344 VITAL STAINING 



have described a technique for vital staining with leucobases of 

 certain basic dyes. Their method of preparation is as follows : 

 To 100 c.c. of 0-01 per cent, dye solution are added 1 to 2-5 c.c. 

 of N/10 sodium hyposulphite and 1 to 4 c.c. of N/lO HCl. The 

 mixture is well stirred with a glass rod and kept at room tem- 

 perature in the dark. According to the dyestuff and the con- 

 centration of the reagents employed, colourless leucobases are 

 formed in from two to twenty-four hours. They resist oxidation 

 by atmospheric oxygen, but are re-oxidised if the medium is made 

 alkaline or exposed to daylight, especially to direct sunlight. 

 Leucobases have been prepared in this way from methylen blue, 

 azure 1, thionine, toluidine blue and brilliant cresyl blue. Mixtures 

 of two leucobases can be employed, e.g. of thionine and azure 1. 

 Staining is carried out by adding one or two drops of the leucobase 

 to cells contained in a drop of physiological saline. The method 

 is described as being suitable for differentiating between different 

 types of cells and for the study of oxydat ion-reduction processes. 

 Roskin and his co-workers have described the reaction of many 

 different kinds of cells to these leucobases, e.g. mast cells of the 

 frog when treated with a mixture of the leucobases of thionine 

 and azure 1 acquire a light blue coloration of their nuclei, rose- 

 coloured cytoiDlasm and an intense red coloration of their 

 granules. 



772. Indirect Vital Staining. Karczag and Paunz {Dent. med. 

 Woch., xlix, 1923, p. 1231) have introduced a technique which 

 they term " indirect vital staining." Dyestuffs (fuchsin S, light 

 green, water blue) are injected subcutaneously in 1-5 per cent, 

 solutions in saline. Rabbits receive in the course of a day 5 grni. 

 of fuchsin, 3 grm. of light green and 4 grm. of water blue. Tissues 

 may be fixed in formalin and frozen sections cut, or fixed in a 

 mixture of formalin and acetic acid and dehydrated and imbedded 

 as soon as possible. The sections are faintly coloured or colourless, 

 as the dyes are changed to colourless carbinol compounds. They 

 are converted to the coloured forms by treating sections for several 

 hours with 0-1 per cent. HCl. 



773. Staining of Intercellular Structures. The matrix of growing 

 bone can be stained by feeding young growing animals with 

 madder or with alizarin. Gottlieb {Anal. Anz., xlvi, 1914, 

 p. 179) injected 1 per cent, solutions of sodium alizarin sulphonate 

 in saline or Ringer solution. The free calcium salts of the new- 

 forming bone are stained, but not the cells. 



Some staining of the elastic fibres of connective tissue occurs 

 with trypan blue. For staining amyloid, Congo red has been 

 employed. 



774. Vital Staining as a Method for Investigating the pH of Cells. 

 Rous {Journ.E.rpt. Med., xli, 1925, pp. 379, 451, 739) has employed 

 litmus and the phthaleins for studying the _pH of the tissues. 



