VITAL STAINING 337 



normal saline, and frozen sections cut and stained with Harris's 

 htcmatoxvlin. Bv careful stainiufT with Hollande's iron-carmine 

 method (see § 259) it is j^ossible to demonstrate mitochoiuh'ia 

 together with the carmine granules. 



For paraffin sections tissues are usually fixed in 85 to 95 per 

 cent, alcohol, 10 per cent, formalin, or in corrosive sublimate. 

 Formal-sublimate (8-5 parts of saturated sublimate to 1 part of 

 40 per cent, formalin) has also been employed. Iodine for the 

 removal of sublimate from sections must be employed with 

 extreme care, or decoloration will ensue. Suitable stains for 

 sections are Mayer's haem-alum, methylen blue or Pappenheim's 

 meth}l green and pj^ronin. Chrome-osmic methods (Altmann 

 and Schultze) for the demonstration of mitochondria have been 

 successfully em])loyed with tissues vitally stained with carmine. 



755. Vital Staining with Carmine in vitro. Carmine is frequently 

 employed for vitally staining tissue cultures. Small drops of a 20 

 per cent, solution of the stock solution should be applied to cultures 

 in the same manner as with trypan blue. They can be fixed in 10 

 per cent, formalin in saline, and stained with Harris's ha^matoxylin 

 or Mayer's hann-alum. 



756. Other Acid Dyes Suitable for Vital Staining. Other acid dyes 

 stated by von Mollendorff to be suitable for intra-vitain staining are 

 trypan red, dianil blue, cinnebar scarlet G, brilliant congo G, Hessisch 

 brilliant purple, vital new orange. 



757. Staining with India Ink. This is particularly suitable for 

 demonstrating the phagocytic properties of cells. It should be 

 injected intravenously. 



(i.) Preparation. (1) Dilute the India ink (Higgins' is usually 

 employed) 10 to 50 per cent, with distilled water. 



(2) Filter through two thicknesses of ordinary filter paper to 

 remove the coarser particles. 



(3) Boil ten minutes to sterilise. 



(ii.) Intravenous Injections. Cappell {loc. cit., 1929) recom- 

 mends for mice (20 grm. per bodyweight) 0-4 c.c. of a 10 per cent, 

 dilution, and for rabbits (1.000 grm. per bodyweight) 2 c.c. of a 

 50 per cent, solution. 



(iii.) Fixation. Since the ink particles are insoluble, any good 

 fixative can be used. In double staining with trypan blue, 

 " Susa " fixation, followed by staining with Rawitz carmine, is 

 recommended. 



(iv.) For Tissue Cultures. The addition of dilute India ink to 

 tissue cultures is a convenient method for investigating the 

 phagocytic properties of cells in vitro. When used with trypan 

 blue it affords a means of distinguishing between phagocytosis 

 and segregation (Ludfokd, loc. cit., 19.'33). 



758. Iron Compounds as Vital Stains. Iron compounds for 

 cytological studies have been employed amongst others by 



