CHLORAZOL PINK Y 



71 



CHLORIDE 



Chlorazol Pink Y, see Thiazine Red R. 



Chloride. In 1908 Macallurn reviewed the 

 older literature and described his silver 

 test for chloride (Macallum, A. B., 

 Ergeb. d. Physiol., 1908, 7, 552-652). 

 The possibility, which has not yet been 

 finally answered, is that at some stage 

 in the technique there is a shift in the 

 position of chloride. The mere applica- 

 tion of the silver reagent may conceiv- 

 ably withdraw chloride from the cell. 

 For these reasons prior treatment of the 

 tissue by the Altmann-Gersh freezing 

 and drying method which reduces the 

 chance of movement of chloride to a 

 minimum is recommended. 



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

 70, 311-329) gives details of the proced- 

 ure on which the following instructions 

 are based. Tissues frozen in liquid air, 

 dried in vacuum, embedded in paraffin 

 and sectioned at 15m are mounted near 

 one edge on chemically clean large cover 

 slips by simply pressing down with a 

 finger, just melting over a flame and 

 pressing down again. Immerse cover- 

 slips with attached sections in anhydrous 

 petroleum ether (b.p. 20-40°C.) freshly 

 distilled over sodium in a watch glass 

 covered by another at all times except 

 during actual manipulations. This re- 

 moves the paraffin. Remove and burn 

 off the ether quickly by a flame and allow 

 to cool to room temperature. Then treat 

 two coverslips with attached sections 

 differently. 



A. Cover for few seconds with drop 

 of 60% aq. silver nitrate diluted with 

 sufficient quantity of cone, phosphoric 

 acid to prevent precipitation of rather 

 large concentrations of phosphates and 

 then saturate with silver chloride. After 

 filtering 2-3 drops aq. dest. are added to 

 every 10 cc. before using. 



B. Cover similarly with: 60% aq. 

 silver nitrate saturated with silver phos- 

 phate and silver chloride and dilute 

 after filtering in the same way. 



Decant fluids from both coverslips. 

 Add to each 1 drop chemically pure 

 glycerin and mount with section plus 

 glycerin down on chemically clean slides. 

 Expose both to carbon arc radiation for 

 same length of time but at a distance 

 not to warm the specimens. Examine 

 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. Dichlor fluorescein 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 cliarged 

 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 Marsh, 

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

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

 ton, A. M., J. Phvsiol., 1937, 00, 167- 

 182). 



(5) Conclusion that in many tissues 

 for all practical purposes all radioactive 

 sodium and radioactive chloride remain 



