,^5 A Study of the Changes in Skins During Their Conversion into Leather A thesis Submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy in the University of Michigan By Anton Augustus Schlichte Reprinted from The Journal of the Americon Leather Chemists Association December-November, 1915 Copyright, 1915 «<''' ^*v Meinen lieben Eltern in Dankbarkeit gewidmet CONTENTS. acknowxkdgkment 3 Introduction 3 Study of Microscopic Changes in Hides during their Conversion INTO Leath er 6 Review of Literature 6 Experimental Work on Technique of Imbedding and Cutting Tissue 7 (a) Paraffine Methods 7 (b) Celloidin Methods 9 (r) Freezing Method 10 Study of Changes in Structure during Tanning 12 Study oe Changes in Volume and Weight of Cai^fskins during the Liming Process 14 Apparatus 15 Experimental Work on Calfskins 17 Attempts to Remedy Thinness of Flank 34 Results 39 Study of Deputation in Sterii^e Limes 42 Review of Literature 42 Experimental Work 43 (a) The Sterilization of Skins 46 (h) Laboratory Tests on Depilation 48 (r) Depilation and Subsequent Tannage 56 Conclusion 60 BiBIJOGRAPH y 62 Gift ba Univertity J ACKNOWLEDGKMKNT. The generosity of Mr. Carl E. Schmidt in establishing a fel- lowship in tanning at the University of Michigan, has enabled the author to pursue for 3 years, the studies outlined in this thesis. Mr. Schmidt has, moreover, given freely of his time and experience and it is a pleasure at this time to express apprecia- tion, not only for the financial assistance rendered but more es- pecially for the able counsel so unstintedly afforded. Acknowledgement is also due Mr. V. A. Wallin for his interest and courtesy in making possible the completion of the experi- ments on heavy leathers. The author wishes to thank sincerely, Prof. A. H. White for his constant interest and hearty co-operation during the entire time consumed by this research. Introduction. The use of hides both as skins and leather for protection against cold and rain, for weapons, or for ornaments, dates back to the remotest history of man. While the hides were tanned in the earlier times with the hair on, methods were soon found to remove it and thus improve the product. The first substance used was probably wood ashes and this continued as the standard for some time. After tanneries were established, for up to this time the tanning was done only on a small scale, new substances were sought for, and lime, one of the oldest depilatory agents, was used. The method followed was to slack the lime in pits and soak the hides in a saturated solution of calcium hydroxide. This method although slightly modified has remained practically the same for centuries. The tanning process was and is in general the following: I. Hides are soaked to remove blood and dirt and to bring them back as nearly as possible to their original condition. 4 A STUDY OF the: CHANGES IN SKINS 2. They are placed in pits containing- milk of lime, bacteria being always present and sulphides being frequently added, for from 3 to 1 8 days until the hair "slips" easily, that is, can be easily removed. 3. They are then bated to remove lime and bring the skin into the desired physical condition. The bate may be either acid or bacterial. 4. The next step is the pickling process in which the skins are treated usually with salt and sulphuric acid. 5. Then follows the tanning process proper, which may be either a mineral or a vegetable tannage. 6. The last step is a tinishing process. The entire tanning process has thus far been outlined to show the dependence of the finished product upon the correct perform- ance of each succeeding step of the process. It is only through tests on the finished leather that the effect of any alteration in any step of the process can be detected. The liming process is the one studied in greatest detail in this paper but any changes due to this operation can be detected only in the finished product. Moreover, the method of soaking must, because of its influence, also be specified. The object of soaking hides is to cause them to resume as nearly as possible their orig- inal clean and pliable condition. This part of the process while not so important when green hides are used becomes a matter of great importance when dried hides are to be investigated. The length of soaking, number of changes of water and the acids or alkalies which may have been added, all have an effect on the final product. Should the hides be soaked too long or should the water not be renewed frequently enough, bacteria multiply and a part or, in extreme cases, all of the hide may be lost. On the other hand acids and alkalies cause swelling of the hide and if too much of either be added the hide will be "plumped" too much.^ This, while not fatal to a good final product, has its disadvantages and as a rule causes trouble. The object of liming is not alone the removal of the hair but also the loosening of the fiber bundles. More surface is thus ex- posed and hence the tanning agents are taken up more readily. ' Jettmar Handbuch, p. 56. A STUDY OF the: CHANGES IN SKINS 5 In the vegetable tannage this makes a heavier leather. In the mineral tannage the loosening of the fiber bundles makes a more pliable leather.- The latter object is of great importance in the chrome tannage which is the method most used in manufacturing light or upper leathers. The hides after soaking are placed in pits in which an excess of calcium hydroxide is always present and to which some sodium or arsenic sulphide may or may not have been added. The hides are ''hauled," that is, taken out and the. lime ''bettered" once a day until the hair "slips" easily. It requires much practice and experience before one is able to tell exactly whether a hide is done or not, and the method is not only unsatisfactory but also very unscientific, for the personal equa- tion of the operator plays too important a part. The hides are then soaked in warm water, paddled and beamed. In the latter • process the skin is placed over a piece of wood semi-circular in cross section and the hair is removed with a blunt knife. The long hairs comes off very easily but the fine or "ground" hair and the pigment, especially in the case of black skins, cause some trouble. Part of the intercellular substance, corium, "scud" or "gneiss" and some lime soaps^ are also removed in this treatment The next operation, "bating," has as its main object complete removal of the lime remaining from the previous treatment. The loosening of the fiber bundles, however, is also materially aided by bating and this, as before mentioned, is, in the case of light leather, of greatest importance. The bates most commonly used owe their activity to bacteria and are frequently more or less un- satisfactory and harmful to the skins. Bates of known bacterial cultures are used somewhat and give good results, but the most common ones consist of organic acids such as butyric, lactic, etc. The skins are then pickled. The pickling consists usually of a treatment with sulphuric acid and salt. One object is to partially reduce the excessive swelling caused by the bate. Another, without doubt, is to furnish some free sulphuric acid which is considered necessary in the subsequent chrome bath. This sul- phuric acid is probably absorbed by the skin and thus carried over to the tan bath. This finishes the treatment received by the ^ Gerber, No. 938, p. 253; Procter's Principles of Leather Manufactur- ing, p. 126. ^ Procter's Principles of Leather Manufacture, p. 136. 6 A STUDY OF the: CHANGES IN SKINS skins in the "Beam House," which is probably the most im-' portant division of the tanning process. The most important part of the beam house work is the hming process, and hence this was made the object of the subsequent investigations. The object was to gain some insight if possible, into this apparently simple but actually very complicated process and to furnish something of practical value to the industry. It seemed obvious that some accurate way of controlling the liming and of judging the product afterward was absolutely necessary in order that improvements could be noted. The ordin- ary method of judging the product by the "feel" left much to be desired and it was recognized that the personal equation had to be eliminated as much as possible if anything of real value were to result. The most natural idea was to obtain some means of following the action of the lime step by step. This could not be accomplished with the naked eye and hence the assistance of the microscope was necessary. A review of the literature at hand showed that although considerable work with the microscope had been done the results were not very satisfactory. Study of Microscopic Changes in Hides During Their Conversion Into Leather. Some of the earliest work on the cutting of sections of leather was done by Kathreiner in 1879.* This work was never pub- lished and on inquiring of Prof. Procter it was found that al- though notes had been preserved, they were in no shape to allow of their being published. Thus we have no authentic record of his work. The next reference found was to some work by Prof. Thomas Palmer.^ He applied his method to determine the pene- tration of vegetable tanning agents. The sections were mostly cut by hand. Some sections after dehydration in alcohol and clearing in clove oil were infiltrated with "Strickers" solution (gum arabic and glycerine 3:1) and cut with a microtome. In some cases he also dehydrated in alcohol two or three hours, cleared in a mixture of cedar oil and benzol, infiltrated in a mix- ture one part 50° melting point and two parts 40° melting point paraffine until transparent, and then changed to a bath of two * Procter's »Leather Industries Laboratory Book, p. 424. ^ Collegium, 1902, p. 325. A STUDY OF THE CHANGES IN SKINS 7 parts 50° melting point and one part 40° melting point paraffine for three or four hours. The mixture of cedar oil and benzol was in the ratio of three to five. He does not tell of any changes noted in the leather, except that the distance to which the tanning agent had penetrated could be noted by differential stains. More- over he does not give the thickness of the sections cut. Procter, in his Leather Industries Laboratory Book, also speaks of cut- ting sections by hand and of cutting sections by means of a micro- tome, but gives no specific directions. M. Henri Boulanger in an elaborate monograph, "Essais du Cuir dans ses Applications industrielles. Memoires publies par la Societe d'Encouragement pour I'industrie national 1907," part of which is published as ''Etude Micrographie du Cuir" in Bulle- tin de la Societe d'Bncouragem,ent in 1908, gives some difections for cutting sections. The pieces to be cut are placed 12 hours in a mixture composed of distilled water 5 grams, pharmaceu- tical glycerine 5 grams, acetone 90 grams. They are then dried and imbedded in hard paraffine and cut. Another method given is to dehydrate in 'gradually increasing alcohol until absolute is used, to place in xylol, then in melted paraffine 38° to 40°. After several days the tissues are cooled, dried 36 or 48 hours, im- bedded in hard paraffine and cut. Although many sections were prepared their thickness is not mentioned. One interesting con- clusion of the author is that the elastic fibers are well preserved and that to them the leather owes its strength and pliability, while the connective tissue has been totally changed. This is a remark- able conclusion when one takes into consideration that less than 3 per cent, of the skin is elastic tissue and more than 95 per cent, is connective tissue.*' Moreover the elastic fibers have little elas- ticity and are the first to rupture when the skin is stretched. Their chief function appears to be that of support.^ Andreis, in an article on "The Process of Liming"® speaks of "taking a transverse section of the hide and noting no horizontal layers or channels." He does not give any methods for cutting the sections and presumably means that they are to be cut by hand. ^ Reimer, Din^ . Poly. Join ., No. 205, p. 149 (1872). ^ Hyde, Diseases of the Skin, p. 22 (1909). ^ JouRNAi,, Am. Leather Chem. Assoc, Vol. VII, p. 609 (1912). 8 A STUDY O? THE CHANGES IN SKINS As none of the references quoted gave definite, concise and adequate directions for preparing sections, experiments were re- sorted to, in order to find a way to prepare good sections by some simple and quick method Having no landmarks to guide us, much time was spent in going astray. At this time certain sec- tions of rocks were being made by grinding and this method ap- peared feasible for leather. The piece of leather to be ground was placed in Canada balsam in a tube in hot water and suction applied. After two to four hours the leather seemed impregnated with balsam and was removed, mounted on a piece of plate glas;3 I inch square and ground with a carborundum wheel until a flat surface was obtained. The piece was then turned over and mounted and the grinding continued, until the section was fairly thin. Then a finer wheel was used, until the section became very thin. It was removed, turned over and mounted on a glass slide and the grinding resumed, until the section was as thin as could be obtained. These sections showed some fiber bundles but were unsatisfactory in three ways. The process was too slow, the sections too thick, and a complete section could not be obtained as some parts were always torn away in grinding. Methods of Imbedding and Cutting Tissues. Leather is harder and tougher than ordinary tissue. It is dense and requires an unusually long period of infiltration. It is tough and offers great resistance to the knife so that the infiltration must be very thorough before good sections can be obtained. Methods of cutting sections after imbedding in paraf¥ine and celloidin and after freezing were studied. Various modifications were tried and those giving best results are described in detail, although they, are merely modifications of methods used in pathology. The method of imbedding in paraf¥ine involves the following steps. I — 95 per cent, alcohol 24 hours, change after 12 hours. 2 — Absolute alcohol 24 hours, change after 12 hours. 3— Xylol, I hour. 4 — Xylol, 2 hours. 5 — Para|fine 42° melted in an oven from 12 to 24 hours depending on size of piece used. 6 — Parafifine 52° from 24 to 48 hours depending on size of the piece. A STUDY OF THE CHANGES IN SKINS 9 The piece is then taken from the molten paraffine and imbedded according to the following procedure. A small dish is greased with tincture of green soap or glycerine and placed in cold water. Clean ^2° paraffine is melted with a free flame and the molten paraffine is allowed to drop into the dish until it has attained a depth greater than the thickness of the piece to be imbedded. As soon as a film of hardened paraffine has formed on the bottom the tissue is removed from its previous bath and placed face down in the dish. The surface of the melted paraffine is now cooled by blowing on it, and as soon as a fairly thick film has formed the whole dish is plunged into ice water. In spite of all precautions taken this method was not satisfac- tory. All attempts to cut sections of a satisfactory thickness were unsuccessful as the tissue was either pulled away from the im- bedding material or was torn by the knife. In fact, no com- plete thin section could be obtained by this method. Other methods based on paraffine infiltration using acetone, clove oil, oil of bergamot and aniline were tried and the results were unsatis- factory. Even infiltration with paraffine in solution, that is, in benzol or xylol, gave poor results. This seems to show that ordinary methods cannot be used and that methods like tlie one used by Boulanger^ ''infiltrating in melted paraffine 15 minutes and then blocking in hard paraffine" cannot give good results. The next method tried was the celloidin method. In this the dehydration was carried over a longer period so that the presence of any moisture was precluded. The procedure was as follows : I — 80 per cent, alcohol 12 hours. 2 — 95 per cent, alcohol 48 hours, changed every 24 hours. 3 — Absolute alcohol 48 hours, changed every 24 hours. 4 — Absolute alcohol and ether (equal parts) 24 hours. 5 — I per cent, celloidin 96 hours. 6 — 2 per cent, celloidin 120 hours. 7 — 5 per cent, celloidin 168 hours. 8 — ID per cent, celloidin 6 days to 2 weeks depending on the size of sample used. In order that a number of pieces might be imbedded simultan- eously they were held in clips tied to strings, which led through holes, in the wooden cover of a shallow dish. This dish was filled ' Bulletin de la Societe d'encouragement, p. 250 (1907). lO A STUDY OF THE CHANGES IN SKINS with a 10 per cent, solution of celloidin, the cover put on and the ether and alcohol allowed to evaporate. The tissues were then cut out, trimmed and mounted on wooden blocks. They were cut in a microtome under a constant flow of 80 per cent, alcohol. The sections obtained by this method, after long practice, were as thin as 5 or 7 microns and averaged about 10 to 18 microns. These could be examined even with an oil immersion lens. This method while giving excellent results had as a very serious dis- advantage the long time necessary for good results. Many at- tempts were made to shorten this method but none gave good results. A very long time is absolutely necessary for perfect infiltration. Believing that the added knowledge gained by experience might after all, enable us to use the paraffine methods, these were again tried. The results while better than those first obtained, still left much to be desired. The leather tore away from the paraffine very easily and it was impossible to cut good sections. The paraffine methods had so many advantages both as to quickness and simplicity, that many modifications of the method previously given, were repeatedly tried. The conclusion finally reached was, that none of the paraffine methods would give good results. Methods using beeswax, gum arabic, etc., were tried and also failed to give good results. The only short method which appeared promising was the freezing method and this was then tried. This method had the obvious advantage of great saving in the time required to obtain complete sections. The method was very simple and, although much practice and experience were necessary before good sections were obtained, the results justified the time spent in acquiring the technique. The sample to be sectioned was cut from calfskins, i by 2 centimeters and if wet, mounted directly. If dry, it was first soaked in water until thoroughly moistened and then mounted upon the base plate of a Bardeen microtome and covered with a thick solution of gum arabic. The liquid carbon dioxide was then turned on and the pieces were frozen very gradually. When the right degree of hardness has once been obtained the piece should never be allowed to warm up and should never be frozen again. This is of great importance as several A STUDY OF the: CHANGES IN SKINS II freezings will cause important changes in the structure/^ The greatest changes are caused by over freezing and if this is done excessively, the piece used may crumble and become worthless. A slight fixation of the tissue in lo per cent, formol" prevents most of the changes due to freezing. The sections when cut are placed in a dish filled with water. They are then transferred by means of a brush or section lifter to a lo per cent, dextrin solution which is kept warm, and floated onto a glass plate, dried a few minutes, soaked in absolute alcohol until clear and coated by pouring over them a i or 2 per cent, solution of celloidin. The plates are placed in warm water and left until the thin celloidin sheet floats off. They can now be handled easily and quite roughly without any danger of damage. They can be stained with alcoholic or aqueous solutions of various dyes. A water solution of eosin and also a double stain, first in Weigert's haematoxylin and then in Van Giesens^^ mixture were used. The latter gave excellent results and enabled one to distinguish the various kinds of fibers present, with great certainty. The sections were not so thin as those obtained by the celloidin method, averaging only 35 to 40 microns, but this was thin enough to allow the use of any high power objectives except the oil immersion. The sections were very delicate and required great care in the preliminary handling. The method requires little time. A skilled operator can fix, freeze, stain and mount a com- plete section in 30 or 40 minutes. In some cases the time required is greater depending on the ease with which the piece in question can be cut. Sections from heavy hides during the liming require much longer, as the tissue is very delicate and flabby and tears very easily. After the hide has been in a tan liquor it can be cut very rapidly. This method has not only the advantage of speed but also is better in that the tissue is practically the same as it was before cutting. The method does not subject the tissue to such severe treatment as for instance the dehydration with- absolute alcohol, which is necessary in the case of the celloidin and paraffine methods. It is true that freezing may dehydrate in a certain sense, but the following immersion in water probably ^•^ Warthin Practical Pathology, p. 215, !• Warthin Practical Pathology, p. 216, '■- Warthin Practical Pathology, p. 260. 12 A STUDY OF the: CHANGES IN SKINS allows the tissue to resume its previous condition. This seems logical, since the freezing lasts a very short time, from 5 to 10 minutes at the most. Study of Changes in Structure During Tanning. Before proceeding to the experimental study of tissues prepared in the laboratory a considerable number of commercial products w^ere examined. Sections were made of various commercial leathers such as dongola, waterproof, wax upper, plow grain, flesh splits, badger sides, oil grain, reliance calf, dull romar sides, kangaroo calf and others. These gave some idea of how a finished leather looked. Through the courtesy of Mr. Carl E. Schmidt of Detroit, pieces were cut daily from a particular calfskin as it went through his tanning process and sent for examination. As soon as these samples were received a section was made by the freezing method and a piece started by the slow celloidin method. This set was kept as a standard of a good product and will be referred to as S-22. This set did not, however, entirely fulfill its purpose, because the necessity of knowing the exact location and orientation of the pieces cut from the hide was not discovered until later. This was shown by an examination of pieces cut in a systematic manner from a finished calfskin (Experiment S-18). Samples were taken from this leather at the butt, right and left flank, and neck and carefully infiltrated with celloidin. Sections were cut both parallel and at right angles to the grain. The sections showed a great difference in structure between pieces cut from the flank and from the butt. These show the flank to have a much smaller amount of connective fibers than the butt and neck. Moreover, the fiber bundles of the flank are further apart and in general form a looser network. The connective tissue is more wrinkled that is, the folds in the individual fiber bundles are more numerous. This apparently accounts, at least partially, for the looseness of the flank* after tanning. This difference between various portions of the same skin was further studied in series S-26, by tanning two pieces of calfskin cut so that one piece was almost entirely flank while the other was along tl\e backbone. Small pieces were cut daily from adja- cent portions of each skin, one from the backbone and the other A STUDY OF the: CHANGKS IN SKINS 13 from the edge of the flank, and sections were prepared by the freezing method using the Van Giesen stain. Sections were cut both parallel and at right angles to the backbone in each instance. These showed the same looseness in structure in the fresh flank as was evident in the finished leather. This subject was pursued still more systematically in Series S-36 where a whole calfskin was trimmed to an approximate rectangle and divided into six pieces. This work is referred to again in the discussion of volume changes where the details are given. Through the courtesy of V. A. Wallin of Grand Rapids, samples of a certain cow hide were sent throughout the tanning process. The pieces were taken so that the sections made therefrom could be cut parallel and at right angles to the backbone. Samples were received during the following stages of the tanning process. All sections were cut by the freezing method. — Washed hide — originally a green salted one. — After 24 hours in lime. — After ^2 hours in lime. — After 96 hours in lime. — Out of hot water. — Into cold water. — Out of cold water. — Out of rockers — in 8 days. — Out of hang yard — in 4 days. — Out of first layer — in 5 days. — Out of second layer — in 9 days. — Out of third layer — in 15 days. — Out of fourth layer — in 22 days. — Out of fifth layer — in 28 days. — Out of wet dip — in 15 days. Out of tempering vats — in 2 days. — Out of bleach. Out of oil wheel. Finished leather. In these various experiments about 125 different blocks were prepared, from which 600 specimens were cut, stained and mounted. A careful examination of all these sections gave, how- ever, rather meager results. The changes from day to day are so gradual and the diflferences in structure of diflferent portions of the same hide are so pronounced that it is extremely difficult No. I. No. 2.- No. 3-- No. 4.- No. 5-- No. 6.- No. 7.- No. 8.- No. 9- No. 10.- No. II.- No. 12.- No. 13.- No. 14.- No. 15-- No. 16.- No. 17.- No. 18.- No. 19.- 14 A STUDY OF THK CHANGES IN SKINS to State the cause for any dififerences observed. The fiber bundles become partially separated into fibrils in the liming process, but it could not be determined whether these fibrils are hollow or solid. In the bark-tanned process the interstices become filled with solid material so that the leather becomes firm and of more uniform structure. In chrome-tanned leather, the interstitial spaces are even greater than in the soaked hide and the fiber bundles and fibrils are sharply defined. The flank is undoubtedly composed of larger fiber bundles than the butt or shoulder and in the former there are larger spaces between these bundles than in the latter. The idea first held that the microscopic method could be used in the tannery, as an accurate check on the process, had to be abandoned. The great difficulty was that in a given skin, pieces from different parts showed such differences in structure that even after long experience one could not tell whether the differ- ences shown were inherent in the skin or due to the influence of the treatment. Changes in Volume of Hides During Liming Process. Changes in the volume of hides in the limes are so great as to be readily noticed. There have, however, been no quantitative data published on this point. The apparatus used for these measurements is shown in Fig. i. It consists of a brass cylinder A, 2.5 inches in diameter and 15 inches high. At the bottom a small brass tube B, 0.25 inch in diameter leads to an upright 100 cc. glass stoppered burette, C. The cover D screws on to the cylinder by means of a very fine thread, which insures a water-tight joint. The cover is divided into 30 equal divisions on the edge K and a vertical mark is made on the cylinder, permitting the cover to be screwed down to a definite point or reading. In the center of the cover a hole is bored into which a pipe with outside threads £ is soldered. Another pipe with inside threads F fits over B. A glass tube G with a fine string around it is inserted into B through F and screws tight by means of F. This joint is also water tight, if the thread has been previously greased. The threads of cover D are also greased, anhydrous lanoline proving a good material for this purpose. • The glass tube G has a mark, to which the liquid A STUDY OF THE) CHANGES IN SKINS 15 used is allowed to ascend before a reading is taken. This is done by keeping burette C filled to a mark, higher than the top of the cylinder. The cylinder is lacquered on the inside and can be used for lime. FIG. 1. To obtain some idea of the accuracy of the measurements made, the following experiments on the volume of a glass stopper are given : Number of test I ll m iv V Zero reading in cc 25.45 58.9 20.70 59.85 57.65 Taken out in cc. 50.00 0.0 50.0 0.0 0.0 Total cc 75.45 58.9 70.70 59.85 57.65 Final reading 34.50 17.9 29.95 18.90 16.65 Volume in cc 40.95 41.00 40.75 40.95 41.00 The average volume was 40.93 cc. and the maximum error was 0.18 cc. in 40.93 cc. equaled 0.44 per cent. This error, however, was relatively small because the stopper could be dried and l6 A STUDY OF THi: CHANGES IN SKINS always brought to a certain condition of surface moisture, not a varying one, as was the case with skins. The removal of liquid which was made necessary whenever the volume exceeded about 50 cc. was best accomplished by means of a pipette. The liquid was drawn up into the burette after a measurement had been taken, by means of a rubber tube attached to the burette and to a suction pump. The procedure was as follows: Some of the liquid to be used was put into the cylinder A and the burette filled by applying suction. The stop cock was closed, the cylinder filled and the cover screwed down to a certain mark. The stop cock was then opened and the liquid allowed to run into the cylinder until the meniscus in the glass tube G was even with the mark. A reading of the burette gave the zero reading. The liquid was sucked back into the burette, the cover taken off, the sample placed in the cylinder and, if the volume was large, some of the liquid was pipetted off. The cylinder was then closed and a read- ing taken as before. This gave a final reading and from it and the zero reading the volume was easily calculated. The skin to be measured was always dried with a moist cotton cloth which was wrung as dry as possible. The skin was always measured in the same liquid in which it was at that stage of the experiment, that is, when in the limes, in lime water ; when in pickle, in pickle solution, etc. Duplicate determinations on pieces of skin whose volume was from 200 to 400 cc. agreed within about i cc. When dry skins were to be measured the cylinder was filled with water. The skin naturally absorbed some water, but if the experiment was performed rapidly, the error was not large, as was shown by the following experiment : Zero reading 56.75 Final reading 14.25 Volume 42.50 after i minute 42.65 after 2 minutes 42.85 after 3 minutes 42.70 after 4 minutes 42.95 after 5 minutes 43.00 after 10 minutes 43-1 after 15 minutes 43.3 after 20 minutes The change in volume due to water absorption is very small. A STUDY OF TPIE CHANGES IN SKINS 17 To test the absolute accuracy of the method this same piece of skin was dried and its volume remeasured. The agreement was within 0.5 cc. One difficulty to be guarded against in measuring dry skins is the tendency of air bubbles to adhere to the hairs. This can be avoided by shaking the whole apparatus just prior to taking measurements. Experimental Work on Voeume Changes of Calfskins. Nine sets of tests involving measurements of volume changes on twenty-one different pieces of skin are reported here. Micro- scopic sections from some of these sets have been referred to previously. The full data are given under the separate headings, but the sets may be outlined as follows : S-25 — A half-calfskin split along the backbone and trimmed, cut into three pieces of about equal size at right angles to the backbone. Carried through the tanning process. Changes in weight of the pieces were also noted and the density at each stage computed. S-26 — A half calfskin as above was carried through the whole process as one unit. Measurements of surface area and weight were included. S-27 — A piece of calfskin carried through the limes only. S-28 — Duplicate of 26. S-29 — A half calfskin was cut parallel to the backbone to make one piece back and the other flank. Carried through the limes. S-31 — Duplicate of 29, but carried only through soaking process. S-36 — A whole calfskin trimmed and cut into six pieces. Pro- tective action of lime soap tested. S-39 — Duplicate of 25. S-39B — Duplicate of 29. Experiment S-25. A dry salted calfskin was split along the backbone and one half was trimmed and cut into three pieces of about equal size at right angles to the backbone. The piece nearest the head was marked I, the middle piece II, and that nearest the tail III. These pieces were soaked in water three days, the water being changed every 12 hours. They were then placed in limes, made by using 5 l8 A STUDY O^ THE CHANGES IN SKINS grams of slaked lime to 400 cc. of water, a portion of old lime being added to inoculate the new lot. It was bated in a bran drench and pickled in salt and sulphuric acid solution made by taking 3 grams of concentrated sulphuric acid and 36 grams of salt in 850 cc. of water. The tannage was by Dennis's one-bath chrome method. In the following tables and curves the changes in volume and weight are calculated on the volume and weight of the skin after J2 hours soaking in water instead of on the dry skin. This follows the usual custom of basing all computations on the wet hide and allows comparison to be made with green hides. EXPERIMENT S-25.— Changes in Density. Condition of skin. Number I Number II Number III In water 72 hours 1.08 i.io 1.12 In lime 24 hours 1.09 i.io 1.13 In lime 48 hours 1.08 1.06 1.06 In lime 72 hours i .08 i .09 1.05 In lime 96 hours 1.05 1.05 1.03 In lime 120 hours 1.06 1.07 1.04 In lime 144 hours 1.07 1.08 i.io In lime 168 hours 1.07 1.08 1.09 In lime 192 hours 1.07 1.07 1.08 Unhaired and fleshed ... 1.07 1.05 Bated 1.04 1.03 Pickled 1.06 1.06 Tanned 1.06 1.07 Experiment S-26. A dry salted calfskin was trimmed and split along the back- bone. It was intended to carry a half -skin through the experi- ment as one piece but the apparatus for volume measurements was found to be too small so it was split into two pieces. The figures for the two pieces have been added together so that the result is reported as if it had been kept as one piece. Measure- ments are reported on volume, weight, area and density through- out the tanning process. These pieces were soaked for 24 hours in water, the latter being changed after 12 hours. The lime used was made by dissolving 5 grams of dry hydrated lime in 400 cc. of water. The pieces were placed in this solution for ten days. The lime was changed every 48 hours and a new lime of the same concentration was prepared. After liming until the hair slipped easily, the skins were placed in warm water for a few minutes, unhaired and fleshed. A STUDY OF TH^ CHANGES IN SKINS 19 fti ^ 'tt 6 vd rO «' CO ''J- TT d -^ Ml fl u be <^ a rO rO rO fO fO rO rO ro M N rO TJ- rO -^ 10 ? a; r^ fe VO vO rO U-) rt 00 ^ V CO Ol (N c< rO rO fO !? c ^ '"' '"' '"' '"' I I I I I I I I I I I u o O 10 M d vd ».■ I^vOvOCOnOO'^ d d ri .r^oN W N J M W lO CO OO" Ov s^t-^vo r;.d tir'-'^ id CO fO lO 00 CS lO w :?5 ^^ rO CO o t^ lO O fO to On CO O t^ o fS 8g ^ cIn n (N t-. o b> (^ M lO lO OO vO k^ l_l 7 T ro ro rj- lO •^ lO lO lO « >o Tt" •^ Cii w a. X 1- s^ . o lo O Tt- (N VO t^ lO 00 o o N o fO w o^ b.5 M VD o h^ h^ KO :tCOr><"0 0'*COf2.«i ■ Ih Wi GJ • ^^ (1) c c a c s w pk a A STUDY OF the: CHANGEIS IN SKINS 21 3 * ?.■" '^n'^*^^ ovono ooovo ion t^aN(?\ ^0"M.vovo »OQ0 ior^t>.t~^t^vo M o ooooooooooo«« a w o S.2 I ■D- >- cd ds •-<' -rj- GO 6 (N o t^ d rA lo rOTj-rf'^a-ioiOiOVOvOvO I I I ft M.5 lO o ? 3 >o »o T rO T 1 1 1 4^ be M -^10*0 a\ o ^H o I rOrO'^rOn rOVOvOOO "^ CO M O u I d "4 00 iOOnO t^ONO -^MvO rO So ^ ^ ^ Tt 00 W fO O •^ lO lO lO O " M O r^ 00 00 « lO >o »o ^ rO t>. »0 O^ vO VO "^ »0 t^ ^ ^ VO vO rf lO t^ ^- 00 rO TT >? fO m rO ,2 (A c« en cr tn en tn ^ 3 3 3 3 3 3 «C 3333000000—, o o o o^^^'a^rt^ '~'^'^ O rJ-00 (NVO O rt ■^00 fSvO CS Tj-vo G\ <-* rJ-_ •j: -^ -^ -^ -jz -^ -^ -^ -^s -^ XI i; -i4 22 A STUDY OF THE CHANGES IN SKINS EXPERIMENT 25.— Density. //£: III :^ I-, I T W^- ^ I^ ■r .SiS li e^ <0 /^7 ^1 /^5 lOi \iL ^i ji/ m / — T-J SoaA *r- 2 j ^ ^' ^' ^ d '■^.r ^T- £/ay. EXPERIMENT 25.— Weight. Per cent, increase on third soaked weight. A STUDY OF the; CHANGES IN SKINS 23 EXPERIMENT 25.— Volume. Per cent, increase on third soaked volume. EXPERIMENT 26.— Volume. Per cent, increase on 24-hour soaked volume. 24 A STUDY O^ THE CHANGE:S IN SKINS EXPERIMENT 26.— Weight. Per cent, increase on 24-hour soaked weight. EXPERIMENT 26.— Density. /./5 IJ 2 /.lO /.09 /.oa /.07 lot U'5 VrxrzXXZ^ w^BL l05-^x;ixL /02^ 101 A STUDY OF the: CHANGES IN SKINS 2$ The samples were bated three hours in an N/io sohition of lactic acid, in a drum. They were pickled about five hours in an N/50 solution of salt and sulphuric acid. They were then placed in a drum with a normal salt solution for one hour and chrome-tanned by the one-bath process. Sections were also cut daily during this series, as has been mentioned in the discussion of the microscopic study. The results are given in the accom- panying table and curves. Experiment S-27. In this test a piece of dry salted calfskin was used. It was soaked in water 24 hours. The water was changed after 12 hours. A lime solution containing 5 grams of slaked lime in 400 cc. of water was used to unhair the skin. The hide was in the limes seven days. Sections and volume measurements were made daily and the results tabulated. EXPERIMENT S-27 Changes in Volume, Weight and Density. Condition of the skin Dry sailed Volume in cc. 83.3 Increase in cc. — 154.6 Per cent, increase —65.0 Weight ing. 92 ■ Increase Per cent, in g. increa.se -169 —64.7 Density 1. 10 In water 24 hours- 237-9 0.0 0.0 26 r 0.0 0.0 1. 10 In lime 24 hours. . In lime 48 hours. . In lime 72 hours. . 335-6 356.2 354-1 97-7 118.2 116.2 41. 1 49.8 48.9 380 374 365 119 113 104 45-6 43-3 39-9 I. 12 1.05 1.03 In lime 144 hours. In lime 168 hours. 342.4 343-6 104.5 105.7 44.0 44-5 360 357 99 96 37-9 36.8 1.05 1.04 Experiment S-28. In this experiment a piece of dry salted calfskin was used. The piece of skin was soaked in water 24 hours, the latter having been changed after 12 hours. The limes used were of the same concentration as in Experiment 26. The pieces of hide were limed 6 days. Volume, weight and area measurements were made daily. The skins were limed, unhaired and fleshed, bated in an N/io solution of lactic acid and followed by a bran in- fusion. They were pickled in an N/50 solution of sulphuric acid and salt and tanned by the method given in the previous experi- ment. The product was fair. Analysis showed 5.6 per cent, ash while a standard sample had 5.0 per cent. ash. The measure- ments taken are in the following table. The increase and per- centage increase in weight and volume are all computed on the volume and weight after 24 hours soaking in water. 26 A STUDY OF THE CHANGES IN SKINS EXPERIMENT 27. Per cent, increase on 24-hour soaked volume and weight. SC "^ TV- y r=; r— "■ 4C ; r''^ - :-.• ■■ ::-•".(- itff 30 rrrr i — ^ " _rrr. ^1*^^ "n^1 Ji 20 m- f ^ ^ -rrr _; '' 10 ^^j r -—J —— --- •^-r -~ ' "Ti 1 :''i : id 00 1-* d\ M 1-^ -^ lO 6 M On lO On r-^ 00 CTn Ov go' -4 tJ- CO Ov rl t^ t^ v£) lO lO 0< O.S t-»iO i-ivOOOGO C^CT^OO t^t^O W2 M O o6vdvdvd u Z, ^ >-i ro to CO I 1 I I ^ I K Sfi M Tt H^ 00 o M lO (N Tf 00 o o r^ lO ^ VO ^ 00 00 <0 fO fO fO <0 rO J? ^ ^u rO O o to r^ On -ii-.J-.»-.3pj33 0033330000 ^ -^00 rf CS VO M ■ - r^ ON M (S !• 1> D 4) D D (U rt rt g 5 S S 6 ? ^ "^ '^ '^ "^ v^ c fl c s c c c oj (U a; S 5 £ A STUDY OF THE CHANGES IN SKINS 37 fcy 1 ' 1 S-2 I lo i-i q CO 00 CO os »o q vq vq ro ■^dvd to—' cJ »ovo c^^ovo I t^CO ONO OnvO t^iO^ I > -r^— — ONOOtN'-'COOt^ W O u I H z W ^. >— » Pi w a. X W lO »0 "^ rO ^ Ji? P5 q qs q d t^0 CJN t^ t^ *q (N rO 00 d d vd I goM rOrJ-ior-.aNfOM t-H q £!irO^ dcd rs rh 0\ ^ >-* 1^ f q rf O »0 00 -^ "^ "? d ^» OO" rO ^ '-' ^' ^' lo lo ^ ^ r-« lo vD »- en (« c/) t« i; rO o >o o >o 00 o fo . 2rirr)dvr^f^!ovy jar, over hydrogen. L,it- Check for Check mu.s gluco.se geliitine Anaerobes Aerobes B. tetanus B. lactici — + + + + + — + + + + + — + -[- + + + A STUDY OF THE CHANGES IN SKINS 47 the solution 24 hours. They were then removed with sterile for- ceps and washed three times by immersion, using a liter of sterile water each time. Then they were planted by means of sterile forceps into 150 cc. of sterile medium, beef tea, gelatine, agar, pea-bean medium, and litmus glucose gelatine being used. The results are shown in the table. Seymour-Jones Test. No. Beef tea Gelatine Pea-bean Agar Although these solutions all showed negative results it is neces- sary, before admitting the test to be conclusive, to show that the amount of mercuric chloride transferred could not have been sufficient to have inhibited the growth of bacteria in the culture media. Mercuric chloride acts as an antiseptic agent even in dilutions of i to 100,000. The samples after washing thoroughly in 3 liters of sterile water had very little mercuric chloride ad- hering. Moreover, the volume of medium used was large, 150 cc. or more in all cases. This again increases the dilution of the mercuric chloride and hence eliminates its antiseptic action. The dilution at which formic acid still exerts its antiseptic action is not known definitely. The fact was taken into consid- eration, however, that the antiseptic action of formic acid is usually less than that of mercuric chloride. If one assumes the piece of hide transferred, to be all formic acid, and then takes into consideration the dilution resulting from the washing in sterile water, one will see that the dilution is more than i to 200,000 and that there is no chance of any antiseptic action inter- fering with the results. In order to check whether the medium was suitable for growing bacteria, bacillus tetanus and bacillus acidi lactici were planted under anaerobic and aerobic conditions respectively. The results were positive in all cases. Since the checks were always positive and the sub-cultures 48 A STUDY OF THE CHANGES IN SKINS from the sterilized hides negative, it was therefore considered proven that the Seymour-Jones method of steriHzation is ef- fective under the given conditions of experiment. Tkst For Bacteria in the: Limes. Since there might be some question as to the presence of bacteria in the hmes, sub-cultures were made on agar, beef tea, pea-bean media and gelatine. All sub-cultures gave positive re- sults. Wood-'* has shown that bacteria of various kinds are present in the limes. Abt^^ has also proven that bacteria are present in the liming process. The medium used in transplanting sub-cultures in all the fol- lowing experiments was slightly more alkaline than that usually employed for this purpose. Expkrimentai, Work — Laboratory Tests on Depilation. The procedure now used was as follows: A 250 cc. Soxhlet flask, provided with a tight cotton plug was sterilized for i minute at 200° C. in a dry heat sterilizer. It was cooled and 175 cc. of water and 50 grams of slaked lime were added. The flask was then autoclaved at 110° C. for 20 minutes and allowed to cool. A piece of hide which had been previously sterilized in a solu- tion (Seymour-Jones) of 1-5,000 mercuric chloride and 0.5 per cent, formic acid, for 24 hours, was added to this solution by means of sterile forceps. This flask was then examined every 2 or 3 days, great care being taken to prevent infection of its con- tents. After II days the hair could be removed with difficulty, but after 13 days the hide could be easily unhaired. In order to prove conclusively that bacteria were absent, sub- cultures of the- lime solution and of hair were made on various media under aerobic and anaerobic conditions. That the med- ium used was suitable for the growth of bacteria was proven by planting test organisms. For this purpose bacillus acidi lacHci and bacillus tetanus were used under aerobic and anaerobic con- ditions, respectively. The results obtained are shown in the fol- lowing table : ^V- ^oc. Chein. Itid., Vol. 29, p. 666. (1910). 2^ Bull. SyAdicat. Gen. Cuirs et Peau.v, p. 416, Nov. 10, 1908. A STUDY OF THE CHANGES IN SKINS 49 Check Test on Sterility of Conditions. Under anaerobic conditions in a Novy j.ir previously exhausted and hydrogen passed in. Beef tea Gelatine Pea-bean lyitmus glucose gelatine I loop of lime solution . — — — — Hair — — — — Checks + + + 4- These results are very satisfactory and show, since all the sub- cultures were negative and the checks positive, that no bacteria capable of growing were present in the limes used. After it had been shown that the Seymour-Jones method of sterilization with mercuric chloride and formic acid was reliable and preliminary tests had shown that it was possible to unhair a skin with sterile limes, the following series of tests was under- taken to study the process more quantitatively. Four different solutions were used to determine how lime alone, and lime with sulphur compounds acted. One flask contained lime only, a sec- ond lime and red arsenic sulphide, a third, lime, red arsenic sul- phide and hair, and the fourth lime and hair. The solutions con- taining hair were boiled vigorously for 45 minutes, before use, with the idea that some hydrolysis of the hair would take place with formation of soluble sulphur compounds and amino acids and that thus the action of an old lime might be simulated. The details of the tests are as follows : Four pieces of dried calfskin 2x3 inches in area were soaked 24 hours in a solution of 0.02 per cent, mercuric chloride and 0.5 per cent, formic acid. Four 250 cc. Soxhlet flasks were plugged with cotton and sterilized for i minute at 200° C. in a dry heat sterilizer. To one of these flasks 50 grams of lime and 175-200 cc. of water were added. To another sterile flask 50 grams of lime, 175-200 cc. of water and 1.5 grams of red arsenic sulphide were added. Then the flasks which were to contain boiled hair were prepared as follows : 200 cc. of water, 50 grams of lime and 10-12 grams of hair clipped from a calfskin, were boiled vigorously for 45 minutes. This solution was placed in a sterile flask. Then 200 cc. of water, 50 grams of lime, 1.5 grams of red arsenic sulphide and 10-12 grams of hair were boiled 45 minutes and put into the fourth sterile flask. Then the four flasks were autoclaved for 20 minutes at 110° C. A piece of 50 A STUDY OF THE CHANGES IN SKINS a o en TJ rt O ?z ^. D O 5S (L) (J o 5 . I « en ^_< O O •- r^
  • -c rt u 3J tn g z: « '" ^ S c O '-n en .-^s 0.0; ID w tn >^ CO ^ ^ S JW cn P^ In u u w w 3 >. fe O rt O s^"^-^. n-j en C O o rt .52 .5 ^O C n S . •So- S iS -, o ^ w o ^y^ •— • OjO 4) CO 7l C ?i tnS ii In « a i^ n O 3 3 Oh u *c3 CO r^ ^ tn-^ Jn ■ «-> ^ CO nS ,^ .-' (U (U fli S 3 i*. Si) 13 cS <« - -^ S ^ . :;: o tn O IT .o « 1_ (U O rr-l O tn 13 frt a O 1^ « Hair sterilized 3 days. No change. No odor. No change. Same. Slight odor of hydro- gen sulphide. Same. About thesame. Slight odor of ammonia and hydrogen sulphide. o ^ >. 2; Xo change, no odor. Hair tight, can be pulled; slight odor. Hair still tight, not much if any odor. Hair slips fairly easy, Corium hard like Number III. Flesh side somewhat swol- len. Very little odor if any. Hair slips very easily. Upper lay er attacked , it can be scraped off. No odor. Same. Same, corium good. O D Q 1 > 5 la mour-Jones solution. No change, no odor. No change, no odor. May be slight odor, hard to tell. Hair can be scraped easily. Corium good and harder than Num- bers I and II. Upper layer not soft like I and II. No odor. Same. Corium firm, same. No odor. Up- per layer still good. Slightlyattacked corium good and fi rm . Q 11 \ § 'S 3 CS u a hours sterilization in Sey- Hair slips very little when pulled strongly. Same. Hair slips more easily. Slight odor of hydrogen sulphide. Hair slips very easily; much of it dissolved. Epidermis attacked; slight hydrogen sul- phide odor. Hair nearly all gone, up- per layer mushy. Co- rium good, odor of hydrogen sulphide. Not much odor of hydro- gen sulphide. Upper layer more attacked than Number I. Patches of it gone. About same. No odor, same. Upper layer al- most all gone. No odor. Corium good. Not so firm. w 1 ;2 > d Into all solutions after 24 No change. ii ii i( li Hair slips some, that is, comes off on scraping. Hair can be scraped easily. Very slight odor epidermis attacked. Not much odor, may be slight hydrogen sul- phide. Upper layer badly aitacked. Hair all gone. Corium still hard and firm. Upper layer badly at- tacked. Corium firm and hard. Very little odor. About same. No odor, same. Upper layer very mushy. No odor corium firm. Flesh side swol- len. ^ 1 54 A STUDY OF THE CHANGES IN SKINS II o > o S CO 3 «^ p2 W H (/3 O M Q c Q W 9 ^ S!: a S a 8&^ (U CO'" " w. O .i; nJ.22 en o '^^ 2 a CO e c ^ §•53 §5;o O eo CO tn en CO o o a; . C to O 3 o) ^^^ a §1 O 3 en O , en 'O (U O en g (U 'O en !Z! o tn C 'O '"^ h3 <1> CO O CO'rt ^ CL, "* r< V- CO . ° s=: « ^ <^ >^ O 3 en ^ Q Oj Spool's a .It % ^ a o 3 Ul ^ « in ffi is ID kH O • •" ,^ B p o CO ^ . iJ". 5i d iH* J^ - CO o &, bc73 o ^" o ^ o " o tuOTj u; v^ en t! 3 *J M) o ^ M3 bicc:: beg •I "C C S .1 3 V. ^ .^ a; en O O 2 ^ •ko ^.'^ 3i^ ir Iff ©as !«)>.eS >. en CO en c O CO'en >.a ^^ 3 o ° acj° biO -^ .S c ^- +j (D 1) 4^ ^ "^ ^«P 2 ^ en g) en M bx;0 _ o o Su o o O W.* g'S *en U Is >^>. fc65 %l 'en W3 _j '^'2 2^ >.n a en a o T3 r^ o ^ CO b£0 aJ U - » o en ^-^ CO b£0 ^U5 A STUDY OF THE CHANGES IN SKINS 55 EXPERIMENT 40.— Check on Steruty of Solutions After Ten Weeks. Number of flask I n m iv Gelatine plates — — — _ Planted 2-12 examined Gelatine tubes — — — — Daily from 2-12 to 2-24 Beef tea tubes — — — — Pea-bean tubes — — — — Checks 4. -f- -f- -|- Anaerobic conditions. Lit- mus glucose gelatine in Planted 2-12 examined Novy jar over hydrogen — — — — 2-26 Checks -f -f -(- -(- EXPERIMENT 40.— Finai, Check on Sterility of Solutions. Number of flask I II m iv Gelatine plates — — — — Planted 3-9 examined Gelatine tubes _ _ _ — Daily after 3-10 to 3-20 Beef tea tubes — — — — Pea-bean tubes — — — — Checks + -j- -J- -|- Anaerobic conditions on lit- mus glucose in a Novy Planted 3-9 jar over hydrogen — — — — Examined 3-23 Checks -^ -^ _(_ -f Experiments 41. As a final check on the previous results the following tests were made: Two flasks were plugged with cotton, steriHzed by- dry heat for i minute at 200° C. To each flask 100 cc. of water and 10 grams of lime were added. They were then autoclaved 20 minutes at 110° C. Two pieces of calfskin 1x2 inches were sterilized 48 hours in a Seymour-Jones solution. The sterile hides were placed in the flask October 30, 1913. The flasks were left undisturbed at room temperature until March 9, 1914, when they were opened and sub-cultures made as in previous experi- ments. All sub-cultures were negative but the checks were posi- tive. The flask, therefore, contained a sterile solution. The skin had changed considerably in its 4 months treatment in sterile lime. The hair was all very loose; the upper layer and the flesh side of the skin had become mushy and swollen. The corium was fairly firm but glassy in appearance. No odor was noticeable. The preceding experiments had all been made on calfskin. In order to check the results on heavier hides a sample of sterilized cowhide was placed in a sterile lime solution Decem- 56 A STUDY OF THE CHANGES IN SKINS -ber 12, 1913. This contained 5 grams of lime and 0.5 gram of sodium sulphide in 100 cc. of water. The hide was examined March 9, 1914. The flask had been left undisturbed during this time. Sub-cultures were made as before, and all results were negative. This showed that the solution was sterile. This hide had also changed materially by its 3 months' treat- ment in sterile lime. All the hair was dissolved. The upper layer had been dissolved and the flesh side' had not only become mushy as in the previous experiment, but in fact it had disappeared, leaving only a firm tough layer of corium, which had apparently suffered very little change. Depilation IN Sterile Solution and Subsequent Tannage. After these preliminary tests of liming under sterile conditions, somewhat larger pieces of hide were used and carried through the entire tanning process. The final products were then com- pared with commercial products obtained by liming in the usual manner. The first experiments were on pieces of cowhide which were tanned subsequently, through the courtesy of Mr. V. A. Wallin, in the Wallin tanneries at Grand Rapids. Experiment 42. Experiments on Cowhides. Two pieces of cowhide about i x 10 feet were sterilized 3 days in Seymour-Jones's solution. The bottles in which the previous solutions had been sterilized had been made of cheap cast glass but had caused much trouble by breaking during ster- ilization, in spite of the utmost precautions taken while heating and cooling them. This difficulty was overcome by using narrow and deep galvanized iron cans 5 centimeters by 42 centimeters by 46 centimeters. Small shelves were arranged so the hides could be suspended over glass rods. The solutions to be used were ster- ihzed in these cans by boiling over a direct flame for 4 to 8 hours. The cover, not fitting tightly, allowed a cloth moistened with 0.02 per cent, mercuric chloride solution to be placed over the can in such a manner that it could be kept sterile. Hide No. i, was limed in a solution containing 5 grams of lime and o.^ gram of sodium sulphide crystals, in 400 cc. of A STUDY OF THE CHANGES IN SKINS 57 water, for 3 days. It was removed while slightly underlimed and placed in a can containing a sterile saturated lime solution and shipped to the Wallin Tannery, Grand Rapids, Michigan, to be put through their regular tanning process. Hide No. 2, was limed in the same kind of a solution as No. i but for 6 days. It was removed and shipped in a sterile saturated lime solution. Sub-cultures made as in previous experiments gave negative results and showed that these hides had been un- haired in sterile solution. The finished sample of leather from hide No. i was returned from the tannery with the comment : **The job seems to be satisfactory. The stock is a little bit snappy on the grain but it is not certain that this has any rela- tion to the liming." The finished sample of leather from hide No. 2 has not yet been returned from the tannery. Experiment S-43. Two pieces of cowhide were received wet and salted. They were washed thoroughly in water and sterilized in Seymour-Jones solution for 48 hours. The limes used were made up by dissolv- ing 5 grams of lime and 0.5 gram of sodium sulphide in 400 cc. of water. They were sterilized by boiling 4 to 8 hours over a free flame. The hides were now treated as follows : One piece was limed 4 days, then placed in a sterile saturated lime solution and shipped to the tannery. The other piece was purposely over- limed and after 7 days in the lime was sent to the tannery in the same way as was the other piece. Sub-cultures from the limes used, were made on various media in the same manner as in previous experiments. All sub-cultures were negative and showed that the solutions had been sterile. The loss of hide substance in the liming process is often con- siderable and an examination was made of some of these lime solutions to determine the amount of hide substance dissolved. For comparison an old lime from the Wallin tannery was also tested. The hide substance in solution was calculated from the content of ammonia shown by the Kjeldahl method. The results are shown in the following table : 58 A STUDY OF THE CHANGES IN SKINS Hide Substance Dissoi^ved in Limes. Ammonia Hide snbstance vSource of lime g. per 1. g. per 1. Remarks Experiment 42 0.1366 0.623 Hide 3 days in Hide #r. 0.1326 sterile lime. Experiment 42 0.3145 i .457 Hide 6 days in Hide ^2. 0.3155 sterile lime. Experiment 43 0.41 14 1909 Hide 4 days in Hide #1. 0.4138 sterile lime. Experiment 43 0.9479 4-376 Hide 7 days in Hide #2. 0.9452 sterile lime. Old lime from Wallin tan 1-439 6.694 1.454 The amount of hide substance dissolved by these sterile limes is less than that shown in the old lime from the tannery but not enough is known of the changes of limes with continued use to warrant a positive conclusion. Experiments on Calfskins. Calfskins received in the dry salted state were unhaired in sterile limes and through the courtesy of Mr. Carl E. Schmidt, tanned by the chrome process in his Detroit tannery. Considerable difficulty had been experienced in handling the can used to contain the sterile limes in the previous experiments, and spots appeared on the hides where they touched the metal. The calfskins were limp enough so that glass vessels could be used. Experiment S-41A. A bottle of about 6 liters capacity was plugged with cotton and sterilized at 200° C. for i minute in a dry heat sterilized. One- half of a small calfskin, weighing 250 grams was sterilized 24 hours in a Seymour-Jones solution. A lime solution containing 5 grams of lime and 0.15 gram of red arsenic sulphide in 400 cc. of water was put into the sterile bottle and this was autoclaved at 110° C. for 20 minutes. The sterile skin was now placed in the sterile lime and left for 9 days. The hair slipped very easily. This skin was sent to the Carl E. Schmidt Tannery at Detroit, Michigan, where it was chrome tanned and finished. The product was of little value. The grain could be peeled off easily. The leather had very little strength and felt very thin. The liming had been allQwed to proceed too long and the skin had become seriously damaged. The grain was also drawn and harsh. A STUDY OF the; CHANGES IN SKINS 59 In subsequent tests on calfskin a large Jena flask of about 15 liters capacity was plugged with sterile cotton. The flask was too large to permit sterilization in a dry heat sterilizer hence it was only washed with distilled water. The cotton plug, after it was made to fit the flask was sterilized for 5 minutes at 200° C. A lime solution was made containing 5 grams of lime and 0.15 gram of red arsenic sulphide per 400 cc. of water, and put into the flask. This was then autoclaved 4 to 5 hours at 110° to 120° C. Experiment 44. One-half of a small calfskin was sterilized 48 hours in Sey- mour-Jones's solution. It was then placed in the sterile lime solution in the sterile flask and allowed to remain 5 days. The hair was partially destroyed and slipped easily, except in certain spots. These spots were very difficult to unhair for some reason. The grain of this leather cracked and scuffed easily and was not satisfactory. Experiment 45. The flask used in Experiment 44 containing the same lime and arsenic sulphide solution, was autoclaved 4 to 5 hours at 110° to 120° C, to insure sterility of the contents. One-half of a dry salted calfskin which had been sterilized for 24 hours in a Sey- mour-Jones solution, was added to the cooled flask with sterile forceps. This skin was limed 6 days. Sub-cultures made pre- cisely as in previous experiments showed negative results and that the limes had been sterile. The skin unhaired easily except in one place an area of about 6 square inches. The white hair on this spot adhered with remarkable tenacity while the black hair on the rest of the skin slipped easily. No explanation of this peculiar occurrence could be suggested at the tannery. The hide felt "full" and the final product was of fair quality. The leather had average tensile strength and a good grain although it did not feel as full as the standard product of the tannery. Stretching did not crack the grain of the final product except on the extreme flank. Experiment 46. To the same lime solution in the flask of Experiment 45 9 grams of red arsenic sulphide, 300 grams of slaked lime and 6o A STUDY OF the: CHANGES IN SKINS enough water were added to bring the contents up to the original vokime. This flask was again autoclaved 4 to 5 hours at 110° to 120° C. to insure steriHty of the contents. The piece of calfskin to be used was sterilized as before in a Seymour-Jones solution for 24 hours. The hair slipped easily after 6 days. Sub-cultures made from this lime solution, on various media as in previous experiments, showed negative results and proved the solution to have been sterile. The final leather felt quite "full" in the judg- ment of the tannery superintendent. It had a good grain and a tensile strength greater than that of the average skin limed in the ordinary manner. It had a very slight harshness which could probably be overcome by modification of the finishing process. The leather felt very full both in the flank and at the backbone. The flank appeared to be better than in the ordinary product. Conclusions. The foregoing paper studies from three different viewpoints the changes taking place in hides during their conversion into leather and particularly during the liming process. A study is made of structural changes throughout the vegetable and mineral tanning processes as shown by the microscope; of gross changes in volume, weight and density of the skin; and of the practi- cability of carrying out the depilation process in sterile solutions. Detailed methods have been worked out for the satisfactory preparation of microscopic sections both by the colloidin and freezing methods. A study of numerous sections of skin shows that the structural changes occurring during depilation and tan- nage are so gradual that only the broad outline can be followed. The inter-fibrillar substance in the bundles of connective tissue dissolves in the liming process and the fiber bundles split up into their component fibrils. The flank is composed of larger, fewer and more irregular fiber bundles with larger interstitial spaces than the better portions of the skin. This gives a partial explan- ation of the poorer quality of flank leather. When dry calfskins are put into water they increase in super- ficial area, thickness and weight but decrease in density. The area remains almost constant during the liming process in bacter- ial limes, but the volume and hence the thickness of the skin in- creases quite consistently and at a decreasing rate during the I A STUDY OF THE CHANGES IN SKINS 6 1 liming process. The weight increases at approximately the same rate but a study of the relationship of weight and volume as shown by the density curves, indicates that the volume increases faster than the weight during the first 4 or 5 days in limes con- taining bacteria so that at the end of this period the hide shows the minimum density which it ever attains in the limes. Within 2 days after this point is reached the volume decreases more rapidly than the weight and the density rises decidedly. The sig- nificance of these points of inflection of the curves is not evident. ' Both volume and weight decrease in the feebly acid bate used but the decrease in weight is greater than that of the volume so that the density falls. In the pickle, conditions are the reverse of those in the bate and the density rises sharply. No great changes in weight volume or density occur during the one bath tannage used. Different pieces of the same skin while the same in general, show decided quantitative variations from each other. The shoulder, back and rump show distinct differences which are not constant in different skins. The flank, however, swells quite con- sistently more than the rest of the skin, both in water and in the limes. These differences may be due to the varying thickness of the skin or to surface conditions such as fat. If the flesh side is painted with a soap solution before immersion in the limes so that an insoluble lime soap is precipitated upon it, the swelling is greatly increased. It has been shown that it is possible to depilate a skin or hide under strictly sterile conditions with lime alone or with the addi- tion of sulphides. The same sterile lime solution can be used to depilate successive pieces of hide. Calfskin kept for 6 months in sterile milk of lime shows a firm though rather glassy corium. A skin kept a similar length of time in sterile milk of Hme con- taining arsenic or sodium sulphide, is completely dissolved. The hair from the skins in the latter solutions is also dissolved com- pletely, while that of the skin in lime alone, appears almost un- changed. Pieces of cowhide and calfskin unhaired under sterile condi- tions have been tanned and finished in commercial tanneries using vegetable and mineral tanning agents, with fair results. It seems entirely probably that with a little more experience in handhng 62 A STUDY OF THE CHANGES IN SKINS Sterile limes a good product, equal in all respects to that produced by the present methods of liming could be obtained. BIBLIOGRAPHY. 1. Jettmar, Handbuch der Chromgerbung (1900). 2. Der Gerber. 3. Procter, The Principles of Leather Manufacture. 4. Procter, The Leather Industries Laboratory Book. 5. Collegium. 6. Bulletin de la Societe d'encouragement pour I'industrie. Nationale. 7. Dinglers Poly technische Journal. 8. Hyde, Diseases of the Skin (1909). 9. JouRNAi, of the American Leather Chemists Association. 10. Warthin, Practical Pathology. 11. Journal of the Society of Chemical Industry. 12. English Patent Number 2873. 13. Villon, Traite de la Fabrication des cuirs. 14. Leather Trades' Review. 15. Bull. Syndicat. Gen. Cuirs et Peaux, p. 416, Nov. 10, 1908. LIBRARY OF CONGRESS 016 055 435 6