VITAL STAINING 335 



suitable for staining sections. Ludford {Wth Sci. Rep. Imj). 

 Cancer Res. Fd., 1932, p. 10!)) recommends Rawitz's carm-alum, 

 made up as described in § 254.. Progressive staining is desirable 

 so as to avoid the necessity for using acid fluids in order to 

 differentiate. 



749. Golgi Apparatus and Trypan Blue Staining. Jasswoin {Zeit. 

 f. Zellforsch., ii, 1925, p. 741), Nassonow (i6i(i., iii, 1926, p. 472), 

 Makarov {Arch. Russes d'Anat., d'Hist. et d'EmbryoL, v, 1926, 

 p. 157) and Glasunow {Zeit. f. Zellforsch., vi, 1928, p. 773) have 

 described a topographical relationship between the Golgi apparatus 

 and dye droplets in a variety of cells. Ludford {loc. cit., 1928) 

 was able to demonstrate the Golgi apparatus in vitally stained 

 kidney cells by the modified Mann-Kopsch method. Small pieces 

 of the vitally stained kidney were cut up with a sharp knife and 

 fixed overnight in corrosive-osmic solution. Then washed for 

 one hour in repeated changes of distilled water. The material 

 was then transferred to 2 per cent, osmic acid, and kept in an 

 incubator at 35° C. for two-and-a-half days : repeatedly washed 

 with warm water, and incubated a further day in distilled water. 



This method gave good results with kidney cells, but was not so 

 satisfactory with liver cells. However, it was found possible to demon- 

 strate trypan blue droplets in the latter by counterstaining sections 

 with neutral red. 



750. Vital Staining with Trypan Blue in vitro. Tissue cultures 

 can be vitally stained either by adding the dyestuff to the medium 

 employed for explantation, or by adding the dye in solution to 

 cultures after growth is well advanced. The dye is usually applied 

 in a 0-5 per cent, solution in Ringer solution, or distilled water. It 

 should be applied by means of a finely pointed pipette. To each 

 coverslip culture add a small drop, just sufficient, on diffusion, to 

 tint the medium a pale blue colour. Before fixation, wash the 

 cultures with Ringer solution. Fixation in " Susa " for ten to 

 twenty minutes followed by staining in Rawitz carmalum (see § 254) 

 for two to four minutes usually gives good preparations. 



For routine method of fixing and staining vitally stained cultures, 

 see Dunn (Arch. f. Zellforsch., xvi, 1934, p. 361). 



751. Vital Staining with Pyrrol Blue (Isamine Blue). This 

 dyestuff was largely used by Goldmann, but has been superseded 

 by trypan blue, which he also introduced as a vital stain. It is 

 deposited at the site of subcutaneous injections, and then goes 

 slowly into solution again giving rise to a general vital coloration. 

 For vitally staining mice, 0-5 to 1-0 c.c. of a 0-5 or 1-0 per cent, 

 solution of the dye should be employed. It is difficult to retain 

 in fixed preparations. 



Cappell (J. Path. Bad., xxxii, 1929, p. 595) recommends Zenker's 

 fluid as the best fixative, but dehydration must be rapid, and the 



