164 VICTOR BURKE 



parts of this technique which are deemed worthy of special 

 attention. 



EFFECT OF SODIUM BICARBONATE WHEN ADDED TO THE PRIMARY 



STAIN 



The addition of a few drops of a strong solution of sodium 

 bicarbonate to the dye on the sUde improves the intensity of 

 the stain in the Gram positive organisms. A few drops of 10 

 per cent lactic acid produces the opposite result. No attempt 

 was made to determine to what these changes are due. We 

 are possibly dealing with changes in osmotic pressure and sub- 

 sequent concentration of the dye in the cell or with a change in 

 the size of the molecule of the iodine-dye precipitate, or simply 

 with a heavier precipitate. The sodium bicarbonate tends to pre- 

 cipitate the dye but the acid does not. The sodium bicarbonate 

 and lactic acid do not produce a permanent change in the cell 

 as is readily shown by exposure of the film to either one of the 

 solutions and then changing the reaction by the addition of the 

 other solution. A film so treated will stain as though the first 

 solution had not been used (Burke, 1921). 



The effect of the sodium bicarbonate is shown in staining films 

 made from old cultures of Gram positive organisms. Such films 

 stamed by the ordinary methods show many organisms which 

 are Gram negative and some which are Gram amphophile or 

 Gram positive. Similar films stained with sodium bicarbonate 

 added to the violet dye will show a larger percentage of Gram 

 positive organisms. Apparently some of the Gram amphophile 

 organisms have absorbed or retained a larger amount of the dye 

 and appear Gram positive. Sodium bicarbonate does not tend 

 to make a naturally Gram negative organism Gram positive. 

 These facts suggest the possibility, not heretofore recognized, 

 that the loss of the Gram positiveness of organisms in old cultures 

 is not entirely due, as formerly assumed, to autolysis and alt era- 

 staining described in this article satisfactory results were obtained with a crystal 

 violet and methyl violet furnished by the National Stain and Reagent Company 

 of Norwood, Ohio; three methyl violet and a gentian violet furnished by the 

 Will Corporation of Rochester, New York; and a methyl violet and gentian 

 violet furnished by the Harmer Laboratories of Philadelphia. 



