TWORTSSTAIN(OLLETTSMODIFICATION)-IndianaUniversity：tworts染色(olletts修改)-印第安那大学.pdfVIP

TWORTS STAIN (OLLETTS MODIFICATION) USED TO DEMONSTRATE: This method is used to demonstrate gram-positive and gram-negative bacteria. RECOMMENDED FIXATIVE: Fixation is done with a formal saline fixative. SPECIMEN THICKNESS PREPARATION: Paraffin embedded tissue section cut at 2-5 microns. MODE OF ACTION: This gram stain works with the dye-lake principal, and then a decolorizer will remove the stain from the gram-negative and not from the gram-positive bacteria, then an application of the second dye will stain the gram-negative bacteria. PURPOSE OF REAGENTS DYES: Aniline crystal violet is the stain solution that will give the gram-positive bacteria its blue coloration. Grams iodine will fomn the dye-lake as in other stains. Acid alcohol will be made with acetic acid in this method and then used as the differentiator. Modified Tworts stain js the second solution added to stain the gram-negative bacteria. SOLUTIONS Aniline Crystal Violet Crystal violet ……………………… 5 gm Absolute alcohol. ………………….10 ml Aniline ……………………………… 2 ml Distilled water …………………… ... 88 ml Modified Tworts Stain (Stock) 0.2% alcoholic neutral red ………… 90 ml 0.2% alcoholic fast green………… ..10 ml Working solution: One volume of the stock solution is diluted with three volume of distilled water. NOTE: The stock alcoholic solution should be of reddish-magenta tint: Too much green will weaken the red bacterial staining; the total dye concentration is less critical. CONTROL TISSUE TO USE: A tissue section containing gram-positive and gram-negative bacteria. COLOR RESULTS: Nuclei-- - -- -- -- - -- -- -- red Cytoplasmlight green Red blood corpuscles green Gram-positive bacteriaDark blue Gram-negative bacteriapink SOURCES OF ERROR TROUBLESHOOTING: As in other Gram stain methods this one will also depend on the decolorization step to achieve a distinction betw