62 THE MITOCHONDRIAL CONSTITUENTS OF PROTOPLASM. 



where the Altmann method and its modifications are useless. Unfortunately it 

 rarely gives good results with embryonic tissues; for these the older osmic acid- 

 containing fixatives arc best adapted. It is often possible to make use of material 

 fixed in the usual way with formalin by starting out with step (2). Moreover, 

 the preparations can be counterstained in a variety of ways (Cowdry, 1916a, p. 441). 



(4) Dubreuil's (1913, p. 74) iron-hematoxylin method for blood-cells: 



Fixation: 



Take up the fluid to be examined in a pipette containinp several times its volume of 0.5 to 1 per cent solution 

 of osmic acid. Transfer to a centrifuge tube. A good fixation is obtained in about an hour. Then add 

 distilled water, centrifuge, and decant. The blood-cells remaining are shaken up with absolute alcohol and 

 then passed into a weak solution of celloidin. A di'op is allowed to spieafl on a slide, which, before complete 

 desiccation, is plunged into 80 per cent alcohol. 



Staining: 



The mitochondria in the cells are then stained with iron hematoxylin (p. 61). 



(5) Bensley's copper chrome hematoxylin method: 



Fixation: 



(1) Flx in either .\ltmann's osmic bichromate mixture (p. .59) or in Bensley's acetic-osmic-bichromate fluid 



(p. t)0) for 12 to 24 hours. 



(2) Wash, dehydrate, clear, embed, and section (p. 59). 



Staining: 



(1) Pass down to water (p. 59). 



(2) Sat. aq. copper acetate, 5 minutes. 



(3) Wash in several changes distilled water, 1 minute. 



(4) 0.5 per cent hematoxylin, 1 minute. (If the copper acetate has not been suflficiently washed out, a black 



precipitate forms in the hematoxylin. The hematoxylin should be well ripened. It may be obtained l)y 

 dilution down from a 10 per cent alcoholic stock solution.) 



(5) Rinse in aq. dest. 



(6) 5 per cent neutral potassium chromate, 1 minute. (The sections should turn a dark blue-black color. 



If they are only a light-blue shade, rinse in aq. dest., place again in the copper acetate, and carry through 

 as just described several times until no increase in color result=.) 



(7) Wash in aq. dest. and return for a few seconds to the copper acetate in order to convert all the dye into the 



copper lake. 



(8) Wash in aq. dest. 



(9) Differentiate under the microscope in Weigert's borax-ferricyanide mi.xture diluted with 2 volumes of 



aq. dest. 



(10) Wash 6 to 8 hours in tap water. 



(11) Dehydrate, clear, and mount in balsam. 



(6) Bensley's (1911, p. 308) neutral safranin method: 



Fixatio7i: 



(1) Fix in chrome subhmate (2.5 per cent potassium bichromate 100 c.c, mercuric chloride 5 gm.) for 24 hours. 



(2) Wash, dehydrate, clear, embed, and section as indicated on page 59. 



Staining: 



(1) Preparation of stain: Add slowly sat. aa. sol. of the color acid, acid violet, to sat. .aq. sol. of the color-base, 



safranin O, contained in a flask until a precipitate no longer forms. The point of neutralization may be 

 roughly determined by dropping a little of the mixture on filter-paper from time to time until the outside 

 red ring of safranin disappears and the whole blot takes on a neutral color. Filter. The filtrate should 

 be as nearly as possible colorless. Dry the precipitate on filter-paper for 12 hoiu-s, collect it, and make 

 a saturated solution of it in absolute alcohol. 



(2) Pass sections down throug' two chanees of toluol and absolute alcohol in order to remove all traces of paraffin 



or toluol, which might interfere with the staining. Then through 95 per cent, 70 per cent, and 50 per 

 cent to aq. dest. (Chrome and osmium fixed material must be bleached in permanganate and oxaUc acid 

 (vide p. 60), and sublimate-fixed tissues must be treated with Lugol's iodine solution for about 10 seconds 

 and washed in aq. dest.) 



(3) Dilute the alcoholic stock solution of the dye with an equal \olume of aq. dest. and stain for from 5 minutes 



to 2 hours. 



(4) Blot quickly with several layers of filter paper. 



(5) Plunge into pure acetone and pass immediately to toluol without waiting to drain. 



(6) Examine under the oil immersion and if necessary differentiate in oil of cloves. If this is not sufficient, the 



slide, after rinsing in absolute alcohol, may be instantaneously flooded with 95 per cent alcohol, and then 

 passed back through absolute alcohol to toluol. 



(7) Wash in two changes of toluol and mount in balsam. 



