Microbial Structures and Staining Reactions 109 



for a few seconds. This will convert the Congo red to a blue color, 

 which appears to be easier on the eyes for some individuals. Sharp 

 boundaries of organisms are characteristic when negative dyes are 

 used, and in studving sizes and shapes of bacteria, particularly 

 the spiral forms, this is a decided advantage. Students should be 

 cautioned not to confuse negative staining with a gram negative 

 reaction; they bear no relationship to each other. 



GRAM STAIN 



The gram stain is by far the most important differential stain 

 employed in bacteriology. The technic was introduced in 1884 

 by Christian Gram, a Danish scientist, after a chance observation 

 that tissues stained by his method could be made to release the 

 dye but the bacteria embedded in the tissue retained their color. 

 Further experimentation revealed that organisms could be divided 

 into GRAM POSITIVE bactcria (those bacteria retaining the dye) and 

 GRAM NEGATIVE bactcria ( those organisms giving up the dye ) when 

 the slide was placed in alcohol. Gram's original method was to 

 treat the smear with crystal violet followed by exposure to a dilute 

 solution of potassium iodide. In order to distinguish the gram 

 negative bacteria which have lost their color after the alcohol 

 treatment, a counterstain of safranin or of carbol fuchsin is em- 

 ployed. Thus, a mixture of gram positive and gram negative 

 organisms after having been subjected to this differential staining 

 procedure will reveal purple cells (the gram positive bacteria) and 

 pink cells ( the gram negative organisms ) . 



The binding effect of the iodine with the crystal violet is less 

 pronounced with the protoplasm comprising the gram negative 

 organisms, and subsequent decolorization in alcohol is more easily 

 accomplished. It should be pointed out, however, that excessive 

 exposure to alcohol will also decolorize gram positive bacteria. 

 The contact periods in each solution varv with the preference of 

 individual laboratories, and procedures designated to acquaint 

 students with the gram stain will outline specific methods. 



There are degrees of gram positiveness and gram negativeness. 

 To minimize the number of stained preparations that are difficult 



