and is then followed by treatment with iodine. After that, alcohol 

 is applied for decolorization; but it proves that certain kinds of 

 bacteria retain the violet stain even after counterstaining with some 

 dye of a different color; while others are readily decolorized by the 

 alcohol, and take the counterstain. According to some, this action 

 is accounted for by assuming that the iodine combines with the 

 methyl violet inside the cell, converting it into a molecule so large 

 as to be unable to pass thru the cell membrane again. It may, on 

 the other hand, be assumed that there is an actual chemical differ- 

 ence between Gram-positive and Gram-negative bacteria, so that 

 the former combine chemically with the iodine and dye, while the 

 latter do not. Neither theory has been definitely proved or dis- 

 proved. 



Evidence is still lacking, in fact, to prove or disprove either the 

 chemical or the physical theory as it relates to general staining. The 

 difference, perhaps, is not one of immense importance. It is fre- 

 quently pointed out that there is no sharp distinction between 

 chemistry and physics, and in such delicate reactions as those in- 

 volved in staining, we may be well in the borderland between the 

 two branches of science, where it is impossible to say that a given 

 phenomenon is purely physical or purely chemical. There are, 

 however, certain chemical principles distinctly different from the 

 physical ones just mentioned, that may well enter into the phe- 

 nomenon of staining; and it is these that are considered most im- 

 portant by the exponents of the chemical theory. 



It is pointed out on behalf of the chemical theory that just be- 

 cause physical forces alone can explain the facts, one is not justified 

 in assuming that chemical unions do not take place when the 

 opportunity for them is present. It is kno^m that some parts of 

 the cell are acid in reaction, others alkaline; and it is a well kno^m 

 chemical principle that the former would tend to combine with the 

 kations in solutions with which they come in contact, the latter 

 with the anions. Now inasmuch as in certain dyes the color exists 

 in the kation (basic dyes) and in others in the anion (acid dyes), it 

 is natural to expect chemical combinations to take place between 

 dye^and tissue, depending upon the reaction of the latter. Argu- 

 ments for the physical theory which exclude chemical action must 

 furnish strong proof that no chemical union occurs; and those who 

 favor the chemical theory claim that such proof is lacking. That 

 the stained tissue does not present any characteristics to the eye 

 not possessed by either tissue or dye before staining does not prove 

 that no new substance has been formed, nor is this claim proved 

 by the fact that sufficiently long action of solvents removes the 

 color. Alcohol and even water are not absolutely inert chemically 

 and may withdraw the dye by chemical instead of physical action ; 

 the very length of time necessary to remove the color completely 

 (sometimes so long as to allow bacterial decomposition of the tissue) 

 indicates that a rather strong union between dye and tissue has 



101 



