HOLLANDE'S FIXATIVE 



157 



HYALURONIC ACID 



methyl violet of the color desired may 

 perhaps be made by the worker himself 

 as a substitute for Hofmann's violet 

 which is in fact the composition of some 

 samples sold as Dahlia and Hofmann's 

 violet. 



Hollande's Fixative. Picric acid, 4.0 gm.; 

 copper acetate, 2.5 gm.; formol, 10 cc; 

 glacial acetic acid, 1.5 cc; aq. dest. 

 100 cc. Recommended for flagellate 

 protozoa (McClung, 1950, p. 445). 



Holmium, see Atomic Weights. 



Holtfreter's Solution, for use in examina- 

 tion of fresh tissues; NacCl, 0.35 gm.; 

 KCl, 0.005 gm.; CaClj, 0.01 gm., 

 NaHCOa-HsO, 0.02 gm., aq. dest. 100 

 cc. (Holtfreter, J., Arch. Entio.-Mech., 

 1931, 124). 



Hookworms. To eliminate opacity in 

 mounts of, see Tahmisian, T. N., Stain 

 Techn., 1945, 20, 26. 



Hormones. Consult volume entitled New 

 and Nonofficial Remedies published 

 each year by the American Medical 

 Association. See Testosterone, Chro- 

 maffin Reaction, Vulpian Reaction, Os- 

 mic Acid. 



Hotchkiss'Method, see Polysaccharides. 



Howell-Jolly Bodies, see Jolly Bodies. 



Huber's Toluidin Blue stain for Nissl bodies 

 (Addison in McClung, p. 150). This 

 much used method is suggested for 

 autopsy material. Fix in 95% alcohol, 

 100 cc; trichloracetic acid (Mallinck- 

 rodt), 1.5 gm.; mercuric chloride (Mal- 

 linckrodt), 3 gm. 2-10 days depending 

 upon size of piece of tissue. Change 

 fixative every 2 days for larger speci- 

 mens. Pour off fluid and store in 95% 

 alcohol until used. Do not take out 

 mercury with iodine. Stain paraffin 

 sections in toluidin blue 15-18 hrs. 

 (Make up solution by adding 1 gm. to 

 500 cc. aq. dest. Heat gently and when 

 it is dissolved add 500 cc. aq. dest.). 

 Pour off stain. Wash in aq. dest. 

 Leave 2 hrs. in lithium carbonate. 

 (Make this by adding 5 gm. to 1000 cc. 

 aq. dest. Boil several minutes. Cool. 

 Filter. To 100 cc. filtrate add 900 cc. 

 aq. dest.) . Differentiate in 70% alcohol 

 5-30 min. Leave flat in 95% alcohol, 

 5-15 min. Dehydrate in absolute, clear 

 in xylol and mount in balsam. 



Humus, see soli. 



Huntoon's Hormone Medium, see Bacteria, 

 Media. 



Hyalin. This is usually easily recognizable 

 in sections stained with Hematoxylin 

 and Eosin or by Phloxin and Methylene 

 Blue, by its affinity for eosin or phloxin. 

 Phosphotungstic Acid Hematoxylin 

 colors it deep blue. A hematoxylin- 

 phloxin method is also recommended 

 by Mallory (p. 207). Fix in alcohol or 

 10% formalin and imbed in paraffin or 



celloidin. Stain in alum hematoxylin, 

 1-5 min. or more. Wash in tap water 

 and stain with 0.5% phloxin in 20% 

 alcohol, 10-30 min. or longer. Wash in 

 tap water and treat for |-1 min. with 

 0.1% aq. lithium carbonate. Wash in 

 tap water, dehydrate, clear and mount. 

 In case of celloidin sections, clear in 

 terpineol or origanum oil from 95% 

 ale Nuclei, blue; fresh hyalin, in- 

 tensely red; older hyalin, pink to 

 colorless. A simple thionin stain is also 

 given by Mallory. It is to stain similar 

 sections for 5-10 min. in 0.5% thionin 

 in 20% ale Differentiate and dehy- 

 drate in 80% alcohol. Then 95% alco- 

 hol, terpineol and terpineol balsam. 

 Nuclei and old hyalin, blue. 

 Hyaluronic Acid. — Written by A. R. Gopal- 

 Ayengar, Barnard Free Skin & Cancer 

 Hospital, St. Louis. (Now at Tata 

 Memorial Hospital, Bombay.) This is 

 a polymer of acetyl glucosamine and 

 glucuronic acid. It occurs in a poly- 

 disperse form in a variety of tissues such 

 as umbilical cord, synovial fluid, 

 vitreous humor, skin, tumors due to 

 virus of leucosis and sarcoma of fowls, 

 and in pleural fluid associated with 

 human mesothelioma. (For an exten- 

 sive treatment of the subject of acid 

 polysaccharides and a comprehensive 

 bibliography, refer to Karl Meyer's re- 

 views on, "Mucolytic enzymes" in 

 Currents in Biochemical Research, In- 

 terscience Publishers, N. Y., 1946; 

 "Mucoids and Glycoproteins" in Ad- 

 vances in Protein Chemistry, Academic 

 Press, N. Y. 1945; "The Chemistry and 

 Biology of Mucopolysaccharides and 

 Glycoproteins" in Cold Spring Harbor 

 Symposia on Quant. Biol., 6, 1938, 91- 

 102.) The enzyme, hyaluronidase, 

 depolymerizes and hydrolyses hyal- 

 uronic acid. It is a Spreading Factor 

 and has been ably presented, along with 

 other spreading factors, by Duran- 

 Reynals, F., Bact. Rev., 1942, 6, 197- 

 252; Meyer, K. and Chaffee, E., Proc. 

 Soc Exp. Biol. & Med., 1940, 43, 487- 

 489; Meyer, K. et al., Proc Soc Exp. 

 Biol. & Med., 1940, 44, 294-296, and 

 others. 



A histochemical method for the dem- 

 onstration of acid polj'saocharides like 

 hyaluronic acid is described bv Hale, 

 C. W., Nature, 1946, 157, 802. the use 

 of metachromatic stains such as tolui- 

 dine blue while satisfactory for sul- 

 phated polysaccharides like chondroitin 

 sulphate is valueless for hyaluronic acid 

 and for related acid polysaccharides 

 which do not stain metachromatically. 

 Fixation of material is an important 

 factor in the retention of hj'aluronic 

 acid for subsequent staining. The or- 



