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.

We claim: 
     
         1 . A kit for automated Gram staining comprising:
 a slow-acting decolorizing formulation in a first container.   
     
     
         2 . The kit of  claim 1 , further comprising:
 a primary stain formulation in a second container   a trapping agent formulation in a third container; and   a counterstain formulation in a fourth container.   
     
     
         3 . The kit of  claim 2 , wherein the first container, second container, third container and fourth container is configured for loading into automated staining equipment. 
     
     
         4 . The kit of  claim 1 , further comprising one or more microscope slides. 
     
     
         5 . The kit of  claim 1 , further comprising one or more control slides. 
     
     
         6 . The kit of  claim 2 , wherein the contents of the first container, second container, third container and fourth container are used to contact a sample in the following order: second container, third container, first container, fourth container; wherein the contents of two containers can be used at the same time. 
     
     
         7 . The kit of  claim 1 , wherein the slow-acting decolorizing formulation comprises 1,2-propanediol or ethylene glycol. 
     
     
         8 . The kit of  claim 1 , wherein the slow-acting decolorizing formulation comprises a mixture of 1,2-propanediol and ethylene glycol. 
     
     
         9 . The kit of  claim 1 , wherein the slow-acting decolorizing formulation comprises a slow-acting decolorizing agent and a fast-acting decolorizing agent. 
     
     
         10 . The kit of  claim 9 , wherein the slow-acting decolorizing formulation comprises at least 80% of the slow-acting decolorizing agent and no more than 20% of the fast acting decolorizing agent. 
     
     
         11 . The kit of  claim 2 , wherein the primary stain formulation comprises crystal violet or Gentian violet. 
     
     
         12 . The kit of  claim 11 , wherein the primary stain formulation further comprises ammonium oxalate. 
     
     
         13 . The kit of  claim 11 , wherein the primary stain formulation further comprises isopropanol, ethanol, and methanol. 
     
     
         14 . The kit of  claim 2 , wherein the trapping agent formulation comprises iodine. 
     
     
         15 . The kit of  claim 14 , wherein the trapping agent formulation further comprises water or alcohol. 
     
     
         16 . The kit of  claim 2 , wherein the counterstain formulation comprises fuchsin, neutral red, and/or safranin O. 
     
     
         17 . The kit of  claim 16 , wherein the counterstain formulation further comprises water. 
     
     
         18 . The kit of  claim 16 , wherein the counterstain formulation further comprises tartrazine 
     
     
         19 . The kit of  claim 16 , wherein the fuchsin is basic fuchsin. 
     
     
         20 . The kit of  claim 16 , wherein the counterstain formulation comprises fuchsin at a concentration of at least 0.1%.