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| Bacterial Stains |
| Gram Stain |
| - | primary stain: crystal violet |
| o | stains both gram-positive & gram-negative bacteria |
| - | mordant: Gram's iodine |
| o | enters bacterial cell & forms iodine-crystal violet complexes |
| - | decolorizer: acetone-alcohol |
| o | dehydrates thick cell wall of gram-positive bacteria; iodine-crystal violet dye complex is trapped in cells → gram-positive cells remain purple |
| o | disrupts outer membrane & partially deteriorates thin cell wall of gram-negative bacteria; iodine-crystal violet dye complex escapes cells → gram-negative cells become clear |
| - | counterstain: safranin |
| o | stains cell wall of cells |
| • | purple of gram-positive cells masks pink color & remain purple |
| • | unstained gram-negative cells become pink |
| - | potential issues |
| o | factors that could cause gram-positive bacteria to appear gram-negative: |
| • | old cultures; damage to cell wall |
| • | overzealous heat fixation |
| • | overzealous decolorization |
 Gram-positive bacteria (Staphylococcus epidermidis) |  Gram-negative bacteria (Escherichia coli) |
| (Ziehl-Neelsen) Acid-Fast Stain |
| - | primary stain: carbolfuchsin |
| o | carbolfuchsin (basic fuchsin in phenol), with mild heating, is able to penetrate the waxy layer of mycolic acids present in the cell walls of acid-fast bacteria, & enters both acid-fast & non-acid-fast bacteria (acid-fast bacteria are difficult to stain as most staining reagents are unable to penetrate the cell wall; phenol & heat assists dye entry into the cell) |
| - | decolorizer: acid-alcohol (3% HCl in ethanol) |
| o | carbolfuchsin remains in acid-fast bacteria as the cell wall is resistant to the acid wash → acid-fast cells remain pink |
| o | without a resistant layer of mycolic acids in the cell wall, the acid wash removes the carbolfuchsin from the non-acid fast bacteria → non-acid-fast cells become clear |
| - | counterstain: methylene blue |
| o | acid-fast cells retain the carbolfuchsin & remain pink |
| o | non-acid-fast cells take up the methylene blue & become blue-green |
| - | acid-fast bacteria: genus Mycobacterium |
| o | genus Nocardia is generally weakly acid-fast, & may not retain the primary stain following the typical acid wash (a weak acid wash (using 1% sulfuric acid) is sometimes used, which will usually allow these bacteria to retain the primary stain while still removing the primary stain from non-acid-fast bacteria) |
| o | common acid-fast bacteria: Mycobacterium tuberculosis, Mycobacterium leprae, & Mycobacterium avium-intracellulare complex |
 Mix of acid-fast & non-acid-fast bacteria (Mycobacterium gordonae & Staphylococcus epidermidis) |
| (Schaeffer-Fulton) Endospore Stain |
| - | primary stain: malachite green |
| o | heating allows malachite green to enter the tough spore coat of endospores... cooling traps the dye inside the spore coat (spore coats, like acid-fast cell walls are resistant to most staining reagents); vegetative cells take up malachite green as well |
| - | water wash |
| o | spore coats of endospores retain stain → endospores remain green |
| o | water washes malachite green from vegetative cells → vegetative cells become clear |
| - | counterstain: safranin |
| o | endospores retain the malachite green & remain green |
| o | vegetative cells take up the safranin & become pink |
| - | common endospore-forming bacteria: genus Bacillus; genus Clostridium |
 Endospore-forming bacterium Bacillus subtilis |
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copyright © 2004, Kevin Kelleher Questions or comments?... drop me an email! |
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