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

Abstract

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.

1 . A composition comprising at least 80% of one or more slow-acting decolorizing agents by total volume of the composition, and no more than 20% of one or more fast-acting decolorizing agents by total volume of the composition. 
     
     
         2 . The composition of  claim 1 , wherein the one or more slow-acting decolorizing agents are selected from the group consisting of 1,2-propanediol, ethylene glycol and combinations thereof. 
     
     
         3 . The composition of  claim 1 , wherein the one or more fast-acting decolorizing agents are selected from the group consisting of propyleneglycol methyl ether, diethyl ether, ethanol, acetone, methanol, 1-propanol, 2-propanol, 1-butanol, and combinations thereof. 
     
     
         4 . The composition of  claim 1 , wherein the one or more fast-acting decolorizing agents comprises a blend of ethanol and acetone. 
     
     
         5 . The composition of  claim 1 , wherein the one or more fast-acting decolorizing agents comprises a blend of ethanol and a second alcohol. 
     
     
         6 . The composition of  claim 1 , wherein the one or more slow-acting decolorizing agents is 1,2-propanediol. 
     
     
         7 . The composition of  claim 1 , wherein the one or more fast-acting decolorizing agents is propyleneglycol methyl ether. 
     
     
         8 . The composition of  claim 1 , wherein the composition comprises 1,2-propanediol and propyleneglycol methyl ether. 
     
     
         9 . The composition of  claim 1 , wherein the composition comprises about 80% of 1,2-propanediol by total volume of the composition and about 20% of propyleneglycol methyl ether by total volume of the composition. 
     
     
         10 . The composition of  claim 1 , further comprising water, a buffer, or mixtures thereof. 
     
     
         11 . The composition of  claim 10 , wherein the composition has a pH ranging from about 3 to about 8. 
     
     
         12 . The composition of  claim 1 , wherein the composition comprises at least 85% of the one or more slow-acting decolorizing agents by total volume of the composition. 
     
     
         13 . The composition of  claim 1 , wherein the composition comprises at least 90% of the one or more slow-acting decolorizing agents by total volume of the composition. 
     
     
         14 . The composition of  claim 1 , wherein the composition consists essentially of at least 80% of one or more slow-acting decolorizing agents by total volume of the composition, and no more than 20% of one or more fast-acting decolorizing agents by total volume of the composition. 
     
     
         15 . The composition of  claim 1 , further comprising a counterstain. 
     
     
         16 . A decolorizing composition comprising a slow-acting decolorizing agent and a second agent selected from the group consisting of water and a buffer, wherein an amount of the second agent ranges from about 0.5% to about 25% by total volume of the decolorizing composition. 
     
     
         17 . The decolorizing composition of  claim 16 , wherein the composition further comprises no more than 20% of a fast-acting decolorizing agent by total volume of the decolorizing composition. 
     
     
         18 . The decolorizing composition of  claim 17 , wherein the fast-acting decolorizing agent is selected from the group consisting of propyleneglycol methyl ether, diethyl ether, ethanol, acetone, methanol, 1-propanol, 2-propanol, 1-butanol, and combinations thereof. 
     
     
         19 . A composition comprising between about 75% to about 99% of one or more slow-acting decolorizing agents and a counterstain. 
     
     
         20 . The composition of  claim 19 , wherein the one or more slow-acting decolorizing agents are selected from the group consisting of 1,2-propanediol, ethylene glycol and combinations thereof.