a gimmick steaming enhances the primary dye’s penetration. 4
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  PRINCIPLE OF SPORESTAINING  A primary stain (malachite green) is used to stain the endospores.  Because endospores resist staining, the malachite green will be forced into the endospores by heating. In this technique heating acts as a mordant.  There is no need of using any decolorizer in this spore staining as the primary dye malachite green bind relatively weakly to the cell wall and spore wall .In fact If washed well with water the dye come right out of cell wall however not from spore wall once the dye is locked in.  Water is used to decolorize the vegetative cells.  As the endospores are resistant to staining, the endospores are equally resistant to de- staining and will retain the primary dye while the vegetative cells will lose the stain.  The addition of a counterstain or secondary stain (safranin) is used to stain the decolorized vegetative cells. 5
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  MATERIALS REQUIRED 1. Slide 2.Loop 3. Spore-forming bacteria 4. Malachite green 5. Safranin 6. Boiling water bath/ Bunsen burner 7. Microscope 8. Immersion oil. 6
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  PROCEDURE 7 1. Prepare smearsof organisms to be tested for presence of endospores on a clean microscope slide and air dry it. 2. Heat fix the smear. 3. Place a small piece of blotting paper (absorbent paper) over the smear, flood it with malachite green and place the slide (smear side up) on a wire gauze on a ring stand. 4. Heat the slide gently till it starts to evaporate (either by putting the slide on a staining rack that has been placed over a boiling water bath or via Bunsen burner).
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  8 5. Remove theheat and reheat the slide as needed to keep the slide steaming for about 3- 5 minutes. as the paper begins to dry add a drop or two of malachite green to keep it moist, but don’t add so much at one time that the temperature is appreciably reduced. 6. Remove the blotting paper and allow the slide to cool to room temperature for 2 minutes. 7. Rinse the slide thoroughly with tap water. 8. Stain the smear with safranin for 2 minutes. 9. Rinse both side of the slide to remove the secondary stain and blot the slide/ air dry. 10. Observe the bacteria under 1000x (oil immersion) total magnification.
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  OBSERVATIONS 1. The sporecan be terminal, central or sub terminal. 2. This may be helpful information in the identification of the unknown. 3. The vegetative cells should appear pink/red (i.e. colour of counter stain), 4. The vegetative cells that contain endospores should stain pink while the spores should be seen as green ellipses within the cells. 5. Mature, free endospores should not be associated with the vegetative bacteria and should be seen as green ellipses. 9
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  REFERENCES 1. Harley andPrescott: Laboratory Exercises in Microbiology, page 58. McGraw Hill, 2002. 2. Hussey, Marise; Zayaitz, Anne (2011-09-29). "Endospore Stain Protocol". American Society for Microbiology. 3. Tankeshwar Acharya: Bacterial spores: structure, importance and examples of spore forming bacteria; (april 28, 2013), Microbiology For Beginners, Structure Of Bacterial Cells, Virulence Factors Of Pathogenic Bacteria. 4. Samiksha S: Spore Staining of Bacteria to Differentiate Bacterial Spore and Vegetative Cell. 10
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