24 STAINING REACTIONS OF BACTERIA 



stains, without mordants. A few instances of elective staining have been described — 

 as, for example, the affinity of picric acid and neutral red for cholera vibrios and 

 kresylechtiviolet for gonococci; but, on the whole, there is so little difference in the 

 behavior of most bacteria to dyes that elective staining methods have not proved of 

 striking value. To this statement there are of course two notable exceptions — the 

 method of Gram and the method of Ziehl-Neelsen. 



The method of Gram illustrates well the fact that the progress which results from 

 a new observation is often in a different direction from that in which the observer was 

 at the time searching. Gram was attempting to develop a method for staining micro- 

 organisms in tissue. The differential staining which he noticed proved to be of great 

 value for the general identification and classification of bacteria. 



The ability to retain dye when stained by the method of Gram is not a property of 

 living cells in general but is almost entirely confined to yeasts and bacteria. All tissue 

 elements appear to be decolorizable save perhaps keratohyalin. Henrici found that 

 sections of vegetable tissue contained no gram positive elements; and in animal tissue 

 — while the nucleus retains the stain somewhat longer than the cytoplasm — all the 

 elements may be ultimately decolorized. Molds stain irregularly, isolated granules in 

 the mycelia retaining the stain, while large areas do not stain at all. Protozoa, spiro- 

 chetes, and malarial parasites are gram negative. It is well known, of course, that the 

 differentiation of bacteria into gram positive and gram negative is not hard and fast, 

 that the gram characteristic of a given organism, like any of its other characteristics, 

 may change with age or be disturbed by variations in environment and in other ways. 

 But within its well-established limitations the method of Gram is an exact one. It is 

 clear that the differential behavior of gram positive and gram negative bacteria must 

 ultimately depend upon difference in chemical or physical characteristics (or both) of 

 the bacteria themselves. A number of such differences have been proved to exist, and 

 for certain others there is strong, if not conclusive, evidence. Of these diiferences, the 

 principal ones are shown in Table I. 



It can be objected that the facts in nature are not quite so clear cut as a table of 

 this sort suggests. Not all gram positive organisms are equally gram positive, and it 

 has been shown that their gram behavior may in different groups of gram positive 

 organisms rest on an entirely different anatomical basis. They therefore differ among 

 themselves and cannot all be classified together. The same thing is doubtless true of 

 the gram negative organisms. Such a statement, for example, as the one that gram 

 positive organisms are as a rule much more susceptible to the bacteriostatic action of 

 basic triphenyl-methane dyes than gram negative' is open to a criticism of this kind, 

 and the criticism has in fact been made.^ Nevertheless, the statement as originally 

 enunciated is correct: with about lo per cent of exceptions, gram positive aerobes, 

 whether spore bearing or not, are extremely susceptible to the bacteriostatic effect of 

 these dyes and, with about lo per cent exceptions, gram negative aerobes are resistant. 

 The original statement of this parallelism was extended by Smith, Eisenberg, Simon, 

 and Wood^ to include about sixty dyes, and the work of these and other investigators 



' Churchman, J. W. : loc. cit. 



= Stearns, A. E., and E. W.: /. Bad., g, 493. 1924. 



3 Simon, C. E., and Wood, M. A.: Am. J. M. Sc, 147, 247, 524. 1914. 



