320 CYTOLOGICAL METHODS. 



than among invertebrates. See also " Charnpy's Fluid " ( 43) and 

 " Altmann's Fluid " ( 42), and 41. 



679. FLEMMING'S Strong Fluid without Acetic Acid, and Iron 

 Haematoxylin. Small organs freshly dissected out in normal saline, 

 or parts of organs cut with safety razor blade, not more than 5 milli- 

 metres in diameter, are placed in about 15 c.c. of one of the above- 

 mentioned fixing fluids, preferably Gatenby's modification. A 

 glass-covered capsule or vial is the best vessel to use. and the material 

 is left for at least twenty hours, and not longer than one week. We 

 find about twenty-four hours gives a satisfactory fixation of most 

 tissues. After fixation the liquid is poured away, and the material 

 is washed for at least two hours, and not necessarily longer than 



/ o 



five, in running tap-water. It is then passed through up-graded 

 alcohols, beginning at 30 per cent., giving the material at least 

 three hours in the strengths 30, 50 and 70 per cent., and overnight 

 in 90 per cent. The pieces of tissue are dehydrated two or three 

 hours in two changes of absolute alcohol, and then transferred to a 

 mixture of half absolute alcohol and half xylol for a quarter of 

 an hour ; then pure xylol, and embedded in wax. Sections are cut 

 from 4 to 8 //, but I generally find 6 pt to be convenient. Leave 

 eight to ten hours in iron alum, twelve to twenty hours in hsema- 

 toxylin. This method gives a delicate and precise stain of the 

 mitochondria (and Golgi apparatus or nebenkern batonettes of male 

 germ-cells only), fat is black, while yolk is generally greenish brown. 

 Especially recommended for germ-cells, and histology of In vert e- 

 brata, but with vertebrate tissues, and especially mammalian 

 material (not embryos), it often gives atrocious results ; for such 

 material, Helly, Zenker, or Regaud's methods are indicated 

 (GATENBY, Quart. Journ. Micr. Sci., 1919). 



Note that Fleraing-witliout-acetic acid is not a suitable fixative for 

 after-staining in Altmann's acid fuchsm. For this the material must be 

 washed' in distilled water for a short time after fixation, and then 

 transferred to 3 per cent, bichromate of potash {or three days ; or the 

 more elaborate mordantage as for Champy-Kull may be used ( 681). 



680. ALTMANN'S Acid Fuchsin and Picric Acid (Die Elementaror- 

 ganismen, Leipzig, 1890). Fix twenty-four hours in mixture of 

 equal parts of 5 per cent, bichromate of potash and 2 per cent, osmic 

 acid. Embed in paraffin, stain sections on slide for one minute 

 over flame, with a solution of 20 grms. (sic) of acid fuchsin in 100 c.c. 

 of aniline oil- water ( 286). Cool, and wash out in a saturated 

 alcoholic solution of picric acid diluted with 2 volumes of water, 



