PHLOXINE-METHYLENE BLUE 



192 



PHOSPHOLIPID CONTENT 



(hypo) 5 min. Wash thoroughly in 

 water. 2.5% aq. phloxine in paraffin 

 over 1 hr. or more. Cool stain, drain 

 and rinse in water. Take 5 cc. 1% methy- 

 lene blue on 1% borax, 5 cc. 1% aq. 

 azure II, add 90 cc. aq. dest., filter onto 

 the sections. Pour on and off several 

 times. After required time differentiate 

 in 100 cc. 95% alcohol plus 2-5 cc. 10% 

 colophony (rosin) in absolute alcohol. 

 Control differentiation under micro- 

 scope. Dehydrate in several changes 

 abs. ale. Clear in xylol and mount in 

 balsam. Nuclei and bacteria, blue; 

 collagen, etc. bright rose. The method 

 yields beautiful preparations of intra- 

 nuclear inclusions in yellow fever and 

 is extensively used for many purposes. 

 Phosphatases. The following accounts 

 have been contributed by Dr. G. Go- 

 mori (May 7, 1946) : 



For Acid, phosphatase based on his paper 

 in Arch. Path., 1941, 32, 189: 



1. Fix thin slices of tissues in ice cold 

 acetone for 24 horns. 



2. Change acetone at room tempera- 

 ture twice for the next 24 hours. 



3. Two changes of benzene, 45 min. 

 each. 



4. Embed in paraffin (not above 56°C. 

 and preferably below), 2 changes, 30 to 

 45 min. each. 



5. Cut sections. Float them on luke- 

 warm (30°C.) water. 



6. Carry sections through xylene and 

 2 alcohols to dist. water. 



7. Incubate in the following solutions 

 for U to 24 hours at 37°C.: 



Molar acetate buffer pH 5* 3 jjarts 



6% lead nitrate 1 part 



Dist. water add slowly, under stir- 

 ring 6 parte 



2% Na glycerophosphate t 3 parts 



* 100 cc. of 13.6% CHsCOONa-SHaO plus 50 cc. 6% 



acetic acid, 

 t Commercial grade (mixture of alpha and beta salts). 



Shake well, let stand for a few hours. 

 Keep in the ice box. Before use, filter 

 a small amount and dilute with 2 to 3 

 parts of dist. water. 



8. Rinse sections thoroughly first in 

 dist. water and afterwards in 2 to 3% 

 acetic acid, followed again by dist. 

 water. 



9. Immerse sections in a solution of 

 yellow ammonium sulfide (10 drops to a 

 Coplin jar) for 1 minute. 



10. Wash. Counterstain as desired. 

 For Alkaline phosphatase: 



1. Fix thin slices of tissues in 80% al- 

 cohol (or absolute acetone). Dehy- 

 drate in 95% and absolute alcohol (or 2 

 changes of absolute acetone), embed 

 through benzene or xylene in paraffin. 

 Cut sections around 6 micra thick. 



2. Run slide through xylene and 2 al- 

 cohols to distilled water. Incubate for 

 1 to 2h at 37 °C. in the following mixture : 



2% sodium gl:,'cerophosphate . 25 cc. 

 2% sodium barbital 25 cc. 



Distilled water 50 cc. 



2% calcium chloride 5 cc. 



2% magnesium sulfate 2 cc. 



Chloroform a few drops 



This solution will keep in the ice box 

 for months. 



3. Rinse slide thoroughly in repeated 

 changes of distilled water. 



4. Immerse slide for 3 minutes in a 

 1 to 2% solution of some cobalt salt 

 (chloride, acetate, sulfate). 



5. Wash thoroughly under the tap. 



6. Immerse slide for 2 minutes in a 

 dilute solution of .yellow ammonium sul- 

 fide (5 to 6 drops to a Coplin jarful of 

 distilled water). Wash under the tap. 



7. Counterstain as desired; dehy- 

 drate, clear and mount. 



Attention is called to the earlier 

 demonstration of phosphatase in bone 

 by Robison (R., Biochem. J., 1923, 17, 

 286-293) and to recent discussion by 

 Blaschko and Jacobson (Bourne, pp. 

 217-221). The distribution of phos- 

 phatase in some normal tissues is indi- 

 cated in colors by Kabat, E. A. and 

 Furth,J.,Am.J. Path., 1941, 17, 303-318. 

 For phosphatase in elementary bodies 

 of vaccinia virus, see Macfarlane, 

 M. G., and Salaman, M. H., Brit. J. 

 Exp. Path., 1938, 19, 184; Hoagland, 

 C. L. et al., J. Exp. Med., 1942, 76, 

 163-173. See Kidney. 



For "localization of different phos- 

 phatases in duodenal epithelium", see 

 Deane, H. W. and Dempsey, E. W., 

 Anat. Rec, 1946, 94, 12-13; "effects of 

 KCN on alkaline phosphatase activity 

 in the kidney and intestine", see Em- 

 mel, V. M., Anat. Rec, 1946, 94, 15; 

 and for "intracellular distribution of 

 alkaline phosphatase activity following 

 various methods of histologic fixation", 

 see Emmel, V. M., Anat. Rec, 1946, 

 94, 92. 



Phosphate Solutions. A method for the 

 direct observation of the effect of 

 buffered phospliate 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). 



