30 rilK STIUCTtUK OK CHHOMOl'HILE CELLS OF TlIF, Xl'-inoiS SVSTK.^L 



injection throufili I he hlood-vcssels ( Shiroko^'oroff) eliniinalrs many very ol).i('('t ion- 

 able artefacts due to faulty penetration. The use of permanganate and oxalic 

 acid (Bcnsley) facilitates the staining of the mitochondria with th(> anilin fuchsin 

 (Altmann), and the counterstaining with methyl green (Gah'ottii permits of the 

 demonstration of the Xissl substance in the same cell with the mitochondria. The 

 fact that \hv method gives good results in the case of other ti.'^sues where the mix- 

 tiu'es of Altmann, Flemming, and others are useless on account of their poor powers 

 of jjenetration, justifies the following detailed statement: 



Fixation: 



Chlorofoiin the aiiiinaL Inject wanned ().8.t ])er eent NaC'l solution into the aorta thi()ujj;li tlic 

 ventricle. If the brain alone i.s to ho studied clamp the descending aoria. If the entire nervous 

 .system is to he fixed, clanip the cd'liac, the renals, the sujierior and inferior mesenteries, the iliacs, 

 and the brachials. Continue the injection until the .salt solution is returned uncoloreil through the 

 juRulars. DuriuK this time lay bare the arch of the aorta and the carotids from connective tissue, 

 so that they mas- expand easily and carry more fluid to the brain, (iravity jiressure of not more tiian 

 (i feet may be employeil. Cut the infeiior vena cava and tlie jufiulars so that the salt solution may 

 run through easily. 



Follow the salt solution with the formalin and bichromate mixture: 3 i)er cent pota.ssium 

 bichromate, 4 parts; neutral foi-malin, 1 part. The potassium bichromate acts l)cst when freshly 

 prei)ared. Neutral formalin is made fi'om the commercial variety by the addition of magnesimn 

 carbonate, a dejMJsit of which sliouUI always remain at the bottom of the formalin bottle. It is 

 imjiortant that the jjressure should be at the maximum when the mixture is first injected, so that 

 the blood-vessels may be fixed in a state of dilation. If the pressure is low when the fixative comes in 

 contact with the vessel-walls they will be fixed in a condition of collapse. It will then l)e difficult, or 

 even iinpo.ssible, to obtain a complete injection. The injection sliould lie continued foi' .-iboul an 

 hour. 



The brain is then dissected out and immersed in the fluid. In the case of the mouse's brain 

 it is sufficient to divide it longitudinally into halves. The fixative must l)e changed every day for 

 4 or 5 days, otherwise il undeigoes a cliange evidenced by a darkening in coloi-. This change is 

 accelerated by light and by h<>at , so that the tissue should be kept in the dark and in a cool place. 

 Fixation may al.so be effected by simple immeision of the tissue in the fixative, instead of by injection, 

 but this procedure is not lecomnunded. 



After this jirolonged fixation th(> tissue is mordanted in a fresh 3 per cent solution of pot;issium 

 bichromate, in which it remains for cS or fl days, changing every second day. 



Wash in running water for 24 hours. The object of this careful washing is to lemovi' most of 

 the formalin and bichromate, for otherwise the tissue will be extremely brittle and hard to cut. 



Dehydration and embeddimj: 



50 per cent alcohol 12 hours; 70 per cent and 9,5 per cent alcohol 24 hours each; absolute alcohol 

 (5 to 12 hours: h;df absolute and xylol (i hours: xylol .3 hours; paraffin ()()° C. ,3 hours; cut in An s(>rial 

 .sections. 



S^lainimj: 



(1) Pa.ss the .sections, mountecl on sli(l(>s. down through toluol, absolute. '.)."). 70. and .")(! per I'cnl 

 idcohol to distille(l watei', 



(2) 1 per cent acpieous >oluliiin of potassium permanganate M) seconds: Imt this tinii' niu>t be 

 iletermined exijerimeiitally. 



(3) 5 per cent aqueous solution of oxalic acid also about 30 seconds, 



(4) Rinse in several changes of distilled water about a minute. lnroiii|ilele washing |irevents 

 the staining with fuchsin. 



(5) Stain in .Vltmann's anilin fuchsin. which is to be made up as follows: .Make a saturated 

 solution of anilin (jil in distilled water by shakingthetwo together (anilin oil goes into solution in water 

 in aliout 1 per cent). Filter and add 20 grams of acid fuch.sin to 100 c.c. of the filtrate. The stain 

 shoulil he ready to use in about 24 hours. It goes bad in about a month. To stain, dry t he slide wil h 

 a towel, excej)! the small area to which the sections are attached. Cover the sections on the slide with 

 a small amount of the st.ain and heat over a sjiirit lamj) until fumes, smelling strongly of anilin oil. 



