334 VITAL STAINING 



Staining occurs after the first injection, and increases in intensity 

 with repeated injections. Fix twenty-four hours after the last 

 injection. 



Fixation and Staining, (a) Frozen Sections. Fix small pieces 

 of tissue in 40 per cent, formalin for twenty minutes to two 

 hours according to size ; or in 10 per cent, formalin in normal 

 saline overnight. Cut sections with a COg freezing microtome. 

 Neutral red, or carmine are good for post-vital staining. Make 

 up neutral red as follows — dissolve 1-0 grm. of neutral red in 

 1,000 c.c. of distilled water, and then add 2 c.c. of a 1 per cent, 

 solution of glacial acetic acid. Stain sections rapidly ; less than 

 a minute is usually sufficient. Rinse with distilled water, and 

 blow off excess water from slide. Bring direct to absolute alcohol, 

 and after clearing in xylol, mount in Canada balsam. For details 

 of frozen section technique see §§ 223-232. 



In many tissues mitochondria can be demonstrated as well as 

 dye droplets by staining sections with Hollande's iron carmine 

 (see § 259). Ludford has employed this method in the study of 

 intra-vitam staining of liver and kidney cells {Proc. R.S.B., 

 ciii, 1928, p. 288). Stain sections for a minute or less in 

 the carmine solution. Blacken them in iron alum, and if 

 necessary leave in this solution till they are sufficiently differen- 

 tiated. Wash in running water. Dip in pyridine, and wash again 

 in running water. Dehydrate, and mount in Canada balsam. In 

 successful preparations the mitochondria are dark brown, and 

 the trypan blue droplets bluish-black to pale blue. 



(b) Paraffin Sections. Trypan blue can be retained in paraffin 

 sections after fixation by various methods. Thus tissues can be 

 fixed in 5 or 10 per cent, formalin, concentrated sublimate solution, 

 or in mixtures of these two (8-5 parts of saturated corrosive sub- 

 limate to 1 part of 40 per cent, formalin). The potassium 

 bichromate technique of Altmann for demonstrating mitochondria 

 has also been successfully employed. Pfuhl (Zeit. f. Zellforsch., 

 xiii, 1931, p. 783), who investigated the action of a large number 

 of fixatives on tissues vitally stained with this dye, came to 

 the conclusion that Heidenhain's " Susa " gave the best results. 

 This fixative is best made up fresh, as described in § 93. 



The amount of sublimate precipitated in the tissues is very 

 slight, so that it is usually unnecessary to employ any methods 

 for removing it. The treatment of vitally stained tissues, or of 

 sections of such tissues, with iodine dissolved in alcohol, for the 

 purpose of removing sublimate should be avoided as the vital 

 staining is seriously impaired. 



After fixation, which should vary from one to twenty-four hours, 

 according to the size of the pieces, they are transferred direct to 

 80 or 90 per cent, alcohol which should be changed several times. 



(c) Staining of Sections. The various carmine stains are specially 



