36 STAINING REACTIONS OF BACTERIA 



feldt/ for example, has shown this to be the case for silver nitrate. Many observers 

 have referred to the fact that stained bacilli may remain actively motile, and they 

 have therefore assumed that the bacteria had been actually stained while alive. This 

 raises the question of the possibility of staining living cells without injuring them. So 

 far as the nucleus is concerned, it has long been held that this structure cannot be 

 stained during life although there is a great deal of evidence contrariwise (e.g.. 

 Churchman).^ That bacteria which are stained, at least in the sense that they appear 

 colored (although it might be objected that the stain was only adhering to the surface 

 and had not penetrated the cell), may grow well has been proved by the experiments 

 of the author.^ I have shown that gram negative organisms deeply stained with gen- 

 tian violet grow luxuriantly when transferred to agar, and by single-cell transplanta- 

 tion have proved that this growth is not due to bacteria which happen to have escaped 

 the stain. Experiments tend to confirm the theory that the penetration of a basic dye 

 into living cells depends on the fact that the dye is dissociated and that, in the form of 

 the free base which predominates at higher pH values, it penetrates very readily, 

 while in the form of salt its penetration may be so slight as to be negligible. Irwin,'' 

 on the basis of experiments with Valonia and Nitella flexilis, has warned against the 

 danger of assuming that the dye which appears to have entered a living cell from a 

 solution is actually the dye present in that solution rather than one of its lower 

 homologues. 



Many observers have noted that the gram positive organisms appear to take up 

 dyes in vivo more readily than the gram negative ones. From this fact it might be 

 argued that their greater sensitivity to the staining and bacteriostatic effect of tri- 

 phenyl-methane dyes was due to their increased permeability. But gram negative 

 and dye-sensitive strains of B. enteritidis and B. coli have been observed which were 



EXPLANATION OF PLATE I 



Photographs, through color screens, of camera lucida drawings in color. The gram positive 

 elements have photographed black; gram negative elements, gray. Magnification X3200. (a). 

 Smear from a suspension of a" 4-hour culture of B. anthracis (American Type Culture Collection 

 No. 10) to which acriviolet has been added. Smear made after 45-minutes exposure to the dye. The 

 organism, sharply gram positive at the beginning of the experiment, has become about 75 per cent 

 gram negative. The gram negative forms are about 40 per cent smaller in diameter than the gram 

 positive. Thesporeisentirely contained within the gram negative portion, {h). Smears from a 4-hour 

 culture of B. anthracis which has been stained by a modification of Burke's method in which the 

 period of exposure to dye and mordant has been greatly shortened and the period of exposure to 

 decolorizer greatly lengthened (10 sec, 10 sec, i min.). A large variety of partial decolorizations is 

 seen, the gram positive material (cortex) being in some cases entirely removed, in other cases per- 

 sisting as plaques, or lumps, or dots among which the gram negative medulla shines through. Notice 

 the terminal caps of gram positive material at a, b, and c. (c). Smear of a mixture of M.frcudenrcichi 

 and B. anthracis stained by a modification of Burke's method (stain 5 sec, iodine 5 sec, decolorizer 

 5 min.). Almost all the individuals of B. anthracis have been completely decolorized; M.frcuden- 

 rcichi has been unaffected. (Photograph made from colored drawings appearing in article by the 

 author, J. Exp. Med., 56, 1007. 1927.) 



' Nyfeldt, A.: Nord. med. Arhv., 50, 184. 1917. 



2 Churchman, J. W., and Russell, D. G.: Proc. Soc. Exper. Biol. 6" Med., xi, 120-24. 1914. 



3 Churchman, J. W., and Kahn, M. C: /. Expcr. Med., 33, 583. 192 1. 

 •« Irwin, M.: Proc. Soc. Exper. Biol, b' Med., 24, 425. 1927. 



