AMNIOTIC FLUID 



12 



AXILIN BLUE 



Amniotic Fluid, technique for study of ab- 

 sorption from, Wislocki, G. B., BulL 

 Johns Hopkins Hospital, 1921 32, 93. 



Anaplasma is a small spherical body found 

 within red blood cells in anaplasmosis 

 diseases. There are two types A margi- 

 nale and A centrale depending upon 

 whether the bodies are situated near 

 the margin or in the centers of the cells. 

 The bodies are supposed to be parasites 

 consisting of nuclear material with little 

 if any cytoplasm. Anaplasmosis is im- 

 portant economically as a group of tick 

 borne diseases of domestic animals. 

 For demonstration stain blood smears 

 by the methods of Giemsa or Wright. 



Anethol is anise camphor suggested as a 

 medium in which to soak tissues before 

 making frozen sections (Stephanow, 

 Zeit. wiss. Mikr., 1900, 17, 181). 



Anethole Clearing Agent.— Written by Joan 

 Haberman, Parkland, Washington. 

 March 10, 1951 — Anethole is a useful 

 agent in clearing segments of brain 

 tissue because of its refractive index. 

 The basic principle is that homogeneous 

 tissue, which has been bleached and 

 dehydrated, may be cleared by placing 

 in an oil of similar refractive index was 

 discussed by Groat, R. A., Stain Techn., 

 1941, 16, 111-117. Brain tissue consists 

 primarily of protein and lipids and is 

 therefore not homogeneous as are other 

 types of tissue such as muscle. Since 

 protein forms the framework of the 

 brain, it necessitates that the lipids be 

 removed to obtain a homogeneous 

 tissue. 



The lipid composition of the brain 

 tissue must be known in order to per- 

 form an adequate extraction. Accord- 

 ing to Koch, as quoted by A. P. 

 Mathews, Physiological Chemistry. 

 Baltimore: Williams and Wilkins Co., 

 1939, the lipid composition is as follows: 

 phospholipids— 27.3% of dry matter; 

 cerebrosides— 13.6% of dry matter; 

 cholesterol— 10.9% of dry matter. The 

 phospholipids, according to G. A. Hill 

 and K. Kelly, Organic Chemistry. 

 Philadelphia: Blakiston Co., 1943, are 

 composed mainly of : lecithin— 48 p. p.m., 

 soluble in ether and in alcohol; cepha- 

 lin — 200 p. p.m., soluble in ether, in- 

 soluble in alcohol; sphingomyelin— 56 

 p. p.m., insolublejin ether, soluble in hot 

 alcohol. These are all insoluble in 

 acetone. The cerebrosides are soluble 

 in hot alcohol, insoluble in ether. 

 Cholesterol can be extracted with ether 

 and alcohol. (The data on solubilities 

 are taken from Harrow, B., Textbook 

 of Biochemistry. Philadelphia: W. B. 

 Saunders Co., 1943). 



Brain tissue must first be dehydrated 

 by using the alcohol series starting at 



35% or higher if one wishes to risk more 

 shrinkage and changing every few hours 

 depending upon the size of the tissue. 

 The lipids are then removed by placing 

 the tissue in 100% alcohol at 60 degrees 

 centigrade for 1-2 days with frequent 

 changes in solution. The remaming 

 lipids are extracted with ether for 2 

 days or longer depending upon the size 

 of the tissue. The procedure results 

 in a tissue that consists largely of pro- 

 tein and gives a refractive index of 

 1.560 as determined on the Abb6 re- 

 fractometer. 



By placing the tissue in an oil of very 

 similar refractive index it will soon be- 

 come transparent when brightly illum- 

 inated. Anethole (C10H12O) having a re- 

 fractive index of 1.5614 was found to 

 be the oil of choice. It is colorless, 

 very slightly soluble in water, soluble in 

 alcohol and ether, and is liquid at ordi- 

 nary temperatures. It can be obtained 

 from the Eastman Kodak Company, 

 Rochester, New York. 



After the tissue has been placed in 

 anethole the container should be left 

 uncovered so that the displaced ether 

 may escape. 



The transparency of the tissue de- 

 pends upon the completeness of the 

 lipid extraction. Larger pieces of 

 tissue such as 4 x 6 x 3 cm must be kept 

 in 100% alcohol for a week and in ether 

 for 1-2 weeks. Frequent changes of 

 solution are advisable. This block of 

 tissue will still appear only translucent 

 when cleared in anethole. However, 

 sections of § to 1 cm. in thickness cut 

 from this block will be sufficiently clear 

 to insure visualization of injected 

 vessels etc. under the binocular with 

 good illumination. It is probable that 

 a large piece of tissue could be cleared 

 satisfactorily if the alcohol and ether 

 extraction were carried out by means of 

 an intermittent siphoning distillation 

 extractor. 



Angina, see Vincent's Angina. 



Angstrom Unit. lA = 0.1 m/* = O.OOOIm = 

 10-' mm. 



Anhydrase, see Carbonic Anhydrase. 



Anilin Blue Alcohol Soluble, see Spirit Blue. 



Anilin Blue, WS (CI, 707)— China blue, 

 cotton blue, marine blue V, soluble blue 

 3M or 2R, water blue (Wasserblau)— A 

 mixture of trisulphonates of di-phenyl 

 rosanilin and tri-phenyl pararosanilin. 

 Conn (p. 135) explains that this desig- 

 nation (like acid fuchsin) applies not 

 to a single compound but to a group 

 of dyes. Anilin blue is, nevertheless, 

 the best stain for Collagenic Fibers and 

 is employed for many other purposes. 



