HYALURONIC ACID 



121 



HYDROGEN ION INDICATORS 



and for related acid polysaccharides 

 which do not stain metachromatically. 

 Fixation of material is an important 

 factor in the retention of hyaluronic 

 acid for subsequent staining. The or- 

 dinary aqueous fixatives containing 

 formalin, while eminently suitable for 

 fixing protein components, tend to dis- 

 solve the hyaluronic acid. To preserve 

 intact hyaluronic acid it is therefore 

 imperative to employ dehydrating fix- 

 ing agents like Carnoy. The material 

 after fixation, dehydration and embed- 

 ding is sectioned in the usual manner 

 and treated with an acid solution of 

 ferric hydroxide. The iron combines 

 with hyaluronic acid but not with the 

 neutral polysaccharides or proteins. 

 The combined iron is then characterized 

 as Prussian blue by treatment with 

 hydrochloric acid and potassium ferro- 

 cyanide. A counter stain like fuchsin 

 is recommended in order to bring out 

 sharply the blue stained acid polysac- 

 charides against a background of red 

 stained cells. 



The detailed outline of the Hale 

 technique is as follows : Fix small pieces 

 of tissue in Carnoy (Abs. alcohol, 6 pts. 

 -f- chloroform, 3 pts. + glacial acetic 

 acid, 1 pt., for | hr. Dehydrate in abs. 

 alcohol, clear, embed in paraffin and 

 section in the usual manner. Mount 

 sections on clean slides without albu- 

 men. Bring sections rapidly to water 

 and flood with a mixture of dialysed 

 iron, 1 vol. and acetic acid (2M), 1 vol., 

 10 min. (Dialyzed iron may be pre- 

 pared by adding ammonia water to a 

 concentrated solution of ferric chloride 

 and dialysing the resulting solution un- 

 til free or nearly free of ammonium 

 salts. It is a dark red liquid easily 

 miscible with water and contains ap- 

 proximately 3.5 per cent Fe, or 5% 

 FejO,. M = Molecular Solution, which 

 see.) Wash well with aq. dest. Flood 

 with a solution containing potassium 

 ferrocyanide (0.02M) and hydrochloric 

 acid (0.14M) — 10 min. Wash with wa- 

 ter and counterstain with appropriate 

 contrasting dye. Dehydrate rapidly, 

 clear in xylol and mount in Canada 

 balsam. 



In order to distinguish hyaluronic 

 acid from other blue staining structures 

 Hale recommends interpolation of 

 another step during the staining proc- 

 ess. The procedure suggested involves 

 use of the specific enzyme-hyaluroni- 

 dase — soon after fixation. The enzyme 

 hydrolyses the hyaluronic acid and 

 prevents the combination of the pol- 

 ysaccharide with iron. Since hyal- 

 uronidase is specific, it has no similar 

 action on other polysaccharides. 



Hyaluronidase is the spreading factor which 

 increases the permeability of connec- 

 tive tissue by reduction in viscosity and 

 by hydrolysis of Hyaluronic Acid. 



Hydrax is a synthetic resin used as a mount- 

 ing medium (Hanna, D., J. Roy. Micr, 

 Soc.,1930, 50, 424-426). 



Hydrogen Acceptors. These are substances 

 like p-amidophenol, p-phenylenedia- 

 mine and resorcin, recommended to 

 strengthen supravital staining of nerve 

 fibers with methylene blue, see Auer- 

 bach's Plexus. 



Hydrogen Ion Indicators. Data contributed 

 by Mr. Lester F. Wicks of The Barnard 

 Free Skin and Cancer Hospital. 



In 1893 Ehrlich injected neutral red 

 in an attempt to determine the reaction 

 about phagocytosed granules. Since 

 then, other workers have applied other 

 dyes, striving to estimate the approxi- 

 mate pH of tissues, of the fluids bathing 

 them, and even of individual cells. 

 Alizarin red and litmus have been much 

 used, the later especially with lower 

 organisms. Thus, Steiglitz applied all 

 three dyes mentioned above to estimate 

 the reaction of living kidney (E. J., 

 Arch. Int. Med., 1924, 33, 483-496) and 

 confirmed the contention that alkaline 

 urine can be formed by an acidic cortex. 

 Harvey and Bensley (B. C. H. and R. 

 R., Biol. Bull., 1912, 23, 225-249) used 

 pH indicators to indicate tliat gastric 

 fluid does not arise directly within the 

 cells of the mucosa. Margaria (R., J. 

 Physiol., 1934, 82, 496-497) injected 

 bromcresol purple and bromphenol blue, 

 and claimed to have measured pH 

 changes upon stretching a muscle. 

 Orr (J. W., J. Path. & Bact., 1937, 44, 

 19-27) employed phenol red to estimate 

 alterations in pH in the skin of tarred 

 mice during carcinogenesis. Chambers 

 and his colleagues have added pH indi- 

 cators to tissue cultures (R., Proc. Roy. 

 Soc, B, 1932, 110, 120-124) and have 

 injected them directly into individual 

 living cells (McClung, pp. 62-109). 

 The most enthusiastic investigator to 

 employ the phthalein and sulphon- 

 phthalein indicators is Rous (P., Sci- 

 ence, 1924, 60, 363: J.A.M.A., 1925, 85, 

 33-35, and many articles in J. E.xp. 

 Med., 1925 to 1927). The literature is 

 extensive but scattered. There are 

 brief reviews by Rous (P., J. Exp. Med., 

 1925, 41, 379-411) and von Mollendorf 

 (W., Ergebn. Physiol., 1920, 18, 141- 

 306). See W. M. Clark in Simmons and 

 Centzkow 161-171. 



It is well to question the dependa- 

 bility of data upon pH of living material 

 as apparently indicated by vital staining 

 methods. Consider the ideal require- 

 ments for such a vital stain. It should 



