By : Michael Titford, Pathology Department, University of South Alabama
The aniline dye industry was created in 1856 when the En- glishman William Perkin prepared the color mauve from ani- line. Dye industries were then formed in several European countries, and each year thousands of new colors and shades were tested on fabrics. Scientists of that period used these new dyes to study the structures of plant and animal tissues. Start- ing in 1880 George Grubler, a German pharmacist, sold bio- logical stains to scientists around the world. Grubler stains earned a reputation for reliability, and many of the staining methods developed at that time stipulated the use of Grubler's dyes, even after the firm was sold to Karl Hollborn who con- tinued Grubler's work and devised new staining methods. The Grubler company continued to prosper until World War I. The Chroma Gesellschaft-Schmid GMBH Company in Germany is a successor of the Grubler company.
The interrupted supply of Grubler stains during World War I prompted the start of the American biological stain industry and subsequently the Biological Stain Commission was formed. (The J Histotechnol 16:155, 1993)
Key words: aniline dyes, Grubler, Hollborn
Just over 100 years have elapsed since the beginning of the biological stain industry. Today, the use of high quality dyes is commonplace in laboratories worldwide. These dyes are commercially available and give standardized results when modern techniques are employed. As simple as this sounds, none of this was possible 100 years ago. In the late 19th cen- tury, investigators who desired optimum staining results pur- chased their stains from the Grubler Company in Leipzig, Germany. Grubler's standards and quality assurance con- vinced scientists of the day that they were getting the best products available in the world.
Today's techniques for the staining of histological sections are the culmination of a series of progressive steps over the past 250 years. Beginning with scientists such as Anton Van Leeuwenhoek (who in 1719 described a method using alco- holic saffron to stain muscle tissue), one begins to appreciate the early efforts to stain biological tissues with a variety of natural coloring agents (1). The subsequent development of the compound microscope in the 1800s hastened the prog- ress and resulted in better techniques of staining tissues sub- mitted for microscopical examination (1). Finally, develop- ment of high power lenses, better knowledge of light optics, and increased resolution of microscopes made it possible for scientists to distinguish cellular details.
Beginning of the Aniline Dye Industry
Before the German dye industry was established, the ani- line dye industry was already a reality in England, where coal gas had been used as a fuel for lighting. The previously unused byproduct of coal gas production, coal tar, was found to be rich in aniline derivatives, and in 1856, William Perkin prepared the color mauve from aniline. Many dyes and other substances were subsequently prepared from coal tar. It was in Germany, however, that the coal tar industry de- veloped to the greatest extent (2). Firms whose names we now associate with pharmaceuticals, such as Hoechst, Bayer, Sandoz and Geigy, developed and tested thousands of new dyes annually (2). Unparalleled growth in industry and sci- entific research resulted. The main use of the dyes then, as now, was in staining fabrics in the textile industry. Such cell biologists as Schmorl, Unna, and Ehrlich searched the rap- idly expanding textile industry for suitable dyes to use in their research (3). Basic fuchsin was made in 1865, aniline blue in 1868, and violet rosaniline in 1874 (1). These dyes were not always suitable for staining biological tissues, as the needs of the textile industry and biologists of the day were quite different; however, it was a beginning.
The Grubler Company
It was against this background that the pharmacist George Grubler started his biological stain company. This single company set the standard in the industry for over 25 years. To appreciate fully the advances made in the field of staining biological specimens, we begin by taking a look at the life of the man behind the industry. George Grubler was born in Meerane, Saxony in 1850, the son of a pharmacist. His education and early career were not unusual, as they followed the usual steps for a pharmacist in Germany at that time. He studied pharmacy at the Univer- sity of Leipzig and graduated during the winter of 1873/74. After further studies, he left the university in 1875 to work as a pharmacist at the Physiological Institute (1878-1880). During this period he submitted a doctoral thesis, "Concern- ing a Crystalline Albumin of Pumpkin Seeds," for which he was awarded a Doctor of Philosophy degree (4).
In 1880, Grubler started a private physiological chemistry laboratory in Leipzig. His primary goal was to provide qual- ity dyes for biological research. It is uncertain who sug- gested this idea to Grubler. Conn maintains that the patholo- gist Carl Weigert recommended the idea, whereas Tutzke credits Grubler's university professors Dreschler and Lud- wig as the inspiration (l, 4). In any case, the laboratory prospered. By 1895, its name was changed to "Physiological Chemistry and Microscopical Chemistry Laboratory" to reflect the increasing involvement of the company with microscopists. In addition to dyes, it sold other items of use to biologists, ie, injection media and glycerin-ether for the differentiation in the Ziehl Neelsen method (5, 6). In 1897 a second concern, a shipping company, was begun to handle the mail order side of the business. This was named "Dr. G. Grubler & Company, Dyes, Chemical Preparations for Microscopy and Bacteriology." The parent Grubler com- pany obtained the dyes from the textile industry and tested them for adequacy in biological staining. Large lots of ac- ceptable batches were purchased, repacked, labeled, and shipped by the second company (4).
Karl Hollborn Purchases Company
Later the same year, Grubler became ill and sold the com- panies to Karl Hollborn. Grubler moved to Dresden and died in Jena in 1915 (4). Fortunately, Karl Hollborn was equally well qualified to run the company, although his interests were different from Grubler's.
Hollborn was born in Hildesheim in 1862. Upon leaving school, he was apprenticed to a pharmacist in Hildesheim. He undertook further studies to become a pharmacist's assis- tant and then attended the University of Leipzig to study pharmacy, graduating in 1888/1889. After working as a phar- macist for a period, he attended the University of Rostock where he completed a doctoral dissertation, "Concerning the Effects of Thionyl Chloride on Several Amino Compounds of the Aromatic Series." In 1894, he was awarded a Doctor of Philosophy degree (4). He worked for a short period in agricultural research and then joined Grubler's laboratory in 1896. He purchased the company in 1897 and changed its name to "Dr. G. Grubler's Microscopical Chemistry and Bacteriological Laboratory (owner, Dr. Karl Hollborn)" (4). Grubler and Hollborn's company stands apart from other commercial suppliers of biological dyes at the time (there were at least seven). The consistency of the dyes that they sold was achieved by expert testing of dye lots obtained from the textile industry. Using "secret" empirical methods, they evaluated dye lots and purchased those suitable for biological staining in large quantities for resale. The German textile in- dustry at that time prepared dyes with varying amounts of different impurities. These impurities could even cause changes in the molecular structure of the dye molecule, which, while having no effect on the staining of fabrics, would have produced variable reactions on tissue sections and smears. The quality control concept of Grubler and Hollborn was a first for the fledgling biological stain indus- try. Consequently, by using these dyes, researchers were able to avoid the costly and time-consuming procedures neces- sary for the evaluation of these new aniline dyes in their own laboratories.
As stated earlier, Grubler and Hollborn had different in- terests. Grubler had concentrated on identifying suitable dye lots and preparing simple stain solutions from them to sell; Hollborn expanded the company and its focus. By working with German scientists of the time, he developed new stain- ing methods. Double stain methods were becoming popular. With Giemsa, Hollborn developed a methylene blue-azure stain whose polychrome properties made it suitable for de- tecting malaria and protozoa. Richard Schaudinn's statement that Grubler's Giemsa dye was successful in the staining of spirochetes further contributed to the commercial success of that product (4). With Paul Unna, Hollborn developed an- other polychrome methylene blue solution, and on his own published a picro-carmine-aniline blue method (4). In the small world of histological staining of that time, the reputa- tion of Grubler-Hollborn dyes (hereafter called Grubler dyes to prevent confusion) advanced as dyes and staining solu- tions were sold to laboratories around the world. Endorse- ments were common. Van Gieson stipulated Grubler's acid fuchsin for his method in 1889 (6). In America, Mallory and Wright wrote in their 1901 edition of Pathological Tech- nique,"Aniline dyes it is extremely important that all ani- line dyes used in histological work be obtained, with possi- bly few exceptions, from Grubler, either directly, or from his authorized agents. In no other way is it possible to obtain with certainty, the results expected" (7). Coplin wrote in his Manual of Pathology in 1911, "One of the great difficul- ties with aniline dyes is the inconsistency of their composi- tion and the unreliability of the many samples placed for sale. For this reason it is recommended that in purchasing aniline dyes, the student should always specify the make of Grubler" (6).
Actually, Grubler dyes were not totally without criticism. Paul Mayer wrote that Grubler's carmine red solution dyed very diffusely, was too expensive, and should be purchased elsewhere (4)! But with few exceptions, the excellent con- sistency and reproducibility of Grubler's products set these above any other available dyes. Not surprisingly, many stain- ing methods published at that period were based on the use of Grubler dyes, even with impurities. Perhaps to add to the confusion of the day, dye lots were sometimes mislabeled or misnamed (Figure 1). Dye staining mechanisms were not fully understood and certain characteristics were often credited to the wrong dye. For example, it was the crystal violet component of gentian violet that was important in the Gram stain. And in Mallory's eosin methylene blue method, it was phloxine that was the correct dye (8). The different companies manufacturing dyes were known to lease patents for dye preparation and then call their dyes by different names to distinguish them from their competitors' products, thus giving rise to synonyms. Further, early scientists using the dyes were known to prepare solutions by estimation rather than by measurement with scales (1). In 1905, Holl- born, busy with the preparation of dyes and stain solutions, sold the shipping side of the company, "Dr. G. Grubler and Co., Dyes, Chemical Preparations for Microscopy and Bac- teriology" to Johannes Schmid. However, dyes were still obtained, prepared, and tested by Hollborn's side of the company.
Start of American Dye Industry
World War I brought to a halt the sale of Grubler dyes to America and England. In America, many laboratories had on hand enough of the dyes to last them many months. The United States Navy, anticipating the creation of military laboratories, purchased much of the available Grubler dyes on the market (9). Even after the war, many scientists re- questing Grubler dyes found supplies to be erratic because of an embargo placed on the German industry by the Allies. During the war, patents for dye production registered by the German Textile Industry in America had been seized and put to use (8). This, coupled with erratic availability of Grubler's dyes, set a pace that hastened the start of the American dye industry. The American dyes were apparently of a different quality than those sold by Grubler. There was much confu- sion. One scientist wrote, "There is at present so much con- fusion in this field that the biologist generally has little idea what actual dye he is buying under any given name" (8). Other problems arose when staining procedures, based on Grubler dyes, were performed with the new American dyes. The new dyes were, in some cases, purer; however, they demonstrated different characteristics.
American scientists became concerned because of the shortage of reliable dyes during and after World War I and the unreliability of the new dyes produced in the United States. They wanted American dyes to be as reliable as the Grubler dyes had been, especially if another war were to break out. About 1920, the Society of American Bacteriolo- gists later known as the American Society of Microbiology) asked Dr. H. J. Conn, chairman of its committee on tech- niques, to examine the subject of dye standardization with a view to certification. With the financial support of the Chemical Foundation, and under the auspices of the Na- tional Research Council, the Committee on Standardization was formed. This later became the Biological Stain Com- mission, a cooperative non-profit organization.
Biological Stain Commission
Starting with a small group of commonly used dyes, Dr. Conn and his associates developed a program for testing dyes that is used up to this day. More than 55 dyes submitted by companies are now tested by the Biological Stain Com- mission (10). The major goals of the Biological Stain Com- mission include education about dyes, testing of dyes, and cooperation with manufacturers to increase dye standardiza- tion. After dye lots are carefully tested, those passing the tests are given certificates of approval. The companies pur- chase these certification labels and attach them to the bottles they sell. It is the dye testing and poundage charges of these certificates, together with publications, that supports the Biological Stain Commission.
Two Grubler Companies
To return to the Grubler Company, in 1921, the mail-order side of the company owned by Schmid split off from the Gru- bler side of the company owned by Hollborn. Because of this split, Schmid's company no longer purchased dyes from Hollborn. This separation created further confusion as there were now two Grubler companies selling dyes. Each com- pany claimed to be the original company, and each claimed to possess the secret testing methods originally used by Gru- bler and Hollborn.
Between the two world wars, to reduce competition and costs, German dye companies had pooled stock and formed a cartel. Each company within the cartel was assigned pro- duction quotas for different dyes. This cartel was known as I.G. Farben. In the early 1930s, Hollborn became the sole distributor of biological dyestuffs for the I.G. Farben indus- try and continued to prosper. The price list "HO-10" dated 1932 gives a long list of dyes, dye solutions, chemicals, indi- cators, mountants, drugs and some biochemicals. Over 500 different dry dyes and dye solutions are listed. Twenty differ- ent hematoxylin solutions are listed.
Many of the dyes listed in Hollborn's "HO-10" price list are no longer used in histology but have interesting names such as night blue, naphthol green, curcumin and indulin. As might be expected, many dyes are no longer available. In 1932, Hollborn's two sons, Rudolph and Hanns, joined the company. The company was then renamed, "Dr. Karl Hollborn and Sons, Formally Dr. G. Grubler's Chemical Laboratory." During this period, Akatos of New York repre- sented the Schmid side of Grubler, while the Hollborn side of the company was represented by Pfaltz and Bauer, also of New York.
The start of World War II again disrupted supplies of dyes from the two Grubler companies. The fledgling American Dye Industry was then better established, and the shortage of German dyes was less of a problem. After the war, the Hollborn side of the company, because of its location in Leipzig, East Germany, developed a limited partnership with the East German government. The company has ceased to exist but it is the intention of Dr. Bernd Hollborn, Karl Holl- born's grandson, to re-establish the company. Following World War II, the Schmid side of the company relocated to Stuttgart in West Germany. For many years it has traded under the name, "Chroma Gesellschaft-Schmid GMBH & Co." They have been represented for the past 30 years by the Roboz company in Washington, D.C.
The history of biological dye production as we approach its centennial makes an interesting study. Early cell biolo- gists and pathologists whose names we associate with certain techniques and methods, such as Mayer and Giemsa, were involved in the evaluation of early Grubler dyes. Today, in the United States and other countries, dusty jars of Grubler dyes are to be found hidden away among dyes of a more mod- ern vintage. Overly zealous histotechnologists and patholo- gists should be discouraged from discarding such dyes as "outdated." An interesting project would be to use these dyes and see if they still work. Perhaps these dyes could be do- nated to the local medical museums for preservation.
Dr. Helmut Rotzsch (Deutsche Bucherei, Leipzig) and Mechthild Hofmann (Deutsche Akademie Der Natur-forscher Leopoldina, Halle) assisted with library research. Donald E. Culberson, PhD, and Frederick H. Kasten, PhD, reviewed the manuscript. Mrs. Betty Clark typed the manuscript.