HYALURONIDASE 



158 



HYDROGEN ION INDICATORS 



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. 

 -H chloroform, 3 pts. + glacial acetic 

 acid, 1 pt., for j 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 fiood 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% 

 Fe20,. 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. 

 Commercial preparations of hyal- 



uronidase from bull testes are available 

 from the Schering Corp., Bloomfield, 

 N. J. Enzyme prepared from certain 

 bacteria apparently have hydrolytic 

 powers different from those of the 

 testicular preparations. 



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 — Written by L. F. 

 Wicks, Veterans Administration Hos- 

 pital, Jefferson Barracks, Missouri. 

 February 1, 1951. — These are also called 

 acid-base indicators and pH indicators. 

 They are dye compounds which are 

 themselves weak acids or weak bases, 

 more usually the latter, and have defi- 

 nite ionization constants. According 

 to the old theory of Ostwald, the color 

 of the indicator in solution depends 

 upon the degree of dissociation and the 

 relative ratio of dissociated and un- 

 dissociated forms. This ratio, and the 

 corresponding shift in color, varies 

 with the concentration of hydrogen ions 

 present, the effect being a composite 

 one. The color change interval will 

 span a certain range, usually of two pH 

 units or less, and it does not require a 

 shift which crosses neutrality. Of the 

 very many dyes and plant coloring 

 matters which alter color with pH, 

 only a few change sufficiently sharply 

 to be of analytical value. 



Acid-base indicators may, of course, 

 be employed for adjusting the pH of a 

 solution. If direct addition is not de- 

 sired, small portions of both liquids may 

 be transferred to a spot plate, or the 

 indicator may be applied in the form 

 of test papers. (Some indicators such 

 as litmus are now rarely employed 

 otherwise. This is partly true also for 

 Congo red and nitrazine.) 



Indicators may also be used to esti- 

 mate the reaction of a solution by the 

 application of a series with different pH 

 ranges. Once roughly determined, 

 there is a procedure ("Gillespie's drop- 

 ratio method") by which a fairly ac- 

 curate pH measurement may be easily 

 made with a single indicator of proper 

 range. 



Perhaps the commonest use for hy- 

 drogen ion indicators by the analytical 

 chemist (who usually prefers the glass 

 electrode for pH measurement and ad- 

 justment), is as an end point device in 

 acid-base titrations. When titrating a 

 weak acid or a weak base, the choice of 



