Staining Filamentous Bacteria Filaments Identifiable by Staining

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Staining Filamentous Bacteria Filaments Identifiable by Staining Staining Filamentous Bacteria Filaments Identifiable by Staining Gram Positive Neisser Positive Microthrix parvicella Microthrix parvicella (granules) Nocardia Nocardia (granules) Nostocoida limicola Nostocoida limicola Type 0041/0675 Type 0092 Type 1851 Type 0914 Tools for Staining • Microscope – Bright field (not phase contrast!) – Preferably 1000x oil immersion objective – Minimum of 200x objective Tools for Staining • Microscope Slides • Clothes pin • Wash bottle • Watch with a second hand • Paper towel • Bon Ami Scouring Powder (optional) Tools for Staining • Gram Stain Kit – Gram Crystal Violet Solution – Gram Iodine Solution – Gram Decolorizing Solution – Gram Safranin Solution Gram Staining Procedure 1. Gram Crystal Violet Solution • Flood slide for 1 minute • Rinse with DI water 2. Gram Iodine Solution • Flood slide for 1 minute • Rinse with DI water 3. Gram Decolorizing Solution • Hold slide at 45 degrees and apply dropwise until blue color stops rinsing off • Blast with DI water to stop reaction, blot dry with paper towel 4. Gram Safranin Solution • Flood slide for 1 minute • Rinse with DI water 5. View Slide at 1000x under bright light (not phase contrast) Tools for Staining • Neisser Stain Kit – Neisser Methyl Blue Solution A – Neisser Crystal Violet Solution B – Neisser Bismark Brown Solution – Transfer pipet – Container for mixing Solutions A and B Neisser Staining Procedure 1. Methyl Blue / Crystal Violet Solution • Mix 2 parts Methly Blue and 1 part Crystal Violet in a small container • Flood slide for 30 seconds • Rinse with DI water 2. Bismark Brown Solution • Flood slide for 1 minute • Rinse with DI water and blot dry (do not rub the slide) 3. View Slide at 1000x bright light (not phase contrast) .
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