PHOSPHATE SOLUTIONS 



268 



PHOSPHOTUNGSTIC ACID 

 HEMATOXYLIN 



208). See method of Lowry, O. H. and 

 Lopez, J. Biol. Chem., 1946, 162, 421- 

 428 for determination of inorganic 

 phosphate in presence of labile phos- 

 phate esters. See Phosphorus. 



Phosphate Solutions. A method for the 

 direct observation of the effect of 

 buffered phosphate solutions on a thin 

 layer of living, vascular tissue in moat 

 chambers introduced into the rabbit's 

 ear is described by Abell, R. G., Anat. 

 Rec, 1935-36, 64, 51-73. 



Phosphine (CI, 793)— leather yellow, xan- 

 thin — a basic xanthene dye used as a 

 microchemical test for nucleoproteins 

 by Schumacher, J., Zentralbl. Bakt., 

 Abt. I. Orig., 1922, 88, 362-366. Phos- 

 phine 3 R is fluorchrome for lipids. 



Phospholipid Content of white blood cells 

 (Boyd, E. M., J. Lab. & Clin. Med., 

 1935-36, 21, 957-962). 



Phosphomolybdic Acid Hematoxylin (Mal- 

 lory's, see McClung, p. 406). Fix in 

 Zenker's fluid, imbed in parafhn and 

 remove mercury with iodine. Rinse in 

 water. Phosphomolybdic acid hema- 

 toxylin at room temperature 12-24 hrs. 

 or at about 54°C. 2-3 hrs. (That is 

 hematoxylin 1 gm., phosphomolybdic 

 acid crystals 2 gm., aq. dest. 100 cc. 

 Requires several weeks to ripen or ripen- 

 ing may be immediate after addition of 

 5 cc. 1% aq. potassium permanganate.) 

 Wash in water. Decolorize in 95% ale. ; 

 dehydrate in abs. Clear in xylol and 

 mount in balsam. Collagenic fibers 

 deep blue. To counterstain first color 

 5-10 min. in 0.5% aq. acid fuchsin, drain 

 and stain directly in the hematoxylin. 



Phosphorescence Microscope, Science 

 (News), 1943, 98, 8 (No. 2547). 



Phosphorus. The histochemical detection 

 of phosphorus is a matter of great im- 

 portance but the techniques are open to 

 much criticism. Lison (pp. 113-120) 

 has reviewed the whole question and 

 advises two techniques as vigorously 

 specific for phosphorus in the ionic 

 form: 



1. Angeli, A., (Riv. di Biol., 1933, 10, 

 702) using plant material treats sections 

 for 20 min. with: ammonium molyb- 

 date, 3 gm.; aq. dest., 20 cc; 30% aq. 

 hydrochloric acid, 20 cc; reduces in 

 N/50 stannous chloride, rinses quickly 

 in aq. dest., washes longer in 2.5% aq. 

 ammonia which results in elements con- 

 taining phosphorus being colored blue 

 green. 



2. Serra, J. A. and Queiros Lopes, A., 

 Portugaliae Acta Biol., 1945, 1, 111- 

 122. Reagents: (A) Fixative. Add few 

 drops glacial acetic acid to mixture 2 

 parts 96% ale and 1 part formalin. 

 (B) Molybdate. 0.5 gra. ammonium 

 molybdate dissolved in 20 cc. aq. 



dest. -f 10 cc. cone. (30%) hydrochloric 

 acid diluted to 50 cc. with aq. dest. 

 (C) Benzidine. 25 mg. dissolved in 5 

 cc. glacial acetic acid diluted to 50 cc. 

 with aq. dest. Fix tissue in "A" and 

 wash well in water. Treat frozen sec- 

 tions, or small pieces, with "B" at 

 10-12° for 2-3 weeks and then at 20-25° 

 for 2-3 days. Cover with drop of "C" 

 for 3 min. Add 2 drops sat. aq. sodium 

 acetate. Mount in glycerol from a 

 supply containing crystals of sodium 

 acetate. Phosphate is revealed by 

 intense blue color. When these authors 

 digest tissue with nuclease to liberate 

 phosphate from nucleic acid this visual- 

 ization of phosphate indicates localiza- 

 tion of the nucleic acid (partially quoted 

 from Glick, p. 35). 



By the titrimetric method of Lind- 

 ner, R. and Kirk, P. L., Microchemie, 

 1937, 22, 300-305 phosphorus can be 

 detected quantitatively in the range 

 0.5-10.0 fjL gm. The whole subject has 

 been reviewed by Glick, D., J. Chem. 

 Education, 1935, 12, 253-259. 

 Phosphotungstic Acid Hematoxylin. (Mal- 

 lory's, see McClung, p. 403) Fix in 

 Zenker's fluid and remove mercury from 

 sections with iodine or 0.5% sodium 

 hyposulphite. Rinse in water. 0.25% 

 aq. potassium permanganate, 5-10 min. 

 Wash in water. 5% aq. oxalic acid, 

 10-20 min. Wash carefully in several 

 changes of water. Phosphotungstic acid 

 hematoxylin, 12-24 hrs. (To make this 

 dissolve 0.1 gm. hematoxylin by heat in 

 50 cc. aq. dest., when cool add 2.0 gm. 

 phosphotungstic acid dissolved in 50 cc. 

 aq. dest. Requires a few weeks to 

 ripen. Ripening can be done at once by 

 addition of 10 cc. 0.25% aq. potassium 

 permanganate). 95% ale, 30 sec; 

 dehydrate quickly in abs. Clear in 

 xylol and mount in balsam. Fibroglia, 

 myoglia, neuroglia and fibrin, deep 

 blue; ground substance, cartilage and 

 bone, yellowish to brownish red; 

 coarse elastic fibers, purple. 



Mullen, J. P. and McCarker, J. C, 

 Am. J. Path., 1941, 17, 289-291 suggest 

 the following procedure for nervous tis- 

 sues fixed in formalin. Tissues stored 

 in 4% aq. formalin for several years give 

 good results. After fixation in 4%, cut 

 blocks 5 mm. or less in thickness. Wash 

 for 6-12 hrs. in running water. Dehy- 

 drate to include 95% alcohol as usual. 

 Complete dehydration in 2 changes n 

 butyl alcohol, 4 hrs. each (but absolute 

 alcohol xylol is satisfactory). Imbed in 

 paraffin directly from n Butyl Alcohol 

 (which see). 



Treat sections for 2 hrs. or longer in 

 following mordant: Dissolve 5 gms. 

 chromium chloride (green crystals ob- 



