METHODS OF SPECIAL STAINING ETC. 665 



20 per cent, formalin and 3 per cent, monochloracetic acid 

 (pU 1-6). 



20 per cent, formalin and 3 per cent, cyanacetic acid (pH 1-3). 



20 per cent, formalin and 2 per cent, trichloracetic acid (jjH 0-6). 



However, a mixture of 20 per cent, formalin and 5 per cent, 

 acetic acid {pH 1-9) does not fix the mitochondria. After fixation, 

 wash rapidly in water, treat six hours with iron alum, stain twelve 

 hovirs with htematoxylin and differentiate with iron alum. 



Kassmaxx [Planta, i, 1926, p. 624) describes, for the purpose of 

 studying mitochondria and plastids, a technique for permitting 

 given cells in living shoots of Cahomha to be kept under continuous 

 observation for months. 



GuiLLiERMOXD (C. R. Acad. ScL, clxx, 1920, p. 1329) recom- 

 mends neutral red and cresyl blue for vitally staining the mito- 

 chondria. The latter stain differentiates the metachromatic 

 bodies from the mitochondria. He also (C. R. Soc. Biol., Ixxxix, 

 1923, p. 527) uses dahlia violet and Janus green. The plastids 

 colour more slowly and less easily than the mitochondria. Aquatic 

 fungi, such as Soprolegnia, are more adaptable to intra-vitam 

 staining than are tissues from higher plants, the cell walls of which 

 are less permeable to the dyes. 



1331. Double Staining of Mitochondria and Starch Grains. 

 Guilliermond recommends iron-alum haematoxylin and iodine, 



osniic acid and iodine, or acid fuchsin, toluidine blue and aurantia 

 (Kull's stain, § 695). 



MiLOViDOV (Arch. Anat. Microsc, xxiv, 1928, p. 9) describes 

 adaptations of Volkonsky's methods. 



(1) Fix in Regaud or cliromo-formol-bichromate. Stain in 

 acid rubin for five minutes at 60 to 80° C, differentiate under the 

 microscope in 5 per cent, alcoholic aurantia and wash in water. 

 Mordant twenty minutes in 2 per cent, aqueous tannin solutions 

 and wash in water. Stain five to ten minutes in 1 per cent, 

 aqueous toluidine blue or methyl green or gentian violet, differen- 

 tiate in alcohol, dehydrate, clear and mount. Mitochondria red, 

 starch grains blue, green or violet. 



(2) Fix in Meves, Regaud or chromo-formol-bichromate, 

 mordant in 3 per cent, iron alum and stain in haematoxylin for 

 twenty-four hours ; differentiate in iron alum and wash well. 

 Mordant in 2 per cent, aqueous tannin for thirty to sixty minutes 

 and wash in distilled water. Then mordant in 1-5 per cent, tartar 

 emetic and wash in water. Stain in 1 per cent, aqueous gentian 

 violet thirty to sixty minutes, differentiate in alcohol, dehydrate, 

 clear and mount. 



1332. Double Staining of Bacteria and Mitochondria in Plant 

 Tissues. The methods are intended for the root nodules of 

 Leguminosic, etc., the leaf tips of Dioscorea and for other tissues 

 containing symbiotic or parasitic bacteria. 



