PEROXYDASE 



265 



PHAGOCYTOSIS 



tap water and dry. Peroxidase granules 

 are colored blue in granular leucocytes 

 and the nuclei orange red. 



4. Nitroprusside-benzidine (Goodpas- 

 ture, E. W., J. Lab. & Clin. Med., 

 1919, 4, 442-444). To make the stain 

 dissolve 0.05 gm. sodium nitroprusside 

 in 2 cc. aq. dest. ; add 100 cc. 95% alco- 

 hol ; 0.05 cc. benzidine C.P.; 0.05 gm. 

 basic fuchsin and 0.5 cc. hydrogen 

 peroxide. Cover well dried blood smear 

 with known amount of stain, 1 min.;add 

 equal volume aq. dest. plus hydrogen 

 peroxide, 3-4 min. ; rinse thoroughly in 

 water and blot dry. Shows many blue 

 granules in granular leucocytes and few 

 in monocytes. Nuclei are colored red. 

 To increase intensity of stain dilute 

 with a little less aq. dest. and stain 

 longer. Method can be used for frozen 

 sections of material fixed in formalin 

 and preserved in 80% ale. A modifica- 

 tion of this stain has been proposed by 

 Beacom (J. Lab. & Clin. Med., 1925-26, 

 11, 1092-1093) with hydrogen peroxide 

 omitted and basic fuchsin doubled. 



5. Benzidine-Giemsa (Armitage, F. 

 L., J. Path., 1939, 49, 579-580). Fix 

 smears in 96% alcohol containing 10% 

 formol freshly made up. Flood with 

 benzidine mixture (0.75 gm. benzidine 

 in 500 cc. 40% ethyl alcohol. Filter. 

 Add 7 cc. 3% HjOi, mix by shaking im- 

 mediately before using) 2 min. for fresh 

 films, longer for older ones. Wash in 

 40% alcohol until definite yellow gran- 

 ules are seen in granular leucocytes. 

 Absolute alcohol and dry in incubator. 

 Counterstain with dilute Giemsa, wash 

 in water, blot and dry. 



6. Benzidine for paraffin sections 

 (McJunkin, F. A., Anat. Rec, 1922-23, 

 24, 67-76). After fixation of small 

 pieces in 10% formalin imbed quickly 

 in paraffin; 70% alcohol, 1 hr. ; acetone, 

 30 min.; benzol, 20 min.; paraffin, 20 

 min. Mount thin sections in usual 

 fashion. Deparaffinize in benzol 20 

 sec, acetone, 10 sec. Water, few sec- 

 onds. Drain off water, apply mixture 

 (80% alcohol, 25 cc. ; benzidine, 0.1 gm. ; 

 hydrogen peroxide, 2 drops) diluted with 

 1 or 2 parts aq. dest., 5 min. Water, 5 

 min.; hematoxylin, 2 min.; water, 1 

 min., 0.1% aq. eosin, 20 sec; 95% 

 alcohol, 30 sec. ; abs. alcohol, 5 sec. Clear 

 in xylol and mount in balsam. 



Note : In above methods a blue 

 counterstain tends to obscure the blue 

 peroxidase reaction. 



7. DCPIP-2, 6-(iichlor-phenol-indo- 

 phenol (Jacoby, F., J. Physiol., 1944, 

 103, Proc. Physiol. Soc. July 29). Fix 

 air dried blood smear in 9 parts abs. ale. 

 and 1 part formol for 2-3 min. Wash in 

 water. Treat smear for 3-5 min. with 



0.5% aq. 2.6-dichlor-phenol-indophenol 

 to every 5 cc. of which 4 drops IIjOi 

 is added prior to use. Wash in water, 

 blot dry and examine. "Peroxidase- 

 positive" granules, deep purple-violet. 

 No precipitation of crystals and gran- 

 ules on smear. Author suggests 0.5% 

 aq. neutral red as a counterstain to be 

 applied after treatment with DCPIP. 

 If smear is to be mounted use neutral 

 balsam. Solution of DCPIP can be 

 stored in ice box for few months. 



Peroxydase, see Peroxidase. 



P6t6rfi, see Double Imbedding, and Osmic 

 Acid Method for nerve fibers. 



Petrunkevitch's Fixatives: Cupric-phenol. 

 Stock solution A = aq. dest., 100 cc; 

 nitric acid (cp. sp. gr. 1.41-1.42), 12 

 cc; Cu(N03)2-3 H^O, 8 gm. Stock 

 solution B = 80% alcohol, 100 cc; 

 phenol crystals, cp. 4 gm.; ether 6 cc. 

 iSmploy 1 part A with 3 parts B. Fix 

 12-24 hrs. Wash in 70% alcohol. 

 Cupric-paranitrophenol. 60% alcohol, 

 100 cc; nitric acid (same), 3 cc; ether 

 5 cc. ; cupric nitrate (same), 2 gm.; 

 paranitrophenol, cp. crystals, 5 gm. 

 Time unspecified. Wash in 70% alco- 

 hol. Said not to harden tissues like 

 ordinary fixatives. May be followed 

 by all common stains. (Petrunkevitch, 

 A., Science, 1933, 77, 117-118). 



Petrunkevitch's Fluid is sat. mercuric 

 chloride in aq. dest., 300 cc, abs. ale, 

 200 cc; acetic acid, 90 cc; and nitric 

 acid, 10 cc. 



pH, see Hydrogen Ion Indicators. 



Phagocytosis. There are numerous methods 

 for the demonstration of this phenome- 

 non from which to choose. 



1. In Vaginal Smears (which see), 

 made after intercourse, neutrophilic 

 leucocytes can be observed in the act of 

 engulfing individual spermatozoa. C. 

 R. Stockard, in Cowdry's Special Cy- 

 tology, 1932, 3, 1611-1629, has described 

 this remarkable process as seen in the 

 living state. "A leucocyte comes in 

 contact with a spermatozoon which with 

 its tail is longer than the leucocyte. 

 The leucocyte by stretching and con- 

 tracting finally takes into itself the 

 entire spermatozoon, the tail being 

 wound in a circular fashion within the 

 cell body." 



2. In temporary mounts of bacteria 

 and Leucocytes (which see) pliagocyto- 

 sis can be followed in detail. Differ- 

 ences in the behavior of neutrophiles 

 from seriously ill and normal persons 

 have been described. 



3. Under Vital Staining will be found 

 many techniques which permit the 

 observation of the phagocytosis of 

 inanimate particulate materials by 

 macrophages. A graphic demonstration 



