DS 22.20-DS 22.21 



DYE STAINS OF SPECIAL APPLICATION 



441 



the high concentration of acid fuclisin will 

 not be maintained if it is poured onto a 

 slide over which are gathered films and 

 droplets of water. Lay the slide on a 

 support of some kind and wave a bunsen 

 flame backward and forward underneath 

 it, until steam rises from the surface of 

 the stain; no bubbles must bo i)ermitted to 

 form. Neither the time of staining nor the 

 exact temperature are in any way critical; 

 as soon as the slide has started to steam, 

 the flame is removed and the slide left 

 until it is cool enough to handle. Then use 

 a jet of water from a wash bottle to re- 

 move the excess acid fuchsin. The sections 

 will now be stained a dense purple red, 

 and are transferred to the solution of 

 toluidine blue in a coplin jar for one to 

 two minutes. The purpose of the toluidine 

 blue is to remove the acid fuchsin from 

 the nuclei and to replace it with the blue 

 stain. After one or two minutes pick out 

 the slide, rinse it in distilled water, and 

 place it in the aurantia until the mito- 

 chondria are differentiated through the 

 removal of the acid fuchsin from the 

 cytoplasm around them, and the sulisti- 

 tution of the clear yellow color of the 

 aurantia for this pink color. This process 

 may only with difficulty be controlled 

 under the microscope. It is better to leave 

 a trial slide in the aurantia for two min- 

 utes, to remove it, dehydrate it in a]:)Solute 

 alcohol for the minimum time possible, 

 and then to transfer it to xylene before 

 examining it under the high power of the 



microscope. If the timing in the toluidine 

 blue and aurantia solutions has been cor- 

 rect, the nuclei will be a vivid, bright 

 blue, the cytoi)lasiu clear yellow, and the 

 mitochondria vivid scarlet. If the nuclei 

 are still purple, and the mitochondria are 

 not clearly differentiated in scarlet, the 

 next trial slide should be left longer in 

 toluidine blue. If, however, the only defect 

 is that the cytoplasm remains a clear pink, 

 the time in aurantia should be increased. 

 By thus juggling the times in the toluidine 

 blue and the aurantia (it is a waste of time 

 to try to control the timing in the acid 

 fuchsin or in the alcohols), it is possible to 

 arrive at a schedule which yields perfect 

 preparations. Once this schedule has been 

 established, it is possible to prepare a long 

 series of slides, in each which there is 

 clear differentiation of nuclei, mito- 

 chondria, and cytoplasm. It must be 

 pointed out that the staining technique of 

 Altmann, which has been used, can also be 

 used to stain bacteria, and that if the 

 sections are mounted on the slide with an 

 excess of either gelatin or egg albumin, 

 and are then ])ermitted to remain damp 

 and warm for 24 hours, bacteria will un- 

 doul)tedly be found, and also that these 

 bacteria have given rise to certain inaccu- 

 rate observations. This can be avoided, 

 however, either by using an adeciuate 

 quantity of a biostatic agent in the 

 adhesive used for the sections, or by avoid- 

 ing leaving them under conditions which 

 will encourage bacterial growth. 



22.21 OTHER TECHNIQUES 



22.21 Altmann 1890 test. 1928 Gatenby and Cowdry Gatenhy and Cowdry 1928, 3.33 



REAGENTS required; A. sat. sol. aniline 100, acid fuchsin 20; B. water 65, sat. sol. picric 

 acid in 95% ale. 35 



method: [sections of F 1700.0000 Altmann 1890 fixed material]-^ water A, on slide, 

 heated to steaming, 1 min. — » B, on slide, heated to steaming, till differentiated — > 

 blot ^ abs. ale, minimum possible time — » balsam, via xylene 



result: mitochondria red on yellow. 



note: This method is sometimes erroneously referred to Zimmerman. For a slight modi- 

 fication of this procedure see DS 21.421 Severinghaus 1932. Kiyono 1914 (236S1, 

 25:481) states that formaldehyde-fixed material t-an be ytained by this technique if 

 the sections are treated 2-3 days at 37°C. in AMS 12.2 Kiyono 1914. For an adapta- 

 tion of this technique to blood smears see DS 2.28 Schmorl 1928. Baker 1932 (19400, 

 30:134) recommends heating tissues intended for this technique with 0.05% to 0.5% 

 parabenzoquinone in isotonic saline for 1 hour prior to fixation. For a detailed de- 

 scription of the preparation of the .4 sol. see under DS 22.20 above. 



22.21 Bailey 1920 11343,42:353 



REAGENTS REQUIRED: A. DS 22.21 Altnuiuu 1890 (sol. A); B. water 100, acid'violet 1, 

 10% sulfuric acid q.s. 



