Gram staining method with improved decolorization of the crystal violet-iodine complex from gram negative bacteria

摘要

Provided herein are methods of staining biological material for the purpose of detecting, and in some examples also identifying, microorganisms. Methods of Gram staining bacteria using a slow-acting decolorizing formulation, such as one that includes 1,2-propandiol or ethylene glycol, can be used to extend the time of the decolorizing step, and thus permit automation of the Gram staining method. Also provided are compositions and kits for performing automated Gram staining on microscope slides.

主权项

1. A method for Gram staining bacteria on a slide using automatic staining equipment, wherein the automatic staining equipment comprises:
carousel means for holding a plurality of slides, each slide having thereon a sample to be stained; means for rotating the carousel means at predetermined speeds; and means for directing and controlling application of reagents onto the slides and samples during rotation of the carousel means, and wherein the method comprises: directing a crystal violet reagent onto the slides in the carousel to contact the bacteria on the slides with the crystal violet reagent; directing water, aqueous buffer, or aqueous detergent, onto the slides in the carousel to rinse the crystal violet reagent from the slides; directing iodine reagent onto the slides in the carousel to contact the bacteria on the slides with the iodine reagent; directing water, aqueous buffer, or aqueous detergent, onto the slides in the carousel to rinse iodine reagent from the slides; directing a slow-acting decolorizing formulation and a counterstain, simultaneously or separately, onto the slides in the carousel, wherein the slow-acting decolorizing formulation comprises a slow acting decolorizing agent selected from the group consisting of 1,2-propanediol, ethylene glycol, and mixtures thereof, and wherein the slow-acting decolorizing formulation takes at least 60 seconds to remove the crystal violet reagent and the iodine reagent from a Gram negative bacteria; and directing water, aqueous buffer, or aqueous detergent, onto the slides in the carousel to rinse the slow-acting decolorizing formulation and the counterstain from the slides, wherein at least 180 seconds elapse between the step of directing a slow-acting decolorizing formulation and a counterstain, simultaneously or separately, onto the slides in the carousel and the step of directing water, aqueous buffer, or aqueous detergent, onto the slides in the carousel to rinse the slow-acting decolorizing formulation and the counterstain from the slides.