294 



Scientific Proceedings (129) 



the dead cells are stained whereas the living cells are not. The 

 differentiation by this method is, however, not so sharp as when 

 Benians' technique is used. In the latter case the dead cells ap- 

 pear a distinct blue color, usually deeper in tint than the sur- 

 rounding film of dye. I have found that with suspensions 

 killed by heat the staining is not intense immediately after kill- 

 ing, but that the cells stain more deeply if allowed to stand 

 several hours after heating; and that they stain more readily 

 after heating to 60° than when heated to 100°. When killed 

 and preserved in formalin the cells do not stain; but dead cells 

 preserved in formalin retain their staining properties. It would 

 seem that autolysis must commence before the cells can stain. 



The metachromatic granules of some diphtheroids and the 

 sporogenous granules of some bacilli are stained by the Congo 

 red even in the living cells. The differentiation of the living 

 and dead cells can not, therefore, be attributed to changes in 

 the permeability of the cell membrane. The stain not only colors 

 the protoplasm of the dead cells but is also concentrated in the 

 film about them. It is possible that the staining may be ex- 

 plained by a loss or change of electrical charge in the cells. 



The films dry rather unevenly, being denser in the middle. It 

 is best to prepare a number of slides and to choose for counting 

 those which show the most uniform films. At least five slides 

 should be counted to compensate for the uneven distribution. 

 The counting is done by means of an eye-piece micrometer ruled 

 in squares and calibrated against a stage micrometer. With 

 suspensions of ten million per c.c. or over I count twenty fields 

 of .01 sq. mm. each from each of five slides. The average 

 deviation of such counts is usually less than 10 per cent. A 

 growth curve obtained by this method of counting was much 

 smoother than those obtained by other methods. A series of 

 comparative counts of a yeast cell suspension by the method 

 described and by the use of a counting chamber of the Helber 

 type showed that the two procedures were about equally ac- 

 curate. But with small bacteria the cells can be seen so much 

 more distinctly in the negatively stained film that a much larger 

 number can be counted without fatigue or eyestrain than is pos- 

 sible with the counting chamber; and I believe that the method 

 will prove correspondingly more accurate with such organisms. 



Broth and peptone solution precipitate the Congo red. The 

 method can not be used with such cultures unless they are first 



