A history of research on yeasts 3: Emil Fischer, Eduard Buchner and their contemporaries, 1880-1900

Through the discovery of Buchner, Biology was relieved of another fragment of mysticism. The splitting up of sugar into CO2 and alcohol is no more the effect of a 'vital principle' than the splitting up of cane sugar by invertase. (Jacques Loeb 1906138 p. 22.) Introduction 363 Pure cultures (clones) 364 New species 367 Yeasts of some traditional fermented drinks 371 Yeast nutrition 373 Cell free fermentation 379 References 383 The first two articles of this series described how yeasts had been shown to be both living organisms and also the cause of alcoholic fermentation7, 8. Between 1835 and 1880, Theodor Schwann (1810–1882) and Louis Pasteur (1822–1895) stood out amongst the scientists responsible for these achievements that were of radical importance for the earliest advances of microbiology and biochemistry. The present article concentrates on the years 1880 to 1900, when pure yeast culturesa were first obtained, many new species were described, cell-free yeast extracts were made that could ferment sugars, and much was learned about sugar metabolism by yeasts. Some of the new yeast species were discovered by examining traditional fermented drinks of various countries. These new developments did not occur in isolation. Important scientific advances in the last years of the nineteenth century included the rediscovery of Mendelian segregation by H. M. de Vries, C. E. Correns and E. von Tschermak10. In the same period, some of the events of fertilization were revealed by Herman Fol76, 77, 78; mitosis and meiosis were accurately described, as by E. A. Strasburger179; and the genetical rôle of the chromosomes began to be understood (138 pp. 188–189). As usual, scientific work was influenced by contemporary political events. In the latter part of the nineteenth century, imperial powers were consolidating their hold on colonies: much of Africa was part of the British, French or German empires. Hence European scientists became interested in tropical diseases; and many microbes from tropical sources, including yeasts, were isolated and studied. In 1897, Ronald Ross, son of an Indian Army officer, entered the Indian medical service and identified in mosquitoes an important stage of the life-cycle of the malaria parasite, Plasmodium falciparum. In physics, the experimental confirmation of Clerk Maxwell's electromagnetic theory of light by Hertz103 led directly to the development of methods of radio communication; Röntgen discovered X-rays167 and Becquerel discovered radioactivity14. There were significant developments in engineering, a number of which facilitated transport and global communications, such as Gottlieb Daimler's invention of the internal combustion engine in 1885; so by 1895, a motor race from Paris to Bordeaux and back (about 1200 km) was possible; the winner, Émile Levassor achieving an average speed of 24 km per hour in his 1.3 litre, 4 HP Panhard et Levassor, fitted with a Daimler engine195. The period was also one of a marked ambivalence toward science among non-scientists and this is reflected in popular fiction. The Scottish novelist, R. L. Stevenson, published his most famous work, The Strange Case of Dr. Jekyll and Mr. Hyde, in 1886. This was science fiction about a case of dual personality, or dissociation: Stevenson described his eponymous protagonist as a distinguished Fellow of the Royal Society of London with his own laboratory. Similarly, H. G. Wells, the leading writer of science fiction and an apostle of science as a source of social improvement, also wrote horror stories about science and scientists, notably The Island of Dr Moreau (1896), and his The War of the Worlds (1898) predicted the use of atomic weapons in warfare. With greatly varied rôles, the protagonists of this period of yeast research were Fischer, Hansen, Buchner and Dienert. Fischer's work was highly innovative, impressive experimentally and had a major impact on both chemistry and biology. Hansen's work was of major importance for yeast research and in brewing. He was very much an 'applied' scientist and made few if any significant fundamental contributions; yet his work was important for the development of fundamental research. Buchner's findings were of considerable importance for the progress of biochemistry, even if (relative to Fischer's work) unimpressive experimentally. The few experiments of Dienert's that are described here were seminal for the study of enzyme induction. At this time, the pace of microbiological research accelerated and a major advance in the development of microbiology made possible experiments on cultures which contain only a single kind of microbe. Pasteur's method of cultivating yeasts had not given pure cultures. He transferred a small portion of a culture by a sterile instrument to sterile liquid medium and, when fresh growth occurred, he used this new growth to inoculate more sterile medium. Pure cultures of moulds and bacteria were obtained before those of yeasts. The great German mycologist, Brefeldb , author of a 15 volume work on mycology26, was one of the first to obtain pure cultures (of higher fungi) by using a dilution method and growing the cultures on solid media made with gelatin. He also published an important paper on methods of studying fungi, in which he stated with great precision the following principles (27 pp. 47–52): (1) inoculation of the medium must be made with a single cell obtained by dilution; (2) freedom from contamination must be checked by microscopical examination; (3) the medium must allow optimum growth of the fungus; (4) the medium should be transparent to allow observation of the germination of a single spore and of subsequent development of the fungus; (5) the culture must be completely protected from external contamination and desiccation. Brefeld's method could not be used to isolate microbes as small as bacteria. However, a successful technique was developed for them in 1878 by Joseph Lister (1827–1912), the English surgeon who introduced aseptic methods to surgery. He constructed a syringe (Figure 1) with a fine screw, which could deliver 0.5 µl of liquid135. He found one µl to be exactly covered by a cover-glass of a particular size. The number of bacteria per µl was equal to the number in the field of view of the microscope, multiplied by the cover-glass area, divided by the area of the field. By correct dilution and taking a small enough sample, there was a good chance of isolating a single bacterium. Lister's syringe. Woodcut 27135 p. 441. The graduated nut (a) revolves on a fine screw on the piston rod (b). The nozzle of glass tube (c) is screwed to the body of the syringe by means of the brass adapter (d), to which the glass tube is secured with gum copal, a water-insoluble resin Joseph Schroeter (1835–1894), a military doctor, Privatdozent at Breslau,c and distinguished mycologist, as well as bacteriologist, was one of the first to differentiate cultures of chromogenic bacteria. He cultured them on solid media, such as starch paste, bread or meat173. Since the bacteria were pigmented, he was able to find colonies that were probably homogeneous. However, the major advance in obtaining pure cultures was made by Robert Koch (1843–1910), one of the greatest bacteriologists of all time, who was also a good amateur photographer. His first solid medium for the study of bacteria, introduced in 1881, was the cut surface of a boiled potato (Figure 2), sliced with a flamed knife, protected from air-borne contamination under a bell-jar and inoculated using a needle116. A pink yeast cultivated on the surface of a potato (from43) Koch was the first to make photomicrographs of bacteria115, for which he made his own photographic plates. These were sheets of glass coated with a solution of silver salts solidified with gelatin. To make the first bacteriological culture plates, Koch substituted nutrient medium for the silver salts. The sterile plates were put under a bell-jar to prevent contamination and were inoculated with a needle or platinum wire; the inoculum was then spread over the surface of the medium. After incubation, bacteria from individual colonies were transferred to slopes of nutrient gelatin (Nährgelatine) in test-tubes plugged with cotton-wool. Koch demonstrated these methods, for which he was gaining world-wide acclaim, at the International Medical Congress of 1881, held in King's College, London. Lister reported Pasteur's dour words of congratulation to Kochd at that meeting: 'C'est un grand progrès, Monsieur'36. Gelatin has the disadvantage of being liquid at 37°C. However, in 1882, Fannie Hesse, the wife of one of Koch's colleagues, suggested using agar, derived from marine algae. She had obtained samples from Batavia, where it was used for jam-making, an activity in which Frau Hesse was presumably engaged too36, 105, 110. Then in 1883, Koch published the poured-plate method, which gave much better separations between adjacent colonies. Instead of streaking the bacteria on the surface of the media, he mixed the inoculum thoroughly with melted gelatin, so that it was evenly distributed in the mixture, which he poured on cold sterile glass plates117. This was one example of the importance of simple innovations. Another was due to Richard Julius Petri (1852–1921), Assistent in Koch's Institute, who described his famous dishe in 1887157. It remained almost unmodified through the twentieth century, apart from the use of plastic, and became the most commonly used piece of microbiological equipment. In 1883, Emil Christian Hansen (1842–1909), who worked at the Carlsberg Laboratory in Copenhagen, developed an effective technique for obtaining pure yeast cultures94, before he knew of Koch's use of solid media98. Working aseptically, he placed a drop of yeast suspension on the underside of a cover-glass (with haematocytometer-like markings) over a moist chamber (Figure 3). If the drop contained, say, 20 cells, a drop of the suspension of the same size was added to 40 ml of water, and 1 ml aliquots of this greatly diluted suspension were introduced into flasks of sterile wort. (Wort is unfermented beer.) When these flasks were left undisturbed, the few (perhaps 3) cells sank to separate positions at the bottom and, after some days, individual growths could be observed. If there was only one growth, this was assumed to be a pure culture98, 108. Hansen's illustration94 of Böttcher's moist chamber; (a) cover-glass, (b) drop of yeast suspension, (c) glass ring, (d) layer of water prevents evaporation Hansen visited Koch's Berlin laboratory in the autumn of 1882185 and, in the light of Koch's work, improved his own method: under the cover-glass, he attached set gelatin containing well-separated cells; only a colony which developed from a single cell was then used to inoculate sterile medium94. In 1882, Hansen's attention had been drawn to the frequent occurrence of a bitter taste and bad odour in Carlsberg beers. By this time he had cultured a number of pure yeast strains from the brewery and was able to attribute the problem to one of them, 'Saccharomyces pastorianus I' (93 p. 414). He isolated four strains of Saccharomyces from the Carlsberg brewery yeast, only one of which, Carlsberg bottom yeast no. 1, gave consistently good beer (98 pp. 15–16). Hansen's method proved of major importance in standardizing yeasts for reliable brewing practice, particularly by making it easier to avoid strains that produce such bad flavours95, 162, 185. Dietrich von Wettstein writes: On November 12, 1883 the Old Carlsberg Brewery started to use in its production Unterhefe Nr. 1. In 1884 the entire production of 200,000 hl beer was based on pure strains of yeast, as was the almost equal quantity manufactured at the New Carlsberg Brewery of Carl Jacobsen. Within a few years the use of clones of bottom fermenting yeast in beer production became the standard procedure throughout the world. By 1892 Pabst, Schlitz and Anheuser-Busch in North America alone manufactured 2.3 million hl with pure yeast strains as did an additional 50 breweries on that continent. ([185] p. 103). Hansen's method of obtaining pure cultures was applied to the production of baker's yeast, too, by M. Delbrück, F. Hayduck and C. Nagel. They worked at the Gärungs-Institutf in Berlin and were involved in developing ways of growing yeast on molasses and ammonium salts, to meet the needs of war-time (1914–1918) shortages of cereal grain. As a result of their work, mentioned in a weekly publication of the German spirit producersg , baker's yeast was propagated industrially, quite early in the twentieth century, by a method based on Hansen's findings (157 p. 423). The development of techniques for producing pure cultures made reliable descriptions of new species practicable. About 130 kinds of yeast were reported or described between 1880 and 19009. Although some had been described previously, often under different names, most were represented as new species. Present-day names are known for only a few and, furthermore, certainty about their identity depends on the existence of well-authenticated strains derived from the original isolates. Table 1 lists species that are still recognized today, but were first described during this period. The authors usually gave some account of the colonies that the yeast formed, of its growth in a liquid medium, the formation of films and pellicles and the microscopical appearance of vegetative cells and filaments. Sometimes asci, ascospores and, for one species, teliospores (Figure 4), were also described, although the sexual significance of neither kind of spore had yet been established. The 1887 English edition of de Bary'sh 1884 text-book on the fungi45 applied 'the term spore quite generally to every single cell which becomes free and is capable of developing directly into a new bion…︁' and distinguished between (a) spores known to develop from sexually fertilized cells, (b) 'spores not sexually developed' and (c) spores classified 'according to their mode of development', such as ascospores (45 p. 129). The ability of the yeasts to ferment glucose, maltose, sucrose or lactose, was often also tested, but few authors gave any account of their methods of testing. Current name Original name and author Isolated from Some information from original description and comments Dipodascus albidus Dipodascus albidus de Lagerheim46 Slime fluxes exuding from trees Descriptions and excellent drawings of filaments and sexual reproduction Dipodascus magnusii Endomyces magnusii Ludwig140 Slime fluxes exuding from trees Descriptions and clear drawings of vegetative cells, hyphae, asci and ascospores Eremothecium coryli Nematospora coryli Peglion15, 155 Diseased hazel nuts (Corylus avellana) Descriptions of cells, with good drawings, including those of asci and ascospores; described the mycelia Filobasidiella neoformans Saccharomyces neoformans Sanfelice171 (1) Lesion of a human tibia by Busse39, 40, but not named; (2) peach juice and named by Sanfelice170 (1) Busse39, 40 described the organism as a yeast and published good drawings of the capsulated cells and also showed them in situ in the tissues; (2) Sanfelice170 also published excellent drawings of the cells Galactomyces citri-aurantii Oidium citri aurantii Ferraris59 Rotting oranges Descriptions of cells, hyphae and arthroconidia Kluyveromyces marxianus Saccharomyces marxianus Hansen95 Grapes (isolated by Louis Marx of Marseilles) Small oval and elongate, sausage-shaped cells; mycelial-like colonies; few reniform, round or oval ascospores ('endospores'); forms up to 1.3% (v/v) ethanol in beer wort; ferments glucose and sucrose which it inverts; does not ferment maltose Metschnikowia bicuspidata Monospora bicuspidata Metschnikoff145 Isolated from a water-flea, Daphnia magna Oval or slightly curved elongate cells, long asci, each containing one needle-shaped ascospore, described and drawn Metschnikowia pulcherrima Torulopsis rosea Berlese22 Ripe grapes Vegetative cells and pulcherrima cells described; fermented grape must Pichia anomala Saccharomyces anomalus Hansen97 Isolated from Bavarian brewer's yeast Oval cells; description and very good drawings of asci and ascospores and their germination; formed pellicle Pichia burtonii Monilia variabilis Lindner134 Food Good drawings of vegetative cells, especially of pseudo-hyphae and septate hyphae Pichia farinosa Saccharomyces farinosus Lindner132 Beer in Gdańsk (then Danzig in Prussia, now Poland) Description and drawings of vegetative cells, pseudohyphae, asci and ascospores Pichia membranifaciens Saccharomyces membranaefaciens Hansen95 Gelatinous mass on roots of elm (Ulmus sp.) attacked by fungi Description of vegetative cells and ascospores ('endospores'); did not ferment glucose, maltose, sucrose or lactose Rhodotorula mucilaginosa Saccharomyces ruber Demme47 Cheese Cells and colonies described; no fermentation Saccharomyces bayanus Saccharomyces bayanus Saccardo169 Turbid beer with bad flavour by Will193, but he did not name the yeast Will193 described and gave good drawings of vegetative cells, pseudohyphae, asci and ascospores; fermented sucrose Saccharomycodes ludwigii Saccharomyces ludwigii Hansen96 Slime flux of an oak (Quercus sp.) Cells ellipsoidal to bottle-shaped or lemon-shaped; new cells separated by forming cross-wall and fission; ascopores formed; fusion of spores observed, but not thought to be sexual; fermented glucose and sucrose, but not maltose or lactose. Hansen later99 changed the name to Saccharomycodes ludwigii Schizosaccharomyces octosporus Schizosaccharomyces octosporus Beijerinck19 Greek currants Descriptions and drawings of cells and their splitting, asci and ascospores; ascospores resistant to desiccation, produced asexually, finding no copulation (19 p. 56); but this was reported by Schiönning172. Beijerinck also recorded that glucose and maltose were fermented (forming ethanol), and were good growth substrates; there was a little growth on mannitol and on glycerol, but none on raffinose, sucrose, lactose, arabinose, galactitol, myo-inositol or quercitol (1-deoxy-muco-inositol). Beijerinck (19 p. 57) drew special attention to the unusual characteristic of being able to ferment maltose, but not sucrose. Schizosaccharomyces pombe Schizosaccharomyces pombe Lindner131 Pombe, an East African millet beer Descriptions and drawings of cells and their splitting, asci and ascospores; glucose, sucrose and maltose fermented. Also drawings of cells by Vorderman187 Sporidiobolus salmonicolor Rhodomyces kochiivon Wettstein186 Human sputum Described cells with measurements, including intercalary teliospores, Dauersporen. Torulaspora delbrueckii Saccharomyces delbrücki Lindner133 Beer Drawing and description of vegetative cells and asci with ascospores; fermented glucose, maltose, and sucrose slowly Trichosporon ovoides Trichosporon ovoides Behrend15 Human white piedra Described cells and filaments Trichosporon pullulans Oidium pullulans Lindner133 Brewery Described and drew vegetative cells and filaments; no fermentation. Xanthophyllomyces dendrorhous Rhodomyces dendrorhous Ludwig141 Exudate of tree Pink slime, composed of chains or branching or budding, free, elliptical cells, 8–10 µm long, 6–7 µm wide Zygosaccharomyces bailii Saccharomyces bailii Lindner132 Beer made from very concentrated wort Drawings of conjugating cells, asci and ascospores; glucose fermented Zygosaccharomyces rouxii Saccharomyces rouxii Boutroux25 Jams and syrups (Boutroux thought it the same as yeast Roux168 had reported contaminating glucose) Vegetative cells described and drawn; glucose fermented, not sucrose Intercalary teliospores, 'Dauersporen', of Sporidiobolus salmonicolor (Rhodomyces kochii), a drawing from von Wettstein's paper of 1885186 In this period, Hansen himself described the following four new species: Kluyveromyces marxianus, Pichia membranifaciens, Saccharomycodes ludwigii and Pichia anomala. At the same time, Lindneri published descriptions of six new species, including Schizosaccharomyces pombe, Torulaspora delbrueckii, Trichosporon pullulans and Zygosaccharomyces bailii. Two famous scientists described new yeast species at this time: Beijerinckj published an account of Schizosaccharomyces octosporus19 and Mechnikovk was the first to give an account of a species of what is now the genus Metschnikowia145. Table 1 summarizes the information published on these and other species at this time. Accounts of the finding and naming of three species of special interest follow. Saccharomyces cerevisiae Although named and described much earlier in the nineteenth century, by both Meyen and Reess, respectively (see7), most yeast taxonomists have designated the main baking and brewing yeast as 'Saccharomyces cerevisiae Meyen ex Hansen'. In 1952, Lodder and Kreger van Rij wrote137: 'Hansen (1883, 1886, 1888)…︁…︁gave a more ample description extended also to physiological characteristics. Reess, who did not make pure cultures, studied quite other yeast strains…︁…︁Reess' descriptions are so very incomplete that Hansen is generally mentioned as the author of…︁…︁Saccharomyces cerevisiae.' Indeed, Meyen146 gave the species its name in 1838, and in 1870 Reess described this fermenting yeast as composed of round or oval cells with tough elastic skins of fungal cellulose163. The cells had maximum diameters of 8–9 µm. Reess accompanied his description with excellent drawings of the cells, as well as asci and ascospores (Figure 5). The chief ways in which Hansen's descriptions of Saccharomyces cerevisiae94 differ from that of Reess were (i) in being based on the study of pure cultures and (ii) in describing the formation and germination of ascospores97. To be consistent, perhaps taxonomists should re-attribute certain species, such as some of the genus Tremella5, 9, as they too were described before the use of pure cultures became practicable.l Drawings of cells of Saccharomyces cerevisiae: Plate I, from Botanische Untersuchungen über die Alkoholgährungspilze by Max Reess, published in 1870163 Filobasidiella (Cryptococcus) neoformans In two papers, published in 1894 and 1895, Bussem illustrated what were almost certainly cells of Filobasidiella neoformans42, 53, 113, 114, 137 taken from a lesion of a woman's tibia (Figure 6)39, 40. He recognized this dangerous pathogen as a yeast and indicated that it was a species of Saccharomyces. Specimens had been sent from the Greifswald University Clinic of Surgery with a covering note, readingn 'Mrs Kapp, age 31, chronic subperiostealo inflammation of tibia (softened sarcomap ?)'. A little later, Busse published a monograph on pathogenic yeasts41. In 1895, Abraham Buschke, then a surgeon at Greifswald, who died in 1943 in Theresienstadt concentration camp114, also reported Busse's yeast38. Sanfeliceq had already, in 1894, isolated a capsulated yeast of the same species from peach juice, naming it Saccharomyces neoformans in 1895170, 171. Subsequently, Vuilleminr named Busse's yeast Cryptococcus hominis188. Some of the earliest published figures showing Filobasidiella neoformans, from Busse40. Busse's Plate 2 shows (Fig. 1) a section of part of a tibia with yeast cells; (Fig. 2) a preparation of inflamed lung tissue; (Fig. 3) yeast cells after culture on potato for 3 days; (Fig. 4) preparation of infected mouse kidney. The preparations in Figs 1, 2 and 4 were stained with Heidenhain's iron haematoxylin and observed using a dry Zeiss apochromatic objective (NA 0.95, f 3 mm); the yeast cells in Fig. 3 were stained with haemalum and carbol fuchsin and observed with an oil immersion Zeiss apochromatic objective (NA 1.30) The clinical importance of Filobasidiella neoformans increased markedly after the 1960s as a result of the widespread clinical use of immunosuppressive methods and, from the 1980s onwards, as a consequence of the AIDS epidemic42, 53. Schizosaccharomyces pombe This yeast was isolated in 1890 from the East African millet beer, called 'pombe', and described by Lindner in 1893131. Pombe was made and widely consumed in the part of Africa east of Lake Tanganyika and west of Zanzibar (then German East Africa and now Tanzania). Richard Francis Burton (1821–1890), the famous explorer and scholar, describes its production and use37. Half of the grain, such as millet, Panicum sp., is soaked in water until it sprouts; then it is mixed with the other half and sometimes with honey. The mixture is boiled twice or thrice, strained through a bag of matting and allowed to ferment. After three days, it tastes like sour beer wort and 'produces an agreeable narcotism'. Lindner's colleague, Zeidler, purified Schizosaccharomyces pombe by growing it in beer wort which was soured with tartaric acid in order to eliminate bacteria.s In his paper, Lindner published descriptions and drawings of the cells, showing their splitting, and also of asci and ascospores (Figure 7); and he reported that this species ferments glucose, sucrose and maltose131. Lindner's drawings of the cells of Schizosaccharomyces pombe131 Also in 1893, Vorderman described the yeast as important in the manufacture of Batavian arrack187, a potent rum-like spirit largely made from fermenting molasses57. Again, the impact of colonialism can be seen, for Vorderman published his observations in a Dutch journal. Batavia is now Jakarta, Java, which was then part of the Dutch East Indies. Beer and wine apart, pombe and arrack were not the only traditional fermented drinks to be examined microbiologically. Others included ginger-beer from England, saké from Japan and kefir from the Caucasus. In 1892, Wardt reported on his investigations into the nature of the ginger-beer plant, 'so often purchased in villages and towns in various parts of the British Isles'191, which produces a slightly alcoholic beverage. Balfouru had suggested previously that the ginger-beer plant was brought to Britain in 1855 by soldiers returning from the Crimean war4. Ginger beer is made by fermenting a mixture of sugar, lemon-juice, potassium hydrogen tartrate and root ginger. The ease of maintaining the 'ginger beer plant' has made it practicable to pass on domestically and provide an exciting 'rediscovery' for generations of school children. Ward, then professor of botany in the Forestry Schoolv of the Royal Indian Engineering College at Egham, England, described the ginger-beer plant as a symbiotic association between a yeast, 'Saccharomyces pyriformis', and a bacterium, 'Bacterium vermiforme', and likened it to the 'kephir-ferment' described below. The present-day identity of Ward's yeast is, however, uncertain (123 p. 507). In 1881, Atkinson,w published his studies on the brewing of saké, a fermented drink made from rice3. The starch of cooked rice is hydrolysed by the action of Aspergillus oryzae and the products of hydrolysis are fermented by yeasts of the genus Saccharomyces118. Atkinson illustrated his account with drawings of the yeasts. His picture of saké brewing, taken from an older Japanese publication, is reproduced here as Figure 8. R. W. Atkinson's illustration of a saké brewery3 taken from a Japanese picture published in 1790 Beijerinck's paper on the fermented milk drink, kefir, appeared in 188917. Kefir is made, mainly in the Caucasus, Balkans and Middle East, from cows', goats' or buffaloes' milk. The milk is fermented by kefir grains, which consist of an astonishingly constant mixture of bacteria and yeasts, embedded in polysaccharide126. Thus, unlike other fermented milk drinks, kefir is not produced by the activity of an evenly distributed microflora, but of the microbes in kefir grains that can be recovered readily after fermentation. Eduard Kern, working in Moscow, had described and drawn the cells of the organisms in kefir grains in 1881109. Eight years later, while studying these grains, Beijerinck17 detected a new enzyme, β-galactosidase (lactase)16, in 'Saccharomyces kefyr', now Kluyveromyces marxianus9. He demonstrated the presence of this enzyme with great ingenuity. For his culture medium, he mixed gelatin with 3% NaCl and 3% lactose. Some of the gelatin also contained a culture of phosphorescent bacteria, Phosphobacterium sp. These bacteria did not use lactose, but grew on glucose or galactose to form a green fluorescent layer. When the medium was inoculated with the kefir yeast, or with one of two other yeasts that used lactose, the fluorescence increased in association with the yeast's growth. A non lactose-using wine yeast did not increase the fluorescence. Hence Beijerinck had demonstrated the existence of a new enzyme comparable to invertase (which had been isolated from yeast by Berthelot in 18608), but one that split lactose. Soon after Beijerinck's study, Mix also published an investigation into 'a kephir-like yeast found in the United States'147. In 1963, La Rivière found the predominant yeast in kefir grains to be Torulaspora (Saccharomyces) delbrueckii, which was associated with Lactobacillus brevis. The two organisms depend on each other for survival in milk125. Further studies on these grains have been made by Marshall and her colleagues143, 160 and on those of Tibix 104. At this time, certain technical innovations much assisted the improved understanding of yeast metabolism. Between 1880 and 1900, new methods were developed for assessing the utilization of different substrates by yeasts and other microbes. Particularly important were the Einhorn tube, the Durham tube and the auxanogram. In 1885, Einhorny described a glass vessel which he used for detecting sugar in diabetic urine by fermentationz , and the great American bacteriologist, Theobald Smith (1895–1934), published a drawing of this kind of vessel (Figure 9) in 1890174. Later, Einhorn (or Smith) tubes were used extensively for detecting fermentations; this was done by yeast taxonomists, such as Stelling-Dekkeraa and Lo