CHLORIDE 



62 



CHLOROTHYMOLS 



immediately the reduced silver by direct 

 illumination or in the dark field. A. 

 shows specifically only the chloride and 

 B. the same amount of chloride plus 

 maximal concentrations of phosphate 

 and some carbonate. 



2. Dichlorjluorescein method (Bens- 

 ley, R. D. and S. H., Anat. Rec, 1935, 

 64, 41-49). For the lung of a rabbit. 

 Inject 1% aq. dichlorfluorescein intra- 

 venously until the animal becomes 

 quite yellow. Then kill it and inject 

 10% aq. silver nitrate or Silver Citrate 

 solution either intratracheally or di- 

 rectly into the lung substance by a hy- 

 podermic syringe until the lung is 

 moderately distended. In about 20 

 min. the color reaction reaches its 

 maximum. The silver chloride becomes 

 pink owing to adsorption of the dichlor- 

 fluorescein on the positively charged 

 silver chloride molecule. Then fix 

 pieces of lung in 10% neutral formalin 

 and make frozen sections. Examine 

 immediately for best color reaction. 

 Dehydrate the sections, clear in absolute 

 alcohol and iso-safrol and mount in bal- 

 sam. The color reaction is not perma- 

 nent but is masked and finally lost by 

 the browning and blackening of the sil- 

 ver. It is not a true microchemical 

 test ; but it does detect the presence of 

 chlorides though they are mobilized by 

 the silver and tend to move to the per- 

 iphery of the cell. The alveolar epi- 

 thelial cells are outlined by pink stip- 

 pling and their cytoplasm is also stippled 

 and the nuclei are richly stippled. 

 Mesothelial and endothelial cells are 

 brilliantly and completely outlined in 

 pink. The technique was first sug- 

 gested by David M. Ritter. 



The location of chloride is a matter of 

 great importance. Lowry, O. H. and 

 Hastings, A. B. in Cowdry's Problems 

 of Ageing, Baltimore : Williams & Wil- 

 kins, 1942, 936 pp. cite the following as 

 evidence for the extracellular position 

 of chloride in skeletal muscle : 



(1) Direct microscopic studies show- 

 ing that chloride is exclusively extra- 

 cellular (Gersh, I., Anat. Rec, 1938, 

 70, 311-329). 



(2) Perfusion experiments showing 

 that chloride can be removed without 

 apparently affecting the intracellular 

 phase (Amberson, W. R. et al.. Am. J. 

 Physiol., 1938, 122, 224-235). 



(3) Variations in amount of chloride 

 and in acid base balances of tissues can 

 only be accounted for by assuming 

 an extracellular position for chloride 

 (Hastings, A. B. and Eichelberger, L., 

 J. Biol. Chem., 1937, 117, 73-93). 



(4) Isolated tissues equilibrated in 

 vivo against solutions of varying chloride 

 concentrations retain chloride in pro- 



portion to the concentration in the 

 medium but at a very much lower level 

 (Fenn, W. O., Cobb, D. M. and IVIarsh, 

 B. S., Am. J. Physiol., 1934. 110, 261- 

 272; Eggleton, M. G. and P. and Hamil- 

 ton, A. M., J. Physiol., 1937, 90, 167- 

 182). 



(5) Conclusion that in many tissues 

 for all practical purposes all radioactive 

 sodium and radioactive chloride remain 

 outside the cells (Manery, J. F. and 

 Bale, W. F., Am. J. Physiol., 1941,132, 

 215-231; Manery, F. W. and Haege, 

 L. F., ibid, 134, 83-93). 



See, however, Heilbrunn, L. V. and 

 Hamilton, P. G., Physiol. Zool., 1942, 

 15, 363-374 for demonstration of chloride 

 in muscle fibers. 



If chloride is always extracellular in all 

 tissues it is possible accurately to meas- 

 ure the amount of extracellular fluid 

 and a new chapter in histochemistry is 

 opened. Lowry and Hastings give an 

 example. If rat muscle is found to 

 contain 10.5 milliequivalents of chloride 

 per kilogram of tissue and the serum of 

 the same animal 105.2 milliequivalents 

 of chloride per kilogram of serum water, 

 in view of the Donnan effect on chloride 

 distribution it can be calculated that a 

 kilogram of extracellular fluid contains 

 109.7 milliequivalents of chloride. Con- 

 sequently the sample of muscle contains 



10 5 



' - X 1000 = 96 gms. of extracellular 



fluid per kilogram. When the extra- 

 cellular fluid contains collagenic and 

 elastic fibers, collagen and elastin must 

 be determined and the necessary correc- 

 tions made as well as for blood and fat 

 when these are present. When the in- 

 tracellular phase is chiefly composed of 

 a single type of cell as in skeletal or 

 cardiac muscle the further evaluation of 

 intracellular components is not diffi- 

 cult. Taking every known precaution, 

 evidence can apparently be collected of 

 the relative composition of extracellular 

 and intracellular phases. 



Chlorophenol Red. See Hydrogen Ion Indi- 

 cators. 



Chlorophyll. The green pigment of plants 

 is a mixture of 2 substances chlorophyll 

 a and b, of which many derivatives are 

 known. In man several fluorescent 

 chloropliyll porphyrins are identifiable 

 in feces and urine. A detailed account 

 of chlorophyll is provided by Rothe- 

 mund, P., in Glasser's Medical Physics, 

 1944, 154-180. 



Chloroplasts, Isolation and collection en 

 masse from spinach leaves by centri- 

 fugation (Menke, W., Zeit. f. Physiol. 

 Chem., 1938-39, 257, 43). 



Chloroprene, see Neoprene. 



Chlorothymols, as preservatives of gelatin. 



