VITAL STAINING 339 



dilution according to Cowdry {Amer. Nat., Ix, 1926, p. 157) will 

 stain initocliondria of human lymphocytes selectively. 



761. Methods for Staining Mitochondria with Janus Green. 

 Supravital. Tease out small fragments of tissue in saline in a 

 small Petri dish. Draw off saline with pipette, and replace with 

 a shallow layer of the dye solution (20000 ^o 5oAoo)- Replace 

 the lid of the dish. With tissues of homoiothermal animals 

 place in incubator at body temperature for ten to twenty minutes, 

 and then mount for examination in saline. 



Supravital staining on prepared slides is especially recom- 

 mended for isolated cells, such as those of the blood and bone 

 marrow. 



SCOTT'S method (Anat. Rec, xxxvii, 1928, p. 233) is carried out as 

 follows : 



Preparation of the Slides. — (i.) Prepare a 1 per cent, solution of the 

 dye in absolute alcohol, and filter after an hour to remove any undis- 

 solved dye. (ii.) Employing a pipette with a bore of approximately 

 1 mm., allow three drops of the dye solution to fall on one end of each 

 of several cleaned slides, (iii.) Smear along the length of each slide 

 with the edge of another slide, as if making blood smears. The alcohol 

 evaporates, leaving a fine, even film of the dyestuff. 



Method of Staining. — (i.) Warm the prepared slides and some cover- 

 slips in the incubator, (ii.) Apply small drops of blood, or suspension of 

 cells, in saline to prepared slides and cover immediately with the warmed 

 cover-glasses. Press gently to spread the drops and seal the edges of 

 the cover-glasses with melted vaseline. Cells of compact tissues can be 

 stained by cutting the latter with a sharp knife, and pressing the cut 

 surface gently on to the prepared slide, then adding a small drop of 

 saline, and applying a cover-glass, (iii.) Examine if possible in a 

 warm chamber (37° C.) or on a warm stage, otherwise it is usually 

 necessary to place the slides in an incubator for a few minutes. 



Fixation of the Vital Staining, (i.) Wipe vaseline from the edge 

 of the cover-glass, (ii.) Remove cover-glass by sliding it towards 

 the nearest edge of the slide. This helps to spread the cells evenly and 

 permits of permanent preparations being made from both cover-glass 

 and slide, (iii.) Dry cover-glass and slide quickly in the air. Rapid 

 drying is essential to avoid distortion of the cells, (iv.) Complete drying, 

 if necessary, in a vacuum desiccator for two to four hours, or by shaking 

 in several changes of anhydrous ether. Treatment with ether is parti- 

 cularly recommended with bone marrow, in order to remove fat as well 

 as moisture, (v.) Clear in xylol, and mount in balsam. 



Intra-vitam Staining. Inject solutions of the dye in dilutions 

 of 1 in 20,000 to 1 in 50,000. After ten to thirty minutes 

 kill the animal, and tease out the tissues in saline. Some authors 

 recommend exposing the tissues to the air for a minute or two 

 before laying on the coverslip. 



Staining by Perfusibn. Kill the animal and inject the dye 

 solution in a dilution of 1 in 15,000 in isotonic salt solution, 

 through the aorta. Then remove tissues and transfer to saline 

 at room temperature (Bexsley, Am. J. Anat.. xiii, 1911-12, 

 p. 297). 



