VITAL STAINING 333 



Diffuse Staining. Rliodaniine B, safranin G and diamant- 

 fuchsin. 



Diffuse Staining with Faint Granule Staining. Crystal violet 6 B, 

 methyl violet 5B, auramine and malachite green. 



Dead cells stain diffusely with most dyestuffs. Intra-vitam 

 staining with trypan blue can therefore be employed for detecting 

 centres of necrosis in the animal body. Dyestuffs which have been 

 segregated by living cells become spread diffusely after cell death 

 except when appropriate iixation has been employed. 



According to Nassonov (Zcit.f. ZeUforsch., xi., 1030, p. 177 ; Profo- 

 plasma, xv., 1932, p. 239), Wallbacii {Zeit. f. ZeUforsch., xiii.. 1931, 

 p. 180) and Ai>exandrov (Protoplastna. xvii., 1932, p. 161), dyestuffs 

 which are sesregated under normal conditions of functional activity, fail 

 to be segregated under certain abnormal metabolic conditions (anaero- 

 biosis and acidosis), and instead stain the nucleus and colour the 

 cytoplasm diffusely. 



747. General Technical Procedure. (1) Sterilise dye solutions 

 before injecting by heating for not less than ten minutes in a 

 test-tube contained in a beaker of boiling water. Heat-labile 

 dyestuffs should be passed through a Bcrkefeld filter. (2) Swab 

 the skin with alcohol where the injection is to be made. (3) Mas- 

 sage the site of subcutaneous injections so as to spread the dye. 

 (4) After staining, tissues can be teased out in Ringer solution, 

 and the cells examined in the living condition. Small pieces of 

 connective tissue should be spread out rapidly on a dry slide 

 and a drop of Ringer solution added immediately, and covered 

 with a coverslip. 



PART II. VITAL STAINING WITH ACID DYES AND RELATED 



SUBSTANCES 



748. Trypan Blue. This is one of the best acid dyes for ititra- 

 vitam staining, and can be well retained in fixed tissues. 



Preparation. Dissolve 0-5 grm. in 100 c.c. of distilled water, 

 saline, or Ringer solution — some workers use a 1 per cent, solution. 

 A solution in distilled water is satisfactory for most purposes. 

 Filter. Heat at temperature of boiling water for ten minutes to 

 sterilise. Old solutions should not be used as alterations in 

 colloidal state occur after a time, and the dye becomes more toxic. 

 It is undesirable to use solutions more than a month old. The dye 

 solution is best kept in an ice safe. 



Methods of Staining and Amounts to Inject. Inject either 

 subcutaneously, intraperitoneally or intravenously. The following 

 amounts are recommended for subcutaneous injections : — 



Mice (per 20 grm. bodyweight) . . 0-5 to 1 c.c. 



Rabbits (per 1.000 grm. bodyweight) . 10 to 15 c.c. 



Frogs (per 20 grm. bodyweight) . . 0-5 to 1 c.c. 



Inject at intervals of three to five days. 



