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Differential staining

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this presentation consist four major staining technique of bacteria

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Differential staining

  1. 1. CONTENT: Introduction Gram stain Acid fast stain Endospore stain Capsuler stain
  2. 2. The gram stain developed by Christian Gram in the 1800’s was the first differential staining technique in use and is still an important tool for distinguish between two main type of bacteria Gram-ve Gram+ve. PROCEDURE:-
  3. 3.  Place slide with heat fixed smear on staining tray.  Gently flood smear with crystal violet and let stand for 1 minute.  Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.  Gently flood the smear with Gram’s iodine and let stand for 1 minute.  Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle. The smear will appear as a purple circle on the slide.  Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.  Immediately rinse with water.  Gently flood with safranin to counter-stain and let stand for 45 seconds.  Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.  Blot dry the slide with bibulous paper.  View the smear using a light-microscope under oil-immersion. 
  4. 4. Gram+ve cell will appear purple and Gram-ve cell appear pink
  5. 5. Acid Fast staining Acid Fast staining is also known as Ziehl Neelsen stain is used to identify specialized bacteria that have waxy mycolic acid in there cell wall. PROCEDURE 1 A slide shaped piece of blotting paper is placed over the bacterial smear. 2 Apply bright pink dye (Ziehl’s carbolfuchsin) on blotting paper and place over a water bath for 3-5 minutes and rinse the slide. 3 Decolorize with acid alcohal 4 Lastly purple counter stain(crystal violet) is applied and slide is rinsed. 5 Acid-fast(waxy)bacterial cell are bright pink and non acid fast bacterial cell are purplish blue.
  6. 6. ENDOSPORE STAINING The endospore stain is used to identify bacteria that can produce endospore or not. The endospore stain is used to identify bacteria that can produce endospore (small,dormant structure alike to bacterial seeds). Forming endospore is very advantages to the bacteria allow to survive under difficult circumstances, including dessication , starvation high heat , exposure to chemicals and radiation. This stain is designed to distinguish between vegetative cell of bacteria from spore.
  7. 7. PROCEDURE: 1 Place bacterial smear on water bath and stain with malachite green. 2 Heat on water bath for 5 minutes. 3 Rinse the slide and a pink counter stain (SAFRANIN) is applied for one minute. 4 After final rinse when viewing slide under oil immersion with a compound light microscope , vegetative cells will appear pink and endospore are stained a bluish green.
  8. 8. CAPSULAR STAINING Bacterial capsular are composed of high molecular weight polysccharides and are associated with virulent and bioflim formation. Capsule are not stain well with crystal violet, methylene blue or other simple stain. Here two method of capsular staining are present following :- 1 Manavel’s staining 2 Hiss method of capsular staining

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