324 CYTOLOGICAL METHODS. 



clear and embed in paraffin. Pass sections on slide down to water ; 

 5 per cent, iron alum at 35 C. for twenty-four hours ; rinse in 

 aq. dest., not tap-water. Stain twenty-four hours in this solution : 

 1 grm. of pure crystals of hsematoxylin in 10 c.c. of absolute alcohol, 

 added to 10 c.c. of glycerine and 80 c.c. of aq. dest. Differentiate in 

 5 per cent, iron alum, watching process under microscope. The 

 main point is to avoid washing out the mordant too much when the 

 slides are being transferred from the iron alum to the hsematoxylin. 

 Permanent stain, very good for vertebrate tissues. See also COWDRY, 

 Amer. Journ. Anat., xix, p. 441. I find ordinary iron hsematoxylin 

 is quite good after Regaud fixation. 



686. BENSLEY-COWDRY Acid Fuchsin and Methyl Green Stain 



(CowcRY, Contrib. Carnegie Inst. Wash., viii, 1918). Fix as for 

 Regaud, either by immersion or injection ; formalin should be 

 neutralised in magnesium carbonate, and, if possible, the fixation 

 should be done in an ice-box, but this is not necessary. Pass sections 

 down to aq. dest. through toluol (or xylol), absolute alcohol, etc., 

 thirty seconds in each ; transfer to 1 per cent, potassium per- 

 manganate for thirty seconds, but time must be determined experi- 

 mentally ; then 5 per cent, oxalic acid for thirty seconds. (Note. 

 The permanganate and oxalic acid may generally be omitted.) 

 Then rinse in several changes of aq. dest. for about one minute 

 (incomplete washing prevents staining in acid fuchsin). Stain in 

 Altmann's fuchsin ( 680) as follows : dry around sections with 

 duster, add stain, warm over spirit lamp until fumes come off ; 

 cool for six minutes ; wipe around sections with duster, rinse off in 

 aq. dest., so that the only remaining stain is in the sections (or a 

 precipitate forms with the methyl green) ; pipette a little 1 per 

 cent, methyl green over the sections for about five seconds at first, 

 modify time as experimentally found convenient ; drain off excess, 

 plunge into 95 per cent, alcohol for a second or two. Rinse in 

 absolute alcohol, clear in toluol, mount in balsam. Difficulties are 

 that the methyl green may remove the fuchsin (due to incomplete 

 chrome mordanting during fixation), or the fuchsin may have over- 

 stained (due to too much mordanting). Sometimes, if the methyl 

 green is too weak, it is better to omit the 95 per cent, alcohol, 

 dehydrating in absolute. The difficulties of this modification of 

 Altmann's stain are easily overcome ; I have used it for a senior 

 histology class, and with success. Like the Champy-Kull method, 

 this stain is not so permanent as Iron Hsematoxylin. See also 

 BENSLEY, Amer. Journ. Anat., xii, p. 308 ; DUESBERG, ibid., xxi, 

 p. 469. 



