224 DYEING 



this will retard or prevent the subsequent loss of methylene blue 

 from chromatin. The dye attaches itself in accordance with its own 

 electric charge and those of the tissue-constituents, and the tannic 

 acid merely traps it. There is here a strong contrast to the action of 

 mordants. When a mordant is used, the distribution of the colour 

 in the finished preparation depends on the affinity of the various 

 tissue-constituents for the metal. 



The use of iodine by Gram ^^^ to hold gentian violet in certain 

 bacteria is the most celebrated example of the use of trapping 

 agents in microtechnique. The method was invented by the 

 Danish pathologist in 1884, by accident. It was his intention to 

 introduce a double-colouring technique for diseased kidneys con- 

 taining casts {Harjicy Under n) in the tubules. He intended that 

 chromatin should be blue with gentian violet and the casts brown 

 with iodine dissolved in potassium iodide solution. Gram's hope 

 was not realized, for the dye disappeared quickly from the sections 

 on subsequent treatment with alcohol. Luckily for the cause of 

 bacteriology, however, he decided to find whether the dye would 

 again be quickly lost if the same method were applied to other 

 organs, and he chose some that were infected with bacteria. The 

 result was startling, for the bacteria were intensely dyed by the 

 gentian violet, while all the tissue-constituents of the host organism 

 lost every trace of blue in the alcohol used for differentiation. Thus 

 the bacteria were rendered more easily visible than had previously 

 been possible. 



Gram found that only certain particular kinds of bacteria lost 

 their blue colour in the alcohol, and this fact became the basis of 

 an important technique for distinguishing bacteria as Gram- 

 positive and Gram-negative. The method is still used in a slightly 

 modified form to the present day. Crystal violet is usually sub- 

 stituted for gentian violet, which is a variable mixture of the former 

 with related dyes. Another dye, of a contrasting colour, is often 

 used subsequently. This tends to disguise or displace the dye 

 trapped by iodine, and the term 'true Gram-positive' is sometimes 

 restricted to those bacteria or other objects that retain the colour 

 of the first dye when the second has been applied, and when 95% 

 alcohol has been allowed to act subsequently for a certain period. 

 There is, however, a large subjective element in all this, for the 

 periods in the various fluids are arbitrarily chosen. 



Most basic dyes can be substituted for gentian violet in Gram's 

 technique, provided that a second dye is not used subsequently. 



