Spore staining methods
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information about endospore and various methods to demonstrate it
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Spore staining methods
- 2. HISTORY Endospores were first described by Cohn (1872) in Bacillus subtilis and later by Koch(1876) in the pathogen, Bacillus anthracis. Cohn demonstrated the heat resistance of endospores in B. subtilis, and Koch described the developmental cycle of spore formation in B. anthracis.
- 3. HISTORY CONTD…. In 1922, Dorner published a method for staining endospores. It employed a lengthy heating step but resulted in differential staining of endospores and vegetative cells in the same sample. Shaeffer and Fulton modified Dorner’s method in 1933 to make the process faster.
- 4. ENDOSPORES….. Endospores are so named because they are formed intracellularly, although they are eventually released from this mother cell or sporangium as free spores. An endospore is a spherical/oval, thick-walled, highly resistant structure formed in certain bacterial species that represent a dormant or resting stage in the growth cycle of an organism. e.g. Bacillus sp., Clostridium sp.
- 5. ENDOSPORES….. Exhaustion of nutrients (Carbon and Nitrogen) Sub-optimal temperatures NOT a method of reproduction The endospore consists of the bacterium's DNA and part of its cytoplasm, surrounded by a very tough outer coating. Triggering factors
- 6. COVERINGS OF SPORE Spore wall : • delicate membrane from which cell wall of future vegetative bacterium develop Spore cortex Spore coat • tough, multi-layered Exosporium
- 7. RESISTANT TO…. Ultraviolet radiation Desiccation High temperature Extreme freezing Chemical disinfectants Starvation DESTROYED BY…. Autoclaving Tyndallisation ENDOSPORES…..
- 8. Germination of spores- Increase in metabolic activity of spore Endospore swells Germ cell appears by rupture or absorption of spore coat Elongates to vegetative bacterium
- 9. Distend bacillary body – Clostridium spp. Do not distend bacillary body – B. anthracis Central – B. anthracis, C. bifermentans Terminal - C. tetani, C. tertium Sub-terminal – C. perfringens (club-shaped) Oval - B. anthracis, C. tertium (tennis racket) Spherical - C. tetani. (drum-stick) SHAPE, POSITION AND SIZE OF SPORE RELATIVE TO PARENT CELL
- 10. SHAPE, POSITION AND SIZE OF SPORE Clostridium species in the pus sample
- 11. Bacillus species Clostridium species Thermobacillus Thermoflavimicrobium Sporolactobacillus Geobacillus Geosporobacter Oxobacter Acetonema – Gram negative bacilli Sporosarcina – Gram positive cocci SPORE FORMING BACTERIA Gram positive bacilli
- 12. METHODS OF DEMONSTRATION OF SPORES
- 13. Endospores are best observed in unstained wet films under the phase contrast microscope. They appear as large, refractile, oval or spherical within the bacterial mother cells or elsewhere free from the bacteria.
- 14. PHASE CONTRAST MICROSCOPY B. thuringiensis by Phase contrast microscopy
- 15. GRAM STAINING METHOD Vegetative cell - violet Spore – unstained clear area Air dry and observe under oil immersion. Safranine, 30 seconds, wash Acetone, water Gram’s iodine, 1 minute, wash Crystal violet, 1 minute, wash Heat fix the bacterial smear.
- 17. MODIFIED ZIEHL-NEELSEN STAINING METHOD Spores – red , Bacteria – blue Counterstain - Loeffler’s methylene blue for 1-2 min Decolourise using 0.25% to 0.5% sulphuric acid for 7-10 min Wash with water Heat the slide intermittently until steam rises for 5 min Cover the slide with carbol fuchsin Heat fix the smear
- 18. DORNER METHOD Vegetative cells – colourless Endospores – red Background – black. Observe under oil immersion lens Dry a thin even film of nigrosin(10%) on the slide Rinse with water and blot dry Decolorize with acid-alcohol for 1 minute Wash with water Cover the smear with carbol fuchsin and intermittently heat the slide for 5 min. Do not allow the stain on the smear to dry Heat fix the slide
- 19. VARIATION IN DORNER METHOD Vegetative cells – colourless Endospores - red Background – black. Observe under oil immersion lens. Mix a loopful of nigrosin on a glass slide with one loopful of the boiled carbol fuchsin-organism suspension and air dry to a thin film. Immerse the tube in a boiling water bath for 10 minutes. Mix an aqueous suspension of bacteria with an equal volume of carbol fuchsin in a test tube.
- 20. SCHAEFFER-FULTON METHOD / MODIFIED ASHBY METHOD Vegetative cell – pink or red and Spore – green. Counterstained with safranine 1 min, vegetative cells stained. After cooling, outer layer makes the spore resistant to the action of decolorizing agent (water), but water can easily decolorize the vegetative cells. Smear taken from the steam bath and allowed to cool. Both the spore and vegetative cells appear green. Smear is heated over a steam bath for 5 min. The primary stain, Malachite Green, is added over the heat fixed bacterial smear
- 23. MOELLER STAIN Spores red; Bacteria-blue. The slide is rinsed with acidified ethanol, and counter-stained with methylene blue for 30 seconds. The slide is then heated over a bunsen burner, or suspended over a hot water bath, covered with a paper towel, and steamed for 3 minutes. Carbol fuchsin is applied to a heat-fixed slide.
- 24. MODIFIED MOELLER STAINING METHOD Rinse thoroughly in running water Differentiate with 2 % sulphuric acid for 5-10 sec. Rinse thoroughly in running water. Flood with un-steamed Kinyoun’s carbol-fuchsin solution containing Tergitol 7, and stain for 3 min. Rinse thoroughly in running water. Immerse in 5% chromic acid solution for 3 min. Fix in absolute methyl alcohol for 1-3 min. Spread a small drop of the specimen on a slide, and allow it to air-dry at room temperature.
- 25. MODIFIED MOELLER STAINING METHOD CONTD… Microscopic examination using oil objective lens Rinse with running water and allow it to air-dry. Counterstain with 0.1 % Loeffler’s methylene blue for 1-2 min. Rinse for 10 sec in running water. Decolorize with 80 % ethanol until removal of excess stain dye from the slides.
- 26. ABBOTT'S METHOD Spores blue, bodies of the bacteria red. Wash in water, dry and mount. Stain for 8-10 seconds in aniline-fuchsin solution. Wash in water. Wash in 95% alcohol containing 0.2 to 0.3% HCl. Wash in water. Stain the slide deeply with methylene-blue, heating until the solution boils.
- 27. HOLBROOK AND ANDERSON LIPID/SPORE STAINING METHOD(1980) Wash in water, dry and examine. Spores-green, vegetative bacilli-red, lipid granules-unstained. Counterstain with 0.5% safranine for 20 sec. Wash film with xylene for 5 sec and blot dry. Stain with 0.3% sudan black B in 70% ethanol for 15 min Stain the film with 5% malachite green for 2 min while the slide is held little above the surface of boiling water in a beaker. Then wash with water and blot dry. Air dry the film and fix with minimal flaming.
- 28. Many treatments are known which destroy the permeability barrier, such as Severe heat fixation (Bartholomew and 'Mittwer, 1950), Acid hydrolysis (Robinow, 1951), Ultraviolet light (Bartholomew and Mittwer, 1952), Mechanical rupture (Fitz-James, 1953; Rode and Foster, 1960). OTHER MODIFICATIONS OF DORNER’S AND SCHAEFFER- FULTON METHODS….
- 30. SUMMARY Method Primary stain Decoloriser Counterstain Interpretation Grams stain Crystal violet Acetone Saffranin Spore-colourless Bacteria-violet Modified Ziehl- Neelsen Stain Carbol fuchsin 0.25%-0.5% H2SO4 Loeffler’s methylene blue Spore-red Bacteria-blue Dorner stain Carbol fuchsin Acid alcohol Nigrosin Spore-red Bacteria-colorless Variation in dorner stain Carbol fuchsin Nigrosin Spore-red Bacteria-colorless Schaeffer-Fulton stain Malachite green Water Safranine Spore-green Bacteria-red Bartholomew and mittwer method Malachite green Water Safranine Spore-green Bacteria-red Abbott's Method Methylene blue Acid alcohol Aniline-fuchsin Spore-blue Bacteria-red Moeller stain Carbol fuchsin Acidified ethanol Methylene blue Spore-red Bacteria-blue Modified moeller stain Kinyoun’s carbol-fuchsin 2%H2SO4 80% ethanol Loeffler’s methylene blue Spore-red Bacteria-blue
- 31. REFERENCES Colour Atlas & Textbook of Diagnostic Microbiology by Koneman Practical Medical Microbiology by Mackie & McCartney Monica Cheesbrough Textbook of Microbiology by Ananthanarayan http://www.microbelibrary.org/component/resource/laboratory-test/3112- endospore-stain-protocol http://chestofbooks.com/health/disease/Pathology/Methods-For-Staining- Spores.html# http://www.generalmicroscience.com/microbial-laboratory- techniques/endospore-staining-by-bartholomew-and-mittwers-method/ M. Hayama, K. Oana, T. Kozakai, S. Umeda, J. Fujimoto, H. Ota, Y. Kawakami Proposal of a simplified technique for staining bacterial spores without applying heat-successful modification of Moeller’s method. Eur J Med Res (2007) 12: 356-359.
Editor's Notes
- Bacteria produce a single endospore internally. The spore is sometimes surrounded by a thin covering known as the exosporium, which overlies the spore coat. The spore coat, which acts like a sieve that excludes large toxic molecules like lysozyme, is resistant to many toxic molecules and may also contain enzymes that are involved in germination. The cortex lies beneath the spore coat and consists of peptidoglycan. The core wall lies beneath the cortex and surrounds the protoplast or core of the endospore. The core contains the spore chromosomal DNA which is encased in chromatin-like proteins known as SASPs (small acid-soluble spore proteins), that protect the spore DNA from UV radiation and heat. The core also contains normal cell structures, such as ribosomes and other enzymes, but is not metabolically active. Up to 20% of the dry weight of the endospore consists of calcium dipicolinate within the core, which is thought to stabilize the DNA. Dipicolinic acid could be responsible for the heat resistance of the spore, and calcium may aid in resistance to heat and oxidizing agents. However, mutants resistant to heat but lacking dipicolinic acid have been isolated, suggesting other mechanisms contributing to heat resistance are also at work.[5] Visualising endospores under the light microscope can be difficult due to the impermeability of the endospore wall to dyes and stains. While the rest of a bacterial cell may stain, the endospore is left colourless. To combat this, a special stain technique called a Moeller stain is used. That allows the endospore to show up as red, while the rest of the cell stains blue. Another staining technique for endospores is the Schaeffer-Fulton stain, which stains endospores green and bacterial bodies red. The arrangement of spore layers is as follows: Exosporium Spore coat Spore cortex Core wall
- Because of their tough protein coats made of keratin, spores are highly resistant to normal staining procedures.
- C. Perfringens also called as C.welchii. Other subterminals- C.sporogenes, c. botulinum, c.novyi type C. other terminals- C. innocuum, C. putrifiucm, C. melanominatum. C.bifermentans and C. sordelli don’t typically bulge. Central – B.cereus, B.megaterium
- It is equivalent to combining Burdon’s stain for lipid with Ashby’s stain for spores.