522 EDMUND NEWTON HARVEY 



3. Mechanis7n of staining animal cells 



Animal cells show the same relations toward neutral red as do 

 plant cells: Paramoecia were placed in the following solutions: 



A. 10 cc. tap water + 0.1 cc. -^^ HCl (j^oo) + 1 drop 0.02 % neutral red. 



B. 10 cc. tap water + 0.1 cc. ^^ NaOH i^o^o) + 1 drop 0.02 % neutral red. 



C. 10 cc. tap water + 1 drop 0.02 % neutral red. 



After 30 minutes the individuals in A are unstained while those 

 in B and C are very deeply stained. In one hour the Paramoecia 

 in A show a faint pink color in some of their vacuoles, and later 

 they become noticeably stained. This is probably due to the 

 fact that the animals are constantly forming new food vacuoles. 

 A small amount of neutral red enters with the fluid of the vacuole. 

 The acid passing in at the same time is neutralized and the dye 

 may then pass the wall of the vacuole and stain certain granular ^ 

 bodies in the protoplasm. Paramoecia stain after some time in 

 acid solutions not because the dye may pass the surface mem- 

 brane but because it is engulfed along with the food of the organ- 

 ism. The food eaten may itself be stained. 



If paramoecia stained in neutral red are centrifuged in an elec- 

 trical centrifuge for one and one half hours it is easy to dif- 

 ferentiate the bodies with which the dye unites. Six more or less 

 distinct zones may be distinguished. These very soon mix again 

 due to the constant rotation of the protoplasm. Their relative 

 volumes are indicated in fig. 1. 



Only two substances in Paramoecium are found stained (1) 

 the food and granules in some of the vacuoles; (2) the minute 

 granules which often form a ring about the food vacuoles. Macro 

 and micronucleus, trichocysts, ciha and the clear fluid portion of 

 the protoplasm of the living organisms are quite unstained. 



The staining of marine eggs is essentially similar to the stain- 

 ing of Paramoecium. Owing to the presence of bicarbonates and 

 phosphates in sea water considerably more acid must be added, 

 than is necessary to change the color of neutral red from yellow 

 to red, before all the dye is actually in the acid condition and 

 consequently, before the dye will fail to enter the eggs. About 

 0.5 cc. T^o HCl to 100 cc. sea water is sufficient to bring about 



