Automated Staining and Decolorization of Biological Material

Abstract

An improved method and apparatus for staining samples of biological material for accurate analysis of the sample. Biological material is applied to a substrate, such as a microscope slide. The biological specimen is then stained with a selected staining composition, which may be gentian violet for a Gram's Stain analysis. The stained biological material is at least partially decolorized and the level of decolorization is analyzed optically. If necessary, the decolorizing step and the optical analysis steps are repeated until a selected level of decolorization is obtained.

1 . (canceled) 
     
     
         2 . An apparatus for detecting the level of staining in a microscope slide, the apparatus comprising:
 a plurality of fluid reservoirs, each containing a fluid, the fluids including at least a stain fluid and a decolorizer fluid,   a slide handling mechanism configured to position and support a microscope slide with a biological specimen thereon for application of fluids from the fluid reservoir for staining of the biological specimen;   a controller coupled to the fluid reservoirs to control flow of the fluids onto the biological specimen on the microscope slide;   a collector configured to at least temporarily collect the decolorizer fluid that runs off the biological specimen;   an electro-optical detector coupled to the controller and arranged to detect a level of decolorization in the decolorizer fluid collected in the collector by comparing a characteristic of the collected decolorizer fluid to a selected threshold that indicates an adequate level of staining in the biological specimen, the detector including:
 a source of radiation arranged to irradiate the collector and the collected decolorizer fluid with radiation of at least one selected wavelength that is absorbed by the stain fluid contained in the collected decolorizer fluid; 
 a radiation detector configured to detect a degree of absorption of the radiation of selected wavelength by the collected decolorizer fluid, the detector coupled to the controller for comparison of the degree of absorption with the selected threshold; and 
 a wavelength-selective filter between the source of radiation and the radiation detector. 
   
     
     
         3 . The apparatus according to  claim 2 , wherein the stain fluid comprises Gentian violet and iodine. 
     
     
         4 . The apparatus according to  claim 2 , wherein the decolorizer fluid is acid alcohol. 
     
     
         5 . The apparatus according to  claim 2 , wherein the selected wavelength of the radiation is that corresponding to a violet portion of the visible spectrum. 
     
     
         6 . The apparatus according to  claim 2 , wherein the radiation detector is a photodiode. 
     
     
         7 . A decolorization module for use in an apparatus for automatically staining a biological specimen on a microscope slide, the apparatus including a plurality of fluid reservoirs, including at least a reservoir of stain fluid and a reservoir of decolorizer, the decolorization module comprising:
 a slide handling mechanism including a microscope slide carrier configured to support the microscope slide with the biological specimen during application of fluid from the fluid reservoirs;   a controller coupled to the fluid reservoirs to control dispensation of at least the stain fluid and the decolorizer onto the biological specimen on the microscope slide on the slide carrier;   a collector configured to at least temporarily collect the decolorizer that runs off the biological specimen;   an electro-optical detector coupled to the controller and arranged to detect a level of decolorization in the decolorizer collected in the collector by comparing a characteristic of the collected decolorizer to a selected threshold that indicates an adequate level of decolorization in the biological specimen, the detector including:
 a source of radiation arranged to irradiate the collected decolorizer with radiation of at least one selected wavelength that is absorbed by the stain fluid contained in the collected decolorizer; 
 a radiation detector configured to detect a degree of absorption of the radiation of selected wavelength by the collected decolorizer, the detector coupled to the controller for comparison of the degree of absorption with the selected threshold; and 
 a wavelength-selective filter between the source of radiation and the detector. 
   
     
     
         8 . The apparatus according to  claim 7 , wherein the stain fluid comprises Gentian violet and iodine, and each is contained in a separate fluid reservoir. 
     
     
         9 . The apparatus according to  claim 7 , wherein the decolorizer is acid alcohol. 
     
     
         10 . The apparatus according to  claim 7 , wherein the selected wavelength of the radiation is that corresponding to a violet portion of the visible spectrum. 
     
     
         11 . The apparatus according to  claim 8 , wherein the radiation detector is a photodiode. 
     
     
         12 . A method of staining and analyzing a biological specimen comprising the steps of:
 applying the biological specimen to a substrate; staining the biological specimen with a selected staining composition;   at least partially decolorizing the stained biological specimen with a decolorizing fluid;   electro-optically detecting a level of staining in the decolorizing fluid; and repeating, if necessary, the decolorizing step and the electro-optically detecting step until a selected level of decolorization is obtained.   
     
     
         2 . The method according to  claim 1  wherein the staining composition is a Gram's stain composition comprising gentian violet and Gram's iodine. 
     
     
         13 . The method according to  claim 12 , wherein the step of applying the biological specimen to the substrate comprises applying the biological specimen to a microscope slide. 
     
     
         14 . The method according to  claim 12 , wherein the decolorizing step comprises washing the biological specimen with acid alcohol. 
     
     
         15 . The method according to  claim 12  further comprising a step of applying a counter-stain composition to the biological specimen after the selected level of decolorization is obtained. 
     
     
         16 . The method according to  claim 12 , wherein the step of electro-optically analyzing the biological specimen comprises the steps of passing radiation of at least one selected frequency through the decolorizing fluid after the decolorizing step; measuring the radiation transmitted through the decolorizing fluid; and comparing the measured transmitted radiation to a threshold decolorization value.