CHLOROTHYMOLS 



63 



CHORIOALLANTOIC MEMBRANE 



glues, starches, etc. (Law, R. S., J. Soc. 

 Chem. Ind., 1941, 60, 66). 



Chocolate Blood Agar, see Bacteria, Media. 



Cholesterol (esters) = cholesterides. In 

 unstained frozen sections mounted in 

 syrup of levulose they show no color of 

 their own; but the Liebermann-Bur- 

 chardt Reaction in frozen sections of 

 formalin fixed tissue is positive. Digi- 

 tonine Reaction in similar sections 

 yields a complex in which the esters, if 

 present, will color with Sudan III and 

 lose birefringence in polarized light. 

 See Lipids tabular analysis, see Schultz 

 test for cholesterol and its esters. 



Cholesterols (free). In unstained frozen 

 section mounted in syrup of levulose, 

 they show no color of their own. Lie- 

 bermann-Burchardt Reaction in frozen 

 sections of formalin fixed tissue is posi- 

 tive: blue, purple or violet then becom- 

 ing green. Digitonine Reaction in simi- 

 lar sections yields strongly birefringent 

 crystals and rosettes which do not stain 

 with Sudan III. See Lipids, tabular 

 analysis. 



Choline. See Florence's Reaction for Semi- 

 nal Stains. 



Choline Deficiency. Use of fluorescence 

 microscopv in (Popper, H. and Chinn, 

 H., Proc. Soc. Exp. Biol. & Med., 1942, 

 49, 202-204). 



Cholinesterase. Important since it cat- 

 alyses hydrolysis of acetylcholine to 

 choline and acetic acid. Histological 

 localization is difficult but Couteaux,R. 

 and Nachmansohn, D., Proc. Soc. Exp. 

 Biol. & Med., 1940, 43, 177-181 found it 

 present in parts of guinea pig's muscle 

 that contained motor-end plates and 

 absent in parts devoid of them. For 

 recent data see Blaschko and Jacobson 

 (Bourne, pp. 221-224). Sharp localiza- 

 tion has been found in the giant nerve 

 fiber of squids (Nachmansohn, D. and 

 Steinbach, H. B., Science, 1942, 95, 

 76-77). Anfinsen, C. B., Lowry, O. H. 

 and Hastings, A. B., J. Cell, and Comp. 

 Physiol., 1942, 20, 231-237 have de- 

 veloped a method whereby the same 

 section of rat brain cortex can be 

 stained for microscopic examination 

 with methyl violet and thereafter used 

 for enzyme measurement. 



Chondriosomes, see Mitochondria. 



Chondriotin Sulphuric Acid. Present in 

 cartilage and bone, stains metachro- 

 matically with basic dyes, described in 

 detail by Lison, L., Arch, de biol., 

 1935, 46, 599-668. See Mucoproteins. 



Chorioallantoic Membrane. 1. Vital stain- 

 ing of virus lesions in membrane (Cooke, 

 J. V. and Blattner, R. J., Proc. Soc. 

 Exp. Biol. & Med., 1940, 43,255-256). 

 Place 1 cc.0.5% aq. trypan blue directly 

 on membrane through window in shell. 

 Rotate egg gently and return to incu- 



bator, 10-30 min. Small lesions require 

 longer time to stain than large ones. 

 Remove membrane, wash it gently in 

 physiological saline and fix flat in 10% 

 formalin, a few minutes. Make up 

 gl3'cerin jelly by soaking 5 gms. gelatin 

 in 44 cc. aq. dest. Then add 50 cc. 

 glycerin and 1 cc. phenol. Heat gently 

 and stir. Flatten membrane on a 

 2 X 2.5 in. slide, warm glycerin jelly to 

 about 70°C. Add drop by drop to mem- 

 brane until well covered. Flame a cover 

 glass and apply with slight pressure until 

 it has begun to set. Remove hardened 

 jelly around edges and seal with balsam. 

 Foci of virus increase are sharply marked 

 by clumps of deep blue stained cells. 



2. Cultivation of microorganisms. 

 The membrane has been shown to be an 

 excellent medium for the cultivation of 

 viruses by Goodpasture, E. W., Wood- 

 ruff, A. M. and Buddingh, G. J., Am. J. 

 Path., 1932, 8, 271-282 and many others. 

 Its usefulness has been extended to 

 Rickettsiae and spirochetes by Good- 

 pasture, E. W., Am. J. Hyg., 1938, 28, 

 111-119, to fungi by Moore, M., Am. 

 J. Path., 1941, 17, 103-125 and to acid- 

 fast bacteria by Moore, M., Am. J. Path., 

 1942, 18, 827-847. This method of inocu- 

 lation has the advantage over laboratory 

 animal inoculation in that lesions will 

 develop in the former within 5-8 days as 

 compared to weeks or months in the 

 latter; most organisms will produce defi- 

 nite and usually characteristic lesions in 

 the chick membrane, whereas they may 

 have no effect on experimental animals, 

 often requiring human subjects; and 

 because the lesions are so readily visible 

 and traceable the chlorioallantois serves 

 well as a means of virulence deter- 

 mination. 



The technique is essentially that of 

 Goodpasture and Buddingh (E. W.and 

 G. J., Am. J. Hyg., 1935, 21, 319-360) 

 with some slight changes. Fertile eggs 

 are incubated 12 days in an electrical 

 thermostat-controlled incubator regu- 

 lated to maintain a temperature of 98 °F. 

 The eggs are turned twice daily. A cm. 

 square window is cut in the shell above 

 the embryo, exposing the chorioallantoic 

 membrane. The position of the em- 

 bryo is determined by candling. The 

 membrane is then inoculated directly 

 with the fungus and the window is cov- 

 ered with a sterile coverslip and sealed 

 with a paraffin-vaseline mixture (9 parts 

 vaseline, 1 part paraffin). yVfter inocu- 

 lation, the eggs are set in a bacteriologic 

 incubator and maintained at a tempera- 

 ture of approximately 33°C., without 

 turning. The membrane is watched 

 daily througli the window. When the 

 inoculated area has shown marked 

 change, the shell is cut below the window 



