PROTOZOA 



203 



PULP OF TEETH 



by Wenrich, D. H., J. Parasitol., 1911, 

 27, 1-28 of alterations in the form of 

 protozoa resulting from variations in 

 microtechnique. 



Protozoa can be beautifully demon- 

 strated by fiuorochromes 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. 

 Protozoa. Media. The following are rec- 

 ommended for intestinal protozoa by Q. 

 M. Geiman (Simmons and Gentzkow, 

 617-619) : 



1. Modification of Cleveland's and 

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

 solve 33 gm. Bacto-Entamoeba medium 

 (Difco) in 1000 cc. aq. dest. Pour in 

 test tubes in amounts sufficient to make 

 medium length slants with no butts. 

 Autoclave, slant, harden at room tem- 

 perature several days. (2) Place few 

 gm. Bacto-Rice-Starch powder (Difco) 

 in 18 X 150 mm. culture tube and steril- 

 ize with tube horizontal in hot air oven 

 160-1S0°C. 1 hr. Repeat twice at daily 

 intervals being careful to avoid chemi- 

 cal changes in the starch occasioned 

 by higher temperatures. (3) Dissolve 

 11.23 gm. Na.HPOi I2H2O + 0.269 gm. 

 KH2PO4 + 8.0 gm. NaCl in aq. dest. 

 to make 1000 cc, autoclave 15 lbs. 20 

 min. and cool. Add 10 parts above 

 solution to 1 part sterile horse serum. 

 Cover f of each slant with this mixture, 

 add 2-3 loopfuls of the sterile starch, 

 incubate 37°C. 24 hrs. to prove sterility. 

 Final pH should be 7-7.2. Store in 

 refrigerator till used. 



2. Boeck and Drbohlav's. Wash 6 

 eggs in 70% alcohol and emulsify con- 

 tents in 75 cc. sterile Locke or Ringer. 

 Distribute in 4 cc. lots in 15 X 150 mm. 

 culture tubes, slant in inspissator and 

 heat 70°C. till mixture solidifies, then 

 autoclave 15 lbs., 20 min. Slant tubes 

 in autoclave, close doors and ports, turn 

 in steam increasing quickly to 15 lbs., 

 for 10 min. Through lower port run in 

 live steam in place of steam-air mixture 

 maintaining constant 15 lbs. pressure. 

 After replacement by steam close lower 

 port and hold 15 lbs. another 15 min. 

 Cut off steam and let cool slowly. 

 Cover each slant with 4 cc. 10:1 Ringer- 



horse serum mixture -f 2 or 3 loopful 

 sterile rice starch. Incubate 37°C., 

 24 hrs. to prove sterility. 



3. Nutrient agar serum-saline. 

 Cover long slants of nutrient agar 

 (Difco. 1.5%) in standard test tubes 

 I to \ with 20:1 sterile Ringer-horse 

 serum mixture. Smaller quantity for 

 intestinal flagellates, larger quantity for 

 TricJiomonas vaginalis. 



4. Trussell and Plass (for Tricho- 

 monas vaginalis). Overlay slants of 

 liver infusion agar (Difco) with a se- 

 lected mixture as for nutrient agar 

 medium. Adjustment of agar and solu- 

 tion by 1 A'' HCl and 0.25% aq. sodium 

 phosphate is suggested, likewise addi- 

 tion of 0.2% aq. dextrose. Incubate 

 37°C., 24 hrs. to prove sterility; store in 

 refrigerator. 



The technique of obtaining cultures 

 of protozoa free from bacteria has been 

 described in a comprehensive fashion 

 by G. W. Kidder in Calkins, G. N. and 

 Summers, F. M., Protozoa in Biological 

 Research. New York: Columbia Uni- 

 versity Press, 1941, 1148 pp. He was 

 concerned mainly with protozoa from 

 natural waters, soil and so forth, closely 

 associated with bacteria throughout 

 their existence. The techniques advo- 

 cated are of 3 types: (1) to get rid of the 

 bacteria by simply washing the pro- 

 tozoa in sterile fluid; (2) to scrape off 

 the adhering bacteria by causing the 

 protozoa to migrate through semi-solid 

 media and (3) to kill off the bacteria by 

 agents non-toxic for the protozoa. The 

 establishment of sterilized protozoa in 

 culture is an essential prerequisite to 

 investigation of their behavior in re- 

 sponse to accessory food factors and 

 nutritional supplements. 



Prussian Blue (CI, 1288) is ferric fcrrocy- 

 anide, a colored salt. It is also known 

 in commerce as Berlin blue, Chinese 

 blue, Paris blue, Milori blue and Steel 

 blue. An aqueous solution of Prussian 

 blue is a good medium for the injection 

 of blood vessels. It contrasts nicely 

 with carmine. The particles of both are 

 sufficiently large to be held witliin the 

 endothelium. Deposition of Prussian 

 blue is useful in the localization of drain- 

 age of Cerebrospinal Fluid (Weed, L. 

 H., J. Med. Res., 1914, 26, 21-117) and 

 in the microchemical demonstration of 

 Iron (Gomori, G., Am. J. Path., 1936, 

 12,655-663). See Berlin Blue. 



Pulp of Teeth. This can be studied in situ 

 in undecalcified teeth or in paraffin or 

 celloidin sections of decalcified ones. 

 See Teeth. If it is to be examined by 

 itself after removal from the teeth and 

 fixation, attempt to preserve its natural 

 elongated shape. Almost all methods 

 available for other soft tissues are ap- 



