PROTHROMBIN 



288 



PROTOZOA. MEDIA 



Prothrombin, rapid micro test (Abramson, 

 D. J. and Weinstein, J. J., AJn. J. Clin. 

 Path. Technical Suppl., 1942, 6, 1-7) : 



1. Make M/40 calcium chloride by 

 dissolving 1.11 gms. anhydrous calcium 

 chloride C.P. in 400 cc. aq. dest. 



2. Make thromboplastin suspension 

 from brain freshly killed rabbit as de- 

 scribed by Quick, A. J. Am. J. Clin. 

 Path., 1940, 10, 222. Dehydrate macer- 

 ated brain in acetone, dry completely, 

 mix with normal saline (0.3 gm. to 5 

 cc.) and incubate at 50°C. 15 min. The 

 supernatant turbid fluid is thromboplas- 

 tin. It must be kept in ice box when 

 not in use. 



3. Measure separately in micro- 

 hemopipettes 10 cc. of calcium chloride 

 sol., of thromboplastin and of blood. 



4. After adding blood, mix thor- 

 oughly with fine glass rod, tilt gently 

 from side to side until gelation begins, 

 then time end point by passing rod 

 through mass. 



Prothrombin time (Sherber, D. A., 

 J. Lab. & Clin. Med., 1940, 26, 1058- 

 1061; and Isenberg, H. D., J. Lab. & 

 Clin. Med., 1951, 37, 807-809). 



Protoporphyrin in Harderian glands, see 

 Porphyrins. 



Protozoa, staining in bulk. (Stone, W. S., 

 J. Lab. & Clin. Med., 1935-36, 21, 839- 

 842) : Suggested for mucous surface 

 protozoa of man and used at Army 

 Medical School. Thoroughly emulsify 

 20 cc. feces in 200 cc. 37°C. physiological 

 saline solution. Allow to stand for 5 

 min. and pour supernatant fluid into 

 two 50 cc. centrifuge tubes. Centri- 

 fuge at 1,850 r.p.m. 5 min. Decant 

 supernatant fluids. Examine residue 

 from one, fresh, and to other add 25 cc. 

 Schaudinn's Fixative. Mix and leave 

 24 hrs. Protozoa in cultures and other 

 fluids are to be concentrated by centri- 

 fugation and fixed in the same way. 

 Between each of following steps centri- 

 fuge organisms and discard supernatant 

 fluid before adding the next. Wash 

 twice in aq. dest. Wash with 70% al- 

 cohol plus sufficient Gram's iodine to 

 make it light brown color, 10 min. 

 Wash 70% alcohol 10 min. Stain 

 Harris' Hematoxylin 1-24 hrs. Wash 

 tap water. Destain by adding 20 cc. 

 acid alcohol (1% HCl in 70%) controlled 

 by microscope. When desired defini- 

 tion is reached add sufficient ammonia 

 water (6 drops NH4OH to 60 cc. aq. 

 dest.) to neutralize acid and give 

 bright blue solution. Wash in tap 

 water. Dehydrate 10 mins. in each of 

 5 alcohols: 70, 95, 95, abs., and abs. 

 Clear in xylol. Mount in balsam. See 

 author's figures. 



Perhaps the best method for concen- 



trating and sectioning protozoa is that 

 of Lucas, M. S., Science, 1929, 70, 482- 

 483. Use a round bottom vial. Let 

 protozoa settle to bottom, pipette off 

 fluid to within 4 mm. of level of top of 

 protozoan mass, then add dilute alco- 

 hol. Next change, pipette off, and add 

 stronger alcohol. Alcohol, xylol, pure 

 xylol, melted paraffin (the vial being 

 held under an electric bulb, etc.) sev- 

 eral changes of each. Finally lower 

 protozoa with as little paraffin as 

 possible into a specially prepared paper 

 tray and harden. 



Levine W. D., Stain Techn., 1939, 

 14, 29-30 suggests following method to 

 make Methylene Blue stains perma- 

 nent : Wash methylene blue stained 

 smears of protozoa repeatedly in aq. 

 dest. 15 min. to 1 hr. Place in tertiary 

 butyl alcohol 1-2 min. then in 3 or more 

 changes 15 min. each. Pass through 

 xylol to balsam or mount directly in 

 balsam. Other dyes like toluidin blue 

 0, nile blue sulfate, eosin Y, ponceau 2R 

 can likewise be retained. 



The protargol method of Bodian has 

 been adjusted to protozoa by Cole, R. M. 

 and Day, M. F., J. Parasitology, 1940, 

 26 Suppl. 29. See also Parasites, 

 Endamoeba Leishmania, Leucocyto- 

 zoa, Malaria, Intestinal Protozoa. Wen- 

 yon, C. M., Protozoology. New York: 

 William Wood, 1926, 1563 pp. is a con- 

 venient book of reference. It gives a 

 fine list of blood protozoa. No investi- 

 gator can afford to ignore the discussion 

 by Wenrich, D. H., J. Parasitol., 1941, 

 27, 1-28 of alterations in the form of 

 protozoa resulting from variations in 

 microtechnique. 



Protozoa can be beautifully demon- 

 strated by fluorochromes showing in 

 ultraviolet light various fluorescent 

 colors (Metcalf, R. L. and Patton, R. 

 L., Stain Techn., 1944, 19, 11-27). 



Obviously the investigation of proto- 

 zoa extends far beyond their identifica- 

 tion in preparations made by various 

 methods. Those dealing with patho- 

 genic protozoa will greatly extend their 

 horizon by consideration of the form 

 and function of these organisms and the 

 ingenious techniques of investigation 

 ably presented in a volume entitled 

 Protozoa in Biological Research edited 

 by Calkins, G. N. and Summers, F. M., 

 New York: Columbia University Press, 

 1941, 1148 pp. See Intestinal Protozoa, 

 Protein Silver. 

 Protozoa. Media. The following are rec- 

 ommended for intestinal protozoa b}' Q. 

 M. Geiman (Simmons and Gentzkow, 

 617-619): 



1. Modification of Cleveland's and 

 Sanders' (for E. histolytica). (1) Dis- 



