UNIVERSITY OF CALIFORNIA PUBLICATIONS IN AGRICULTURAL SCIENCES Vol. 4, No. 1, pp. 1-66, pis. 1-2, 15 text-figures December 31, 1918 THE FERMENTATION ORGANISMS OF CALIFORNIA GRAPES 1 BY W. V. CEUESS CONTENTS PAGE Introduction 3 I. General discussion of grape organisms 3 Molds 4 Penicillium 4 Aspergillus niger 4 Oidium 4 Borytis cinerea 6 Mucor : 6 Monilia 6 True Yeasts 6 S. ellipsoideus 6 S. cerevisiae 8 S. malei 8 S. pastorianus 8 S. anomalus 8 S. ludiwigii 9 S. marxianus 9 Psuedo Yeasts 9 Apiculatus 9 Mycoderma 9 Torula 10 1 1 . Properties of molds and bacteria from California grapes 10 Sources of cultures 10 Molds 12 Penicillium 12 Olive color penicillium species 12 Aspergillus 12 Mucor 14 Dematium 14 Monilia 14 Green molds (unidentified) 14 Bacteria 14 i The writer wishes to thank Professor F. T. Bioletti for his helpful sug- gestions for carrying out these investigations and preparation of the manuscript. University of California Publications in Agricultural Sciences [Vol.4 PAGE III. Characteristics of yeasts from California grapes 16 Methods of study 16 Morphology 16 Fermentation tests 16 Yeasts studied 18 Apiculatus yeasts 18 Organism 35 (Apiculatus) morphology 18 Organism 72 (Apiculatus) morphology 19 Organisms 35 and 37, rates of fermentation 19 Mycoderma forms 22 Organism 65, morphology 22 Organism 68, morphology 23 Organism 70, morphology 23 Organism 71, morphology 24 Organism 73, morphology 24 Organism 76, morphology 25 Organism 78, morphology 25 Fermentation tests of Mycoderma forms 26 Torula forms 30 Organism 37, morphology 30 Organism 77, morphology 31 Fermentation tests of torula yeast 32 S. Pastorianus and Willia yeast 34 Organism 36, (S. Pastorianus) morphology 34 Organism 69 (Willia species) morphology 35 Fermentation tests of S. Pastorianus and Willia 35 True wine yeasts, S. Ellipsoideus 38 Organism 64, morphology 38 Organism 66, morphology 39 Organism 67, morphology 39 Organism 74, morphology 40 Organism 75, morphology 40 Organism 79, morphology 41 Fermentation records of true wine yeasts at 33° C 41 Attenuation and alcohol formation at 33° C 44 Comparison of fermentations at 33°C and 24°C (all varieties).... 46 IV. Influence of locality on the character of the micro-organisms on grapes.. 50 Methods of taking samples 51 Methods of counting living cells on grapes 51 Results of countings 52 Micro-organisms on grapes from Davis 52 Micro-organisms on grapes from El Centro 52 Micro-organisms on grapes from Fresno 52 Micro-organisms on grapes from Martinez 53 Micro-organisms on grapes from Ripon 53 Yeasts from the Tulare experiment station 53 Discussion of results 53 V. Influence of the stage, of ripeness on the character of the micro-organisms on grapes 54 Sampling 54 Results 55 1918] Cruess: Fermentation Organisms of California Grapes 3 PAGE VI. Changes in the numbers and character of the micro-organisms on grapes during shipment from vineyard to cellar 56 VII. Character and number of micro-organisms on grapes as received at the winery during the seasons of 1911 and 1912 57 VIII. Experiments upon the control of micro-organisms on grapes for wine making 59 Before shipment 59 After arrival at cellar 62 Summary of parts II - VIII (inclusive) 63 INTRODUCTION The manufacture of wine, grape-juice, and raisins and the shipping of grapes depend very largely upon the control of micro-organisms, particularly of those occurring naturally upon the fruit. This eco- nomic fact and the scientific interest of the subject led to a study of the micro-organisms occurring on the grapes of California. The investigation was qualitative and quantitative, covering the effect on type and number of micro-organisms of (a) locality, (6) degree of ripeness, (c) shipment from vineyard to winery. It included studies of (d) the micro-organisms normally found on grapes as received at the winery, (e) their control in fermentation, and (/) their morpho- logical and physiological characteristics. I. GENERAL DISCUSSION OF GRAPE MICRO-ORGANISMS Several groups of micro-organisms are normally found on grapes. These may be classified under the general terms : (1) Budding fungi, including molds, true yeasts which form spores, and pseudo-yeasts, which do not form spores; and (2) fission fungi, including bacteria (non-motile rods), bacilli (motile rods), the various forms of Coc- caceae, and the fission yeasts. Representatives of all the above groups except the bacilli, Coc- caceae, and fission yeasts were found in California grapes. Most attention has been given to the fermentation organisms: i.e., yeasts. The molds and bacteria have in most cases been merely listed. The more important forms of organisms occurring on grapes are the following: University of California Publications in Agricultural Sciences [Vol. 4 Molds Penicillium. — This group includes a large number of varieties, which are the most widely distributed and common of all the molds. They are characterized by the method of formation and grouping of conidia shown in a typical manner in figure 1. The most common mold of this group met with on grapes is Peni- cillium glaucum or Penicillium expansum. It is the common green mold and is the cause of moldy flavors in grapes, barrels, etc. During the first stages of growth it appears as a cottony, white mass of mycelial threads. These white threads soon develop fructifi- cations bearing large numbers of conidia that are green in young cultures and brown in old. These give a powdery appearance to the culture. The individual conidiophores are branching and present a broomlike appearance under the microscope. It does not carry on alcoholic fermentation, but may destroy sugar by oxidation to C0 2 and H 2 or the formation of penicillic acid. Aspergillus niger. — This is a black mold of very common occur- rence on California grapes (see fig. 1). On grapes and must it forms a white mycelium from which spring short rods bearing aggregations of black cells. These conidiophores are not branching, thus differing from penicillium. These groups of cells are easily discernible with the unaided eye. On the grapes of some districts the spores or conidia may be broken away from the main growth of the mold during pick- ing and rise as a black dust. At present it is not held to be very harmful in wine making. Oidium or Powdery Mildew of the Vine. — This fungus is of more concern to the grape grower than it is to the wine maker and will be found fully described in Bulletin 186 2 of the University of California Agricultural Experiment Station (see fig. 1). This mold may pre- vent the grapes reaching maturity or may cause them to crack and thus to be liable to attack by penicillium. In both cases, the grapes affected may become unfit for wine making. The microscopical appear- ance of the summer form of the powdery mildew is shown in figure 1. It occurs most commonly as a downy white growth on the leaves, canes, and grapes during moist or foggy weather and is most prevalent dur- ing early summer. It forms winter spores or perithecia on the canes, in which form the organism lives through the winter. zBioletti, V. T. (Oidium or Powdery Mildew of the Vine), Univ. Calif. Exp. Si;,.. Bull. ISO, pp. 317-327, 1907. 1918] Cruess: Fermentation Organisms of California Grapes / FZNICILLIUM k80O Z.ftSPEiRGt LL 1/5*500 3-BOTFtVTUSxdOO -f MUCOR* /SO- SOIDI UM x dOO €>E>FKO VV/V MOL 0*800 Figure 1 Molds from California grapes: 1. Penicillium variety from California grapes, magnified 800 diameters. 2. Aspergillus variety from California grapes; X 800. 3. Botrytis cinerea from California grapes; X 800. 4. Mucor variety from California grapes; X 150. 5. Oidium or Powdery Mildew from California vine, summer form; X 800. 6. Brown mold, parasitic fungus from California grapes; X 800. Species and variety not determined. 6 University of California Publications in Agricultural Sciences [Vol. 4 Botrytis cinerea. — This mold is a parasite or facultative sapro- phyte occurring on grapes in moist climates (see fig. 1). It rarely develops on California grapes during the wine making season but is often found on grapes left on the vine during the winter. It appears as a gray, matted growth on the surface of the grapes. The indi- vidual fructifications may be seen with the naked eye on short upright conidiophores. Under the microscope, the conidia may be seen in grapelike clusters. The mold did not grow readily in culture media, but will develop profusely on infected grapes in a moist jar. In Europe, the Botrytis is considered beneficial in the Sauternes district, because it causes a concentration of the sugar content of the grapes by favoring evaporation of the moisture. It produces also an oxydase that acts vigorously upon the color of the grapes or wine. Mucor. — Mucor mold will nearly always be found in an examina- tion of California grapes (see fig. 1). Since it develops very slowly in comparison with the commoner molds and yeasts found on grapes it is not probable that it does very much harm in wine making. In pure cultures, it produces a gray filamentous mass of mycelial threads, from which upright rods are given off, bearing at their upper- most ends spherical sporangia filled with large numbers of spores. These sporangia are easily seen with the unaided eye. Most of the mucor molds are capable of forming yeastlike cells and carrying on a feeble alcoholic fermentation in sugary liquids. Monilia. — The monilia molds and especially Monilia Candida occur very commonly on fruits of all kinds (see fig. 2). Most of the grapes examined in 1912 bore considerable numbers of the cells of this organism. In young cultures, it forms colorless yeastlike cells and gives a feeble fermentation. The fermentation is followed by a moldlike growth on the surface of the grape must, etc., which in old cultures becomes olive green in spots. True Yeasts Culture Yeasts. — (1) Saccharomyces ellipsoideus (True Wine Yeast). In general, the ellipsoideus yeasts are characterized by rapid growth in grape must with the production of a strong fermentation, yielding 10% to 16% of alcohol. They differ from the beer yeasts, 8. cerevisiae, principally in their higher alcohol-forming power and 1918] Cruess: Fermentation Organisms of California Grapes ill the flavor of the fermented liquids, the wine yeast giving a vinous flavor to fermented liquids and the beer yeast a beer flavor. In grape must, the S. ellipsoideus forms a cloudy growth and a pasty or gran- ular sediment in the bottom of the container during the main fer- mentation. After fermentation is complete the suspended cells settle I.DEMflTIUMxSOa % MONILW x 800 3. VI A/e6/7/=f BftCT£/?//9x/OCO ^ TOURNC BnCTOf/ffx/OOO. Figure 2 Molds and yeasts from California grapes: 1. Dematium variety from California grapes; X 800. 2. Monilia variety from California grapes; X 800. 3. Vinegar bacteria from California grapes; X 1000. 4. Tourne bacteria from California wine; X 1000. out, giving a clear liquid and compact sediment. Under suitable con- ditions spores are formed. The usual shape of the cells is shortly ellipsoidal, although this will vary from spherical to elongate, with the different varieties. The shape of the spores is spherical. The appearance of several specimens of S. ellipsoideus from California grapes and of cells containing spores will be found in figures 5 and 6. 8 University of California Publications in Agricultural Sciences [Vol. 4 The main part of all wine fermentations is carried on by this yeast. There are a great many different varieties in this group and these vary considerably in their suitability for wine-making purposes. Modern methods of wine making aim to make use of the most desirable varieties. 2. Saccharomyces cerevisiae (Beer Yeast). — It is possible that these yeasts occur on grapes to a more or less limited extent, depend- ing upon the proximity to vineyards of breweries or distilleries using these yeasts. No reference has been found in the literature describing their occurrence on grapes and none have been found on California grapes. There is probably little likelihood of their being present on grapes in sufficient numbers to influence in any way the fermenta- tions. This yeast is worthy of mention in connection with wine mak- ing, however, because it has been used in the past to start wine fer- mentations. "Where brewery yeast is so used, it produces a wine of beerlike flavor and low alcohol content and with an excess of unfer- mented sugar. Such a wine is not palatable and is very liable to be attacked by bacteria and to be lost through bacterial decomposition. 3. Saccharomyces malei. — This yeast occurs on apples and is similar to 8. ellipsoideus, but usually forms less alcohol. It was not found on any samples of grapes examined, but probably occurs occa- sionally on grapes. Wild Yeasts. — (1) Saccharomyces pastorianus. — This group of yeasts is characterized by its elongate or sausage-shaped appearance and its ability to form spores. Members of this group of yeasts have been found on two samples of California grapes. They form small amounts of alcohol in grape must and at the same time usually pro- duce undesirable flavors and odors — usually a bitter flavor. The mic- roscopical appearance of a culture of S. pastorianus yeast from Cali- fornia grapes is shown in figures 3 and 6. 2. Saccharomyces anomalus (Willia Yeasts). — The yeasts of this group are characterized by the production of hat-shaped spores. The appearance of such spores is shown in figure 7. The members of this group grow rapidly in must and form a wrinkled film. They carry on a weak fermentation with the production of small amounts of alcohol. Liquids fermented by these yeasts are high in aromatic com- pounds of various sorts and for this reason their use has been sug- gested as ;i means of flavoring various fermented beverages. Cells from a ei i H me isolated from California grapes are shown magnified 1000 diameters in figures 3 and 6. 1918] Cruess: Fermentation Organisms of California Grapes 9 3. Saccharomyces ludwigii. — This yeast has been found on grapes grown in Europe. In microscopical appearance it resembles the S. apiculatus yeast, but is much larger. It was not found on Cali- fornia grapes. 4. Saccharomyces marxianus. — This yeast has been reported as being present on grapes. It is recognized by its kidney-shaped spores. It was not found on the samples of California grapes examined. Pseudo-Yeasts Apiculatus Yeast. — The apiculatus yeast (Saccharomyces apicu- latus, Hansenia apiculata) is recognized by the peculiarly pointed ap- pearance of many of its cells (fig. 3) . According to most authorities it does not form spores and so is placed in the group of pseudo-yeasts. According to Lindner, however, it forms spores in drop cultures, one spore per cell. It carries on a feeble bottom fermentation in grape must but does not have the power to ferment saccharose, maltose, or lactose. In must it gives from 0% to 6% alcohol and at the same time produces fruity flavors and odors. It settles more slowly and less completely than 8. ellipsoideus. Apiculatus yeast probably does more harm in wine making than all other varieties of wild yeasts com- bined, because of its large numbers on grapes and its very rapid development after the grapes are crushed. It develops so rapidly that the first stages of most natural wine fermentations are carried on by this yeast and the preliminary fermentation of grapes is often spoken of as the "apiculate" stage. During this preliminary fermentation it produces undesirable flavors and aromas, destroys yeast food that should have gone to the true wine yeast, forms compounds deleterious to the vigorous development of the true wine yeast and gives a great many cells of low specific gravity that settle out slowly after the main fermentation is over. After the S. ellipsoideus yeast has formed 8 to 10 per cent alcohol the Apiculatus yeast is killed and will not be found in the living state in the finished wine. Mycoderma Types. — The Mycoderma forms are known to all wine makers as "wine flowers" (fig. 4). They are present in considerable numbers on uncrushed grapes and have been found on nearly all samples of California grapes so far examined. In pure cultures in liquids, they appear as white films, usually more or less deeply wrinkled. From time to time portions of the pellicle are detached and fall to the bottom giving in time a large amount of sediment. A 10 University of California Publications in Agricultural Sciences [Vol.4 feeble fermentation is carried on at the surface of the liquid, pro- vided that it contains fermentable sugar. Under the microscope the yeast appears as cells of irregular shape and size, the usual form, how- ever, being elongate or sausage-shaped. The cells tend to adhere together in groups and chains. It is thought that the cells are main- tained at the surface of the liquid by means of small bubbles of air occluded between them. In pure cultures in grape must, the Mycoderma yeasts are capable of destroying varying amounts of sugar with the production of small amounts of alcohol, in most cases, and obnoxious flavors and aromas. It is probable that in many natural wine fermentations they are more or less active at the beginning of the fermentation. They undoubtedly cause trouble in tanks of wine that are not kept well filled during storage. They are aerobic. Torula Yeasts. — Under the heading of Torula are placed most of the yeasts that do not fit into other groups. Consequently, this name covers yeasts of wide variation in properties. As a group they do not form spores; they form small amounts of alcohol, do not normally develop as a film growth, and the form considered as typical is spher- ical, although there is considerable variation from the typical form. They are found on grapes fairly commonly, though less abundantly than 8. apiculatus and Mycoderma. Two forms from California grapes are shown in figure 3. II. PROPERTIES OF MOLDS AND BACTERIA FROM CALIFORNIA GRAPES Sources of Cultures In 1911 micro-organisms were isolated from grapes received at the winery of J. E. Colton, Martinez, California, and from grapes picked at the University Farm, Davis. Samples were obtained in 1912 from Fresno and El Centro. The properties of these micro-organisms were studied more or less in detail. Most attention was paid to the or- ganisms and characteristics that were of importance in wine making. Method of Separation. — The materials used in the separation of the various types of organisms present on the grapes were sterile grape must agar, sterile grape must, petri dishes, and a small platinum inoculating rod. The grape must agar was made by dissolving 20 grams of agar agar in 1000 c.c. of boiling water to which was added 60 c.c. of grape must after the agar had dissolved. It is necessary 1918] Cruess: Fermentation Organisms of California Grapes 11 Figure 3 Yeasts from California grapes magnified 1000 diameters: 35. S. apiculatus from Davis grapes. 72. S. apiculatus from Acampo grapes. 37. Torula from Davis grapes. 77. Torula from Contra Costa County grapes. 36. S. pastorianus from Davis grapes. 69. Willia species -from Acampo grapes. 12 University of California Publications in Agricultural Sciences [Vol. 4 to use this high diluation of must in the agar in order that the agar will not be hydrolyzed by the acid of the must. At this dilution the agar contains about .05 per cent acid as tartaric. This should permit the growth of all forms. The hot agar solution was filtered through cotton wool and filled in 10 c.c. portions into test tubes plugged with cotton wool. It was sterilized at 15 pounds pressure in an autoclave. The grape must was clarified by boiling with the white of egg and filtration. The clear must was filled into plugged test tubes and sterilized at 100° C. The Petri dishes were sterilized in packages of three by dry heat. The type of dish used is shown in figure 3. Samples of the grapes received at the winery were taken from the center of the boxes and crushed into sterile containers. A small amount of the must was in each case transferred by a sterile platinum wire to a tube of melted agar kept at 40° C to 45° C, and further dilutions were made by transfer to other tubes of agar. The organisms were separated by growth on agar must. They were purified by replating on the same medium. Subcultures were then made in sterile must, on agar slants and permanent stock cultures were made in sterile 10 per cent cane sugar in Freudenreich flasks. The pure cultures obtained in this way are discussed in groups, the members of each group having certain characteristics in common. No attempt has been made so far to study the molds and bacteria in detail and for this reason they have simply been listed with a few words of explanation. Molds Penicillium Species (probably Penicillium expansum) . — Found on nearly all samples examined ; produces fructifications bearing numer- ous round conidia as shown in figure 1 ; forms sclerotia, resistant vegetative forms, in old cultures; no fermentation in must, but grows vigorously in all culture media tested; giving a characteristic moldy odor. Penicillium Species of Olive Green Color. — Forms ellipsoidal coni- dia ; very common on California grapes and also found in samples of pasteurized unfermented grape juice that had molded after pasteuri- zation. Aspergillus. — Two forms: (a) produces small, smooth, black coni- r /a/ HOIIR^ w> > 1 35 74, 3 3$.-" jg s ^ 8.--' '* s* T FFRMFNTf\ ITIOA/^ RT H'' tt°r in MUST Z5%i <=$/=) I l\ng. 5 -* fe . H FrtiMFNTflTlC^ )NS B T33C 3 CD 5 53" IN MUST £S i3% / 3FILI ING. Z CO „+*' / O * ^1 — , J - ^s' 1 *0 * £ Til FFfrMFNTFITl oaas R T 3 t CD 1 33Y? IN F>FFR V \/OF{l £ ^ 1 8 75? 7A ^z— -3£-- -as ; 5 § -f 1 _!ozi£r/?A7/r/ VTFF ^ION. S AT 3 »*«» o CVW7 n pr xr^i 1ST *bOL. Z ^ 1 8 o ~Z>5 — « >J ^"^ r//V^" //v HOUF 'S »- r 3 O 8 /J £0 A £0 * 00 z 40 Z ?6 3 HO 3i so ^ 00 J 1*3 Fig. 7. — Fermentation curves of S. apiculatus yeasts from California grapes. TABLE 2 Yeasts Numbers 35 and 72 (S. apiculatus). Attenuation and Yields Alcohol in Must, Beer Wort, Dextrose, Saccharose, and Lactose Solutions Yeast Xo. 72 72 72 72 72 72 Attenuation in grams per 100 c.c. Medium Must of 23° Balling 3.81 Must of 29?75 Balling 1.20 Beer wort of 14?56 Balling 1.61 Dextrose solution, 14?9 Ball. 2.73 Saccharose sol., 13?73 Ball. .78 Lactose solution, 15?16 Ball. .31 Theoretical yield of alcohol, per cent 2.50 .79 1.11 1.79 .53 .20 Observed yield of alcohol, per cent 1.00 .30 .30 .65 .00 .00 35 Must of 23° Balling 8.50 35 Must of 29?75 Balling 6.15 35 Beer wort of 14?56 Balling 1.35 35 Dextrose solution, 14?9 Ball. 3.25 35 Saccharose sol., 13? 73 Ball. .00 35 Lactose solution, 15?16 Ball. .00 5.58 4.03 .93 2.13 .00 .00 3.05 2.35 .65 .00 .00 Per cent of theoretical yield ob- yield obtained 40.0 37.9 27.6 36.3 .00 .00 54.5 58.3 30.5 22 University of California Publications in Agricultural Sciences [Vol. 4 The results show that in most cases, less than 50% of the alcohol, theoretically obtainable from the sugar fermented, was actually formed by the two yeasts. For example, yeast no. 35 destroyed 3.81% sugar and should have produced 2.5% alcohol in must of 23° Balling, while the actual yield was only 1% or 40% of the amount that was theo- retically possible. True wine yeasts of the 8. ellipsoideus type pro- duce at least 90% of the theoretical yield in grape must. Since the yields of alcohol are so low and the flavor of the fer- mented liquids is not desirable, the two yeasts may be considered of little use for industrial purposes. Their importance lies in their power to cause trouble in wine making by growing in natural fer- mentations before the true wine yeast has developed. In this way they may injure the flavor and clearing quality of the wine and favor "stuck" tanks by the production of compounds that are injurious to the 8. ellipsoideus yeast. Because of their very common occurrence in large numbers on California grapes, measures should always be taken to discourage their growth. MYCODERMA FORMS FROM CALIFORNIA GRAPES Under Mycoderma forms, seven film forming organisms have been described in the following pages. Some of these organisms are yeast- like in appearance and in their fermentation properties ; other re- semble molds more closely than they do yeasts. All have the common properties of forming heavy films on nutrient liquids and of not forming spores. Organism 65. Mycoderma from Acampo Grapes. Morphology. — Microscopical Appearance. — Sausage-shape to fila- mentous. Tends to grow in mycelial threads (fig. 4). Size. — Average, 9^ x 2.5/x. Maximum, 40/x x 2.5/x. Minimum, 2/x x 1.5/x. Spore Formation. — No spores at 22° C and 28° C. Colonies on Agar Agar. — Small and growth slow. Powdery ap- pearance. Color, white. Flat. Growth at surface only. Edges made up of mycelial threads. Under low power, colonies are made up of chains of long cells. These spread over the surface of the agar. Colonies on Gelatin. — More vigorous than on agar. Concentric ring growth. Powdery white. Flat. Under microscope, same as on agar. Gelatin slowly liquefied. 1918] Cruess: Fermentation Organisms of California Grapes 23 Growth on Liquid Media. — Grows profusely on surface of grape must, beer wort, dextrose, saccharose and lactose solutions, but does not produce visible fermentation in any of these liquids. Film remains permanently on the surface. Does not sink. Film is very tough in texture. Organism 68. Mycoderma from Grapes Grown at Acampo, California Morphology. — Microscopical Appearance. — Predominating form sausage-shaped. Cells vary from spherical to long-rod shaped cells (fig. 4). Size. — Average, 7.5/x x 3.75/x. Maximum, 15/* x 3.75/x. Minimum, 3/x x S/x. Spore Formation. — No spores formed. at 22° C or 28° C. Colonies on Agar Agar. — Large. Edges entire. Waxy luster. Centers of colonies slightly elevated. Edges entire. Opaque. Under low power of microscope, internal structure granular and edges are surrounded by long chains of cells of ellipsoidal shape. Colonies on Gelatin. Filmy, translucent colonies, that rapidly cover the whole surface. Under microscope the colonies are made up of chains of cells. Growth in Liquid Media. — Heavy growth in grape must, beer wort, and dextrose with formation of large sediment, the volume of which may be equal to 10 per cent of the total volume of the liquid. Growth slow in lactose and saccharose solutions. Visible fermentation very slow in grape must, beer wort and dextrose solution at first, but finally becomes vigorous in must and dextrose solutions. No visible fermenta- tion in lactose and saccharose. Organism 70. Mycoderma from Acampo Grapes Morphology. — Microscopical Appearance. — Usual form, sausage- shape, but vary from spherical to sausage-shaped. Cells are usually grouped in branching chains (fig. 4). Size. — Average, 7/* x 2/x. Maximum, 18/x. x 2/x. Minimum, 1.5/x x 1.5^. Spore Formation. — No spores formed at 22° C and 28° C. Colonies on Agar Agar. Large. Center elevated. Surface cor- rugated by ridges radiating from centers of colonies. Centers of colonies brown ; edges yellowish white. Edges indented. Low power of microscope shows chains of cells growing from edges of colonies; this growth is beneath the surface of the agar (see plate 2). 24 University of California Publications in Agricultural Sciences [Vol.4 Colonies on Gelatin. — Large. Centers convex. Colonies flatten near edges and become filmy in appearance. Edges entire under microscope. Gelatin slowly liquefied. Growth in Liquid Media. — Heavy, wrinkled surface growth w r hich frequently falls to bottom forming voluminous sediment. Fermenta- tion slow. No fermentation in lactose. Organism 71. Mycoderma from Contra Costa County Grapes Morphology. — Microscopical Appearance. — Predominating form sausage-shaped. Varies from spherical to sausage-shape (fig. 4). Size. — Average, 6.9/x x 2/x. Maximum, 18/x x 2/x. Minimum, 1.5/X X 1.5/X. Spore Formation.— No spores at 22° C or 28° C. Colonies on Agar Agar. — Large. Flat. Spreading. Edges lobate. Edges entire under microscope (see plate 2). Colonies on Gelatin. — Flat. Filmy. Translucent. Rapidly cover entire surface of the gelatin. Slow liquefaction (see plate 2). Growth in Liquid Media. — A wrinkled, loosely coherent film on all liquids tested. Films frequently sink and are quickly replaced by new growth. Fermentation in must, beer wort, and dextrose fairly vigor- ous, but extends over long period. No perceptible fermentation in sac- charose and lactose. Organism 73. Mycoderma from Grapes Grown at Acampo Morphology. — Microscopical Appearance. — Usual form sausage- shaped. Varies from spherical to sausage-shaped (fig. 4). Size. — Average, 6.7/*. x 2.5/x. Maximum, 18/x x 3/x. Minimum, 3/x x 1.5/*. Spore Formation. — No spore formation at 22° C and 28° C. Colonies on Agar Agar. — Centers elevated. Edges flat and ser- rated. Shape irregular. Under the microscope chains of cells are seen to radiate from the edges of the colonies. The chains are made up of long cells with pairs of short ellipsoidal cells at the junctures of the long cells. Colonies on Gelatin. — Flat. Smooth. Filmy. Translucent. Gela- liti rapidly covered by a filamentous growth. Gelatin is slowly softened to a syrupy consistency. Growth in Liquid Media. — Growth vigorous in all liquid media tested. Vigorous fermentation in grape must and dextrose solution. 1918] Cruess: Fermentation Organisms of California Grapes 25 During fermentation a heavy wrinkled film is formed with a moderate amount of sediment growth. After fermentation most of the film growth settles to the bottom. Fermentation is more vigorous and film formation is not so well developed as in no. 70. Organism 76. Mycoderma from Grapes Grown at Acampo Morphology. — Microscopical Appearance. — Sausage-shaped to fila- mentous (fig. 4). Size. — Average, 10.5/x x 2.1/x. Maximum, 75/*. x 2.5/*. Minimum, 3/x x 2>fx. Spore Formation. — No spores formed at 22° C or 28° C. Colonies on Agar Agar. — Large. Flat. Surface smooth. Centers slightly elevated. Edges smooth and entire under microscope. Colonies on Gelatin. — Large. Resinous. Translucent. Surface of colonies deeply corrugated. Mycelial projections from edges visible to naked eye. Surface glistening and slimy. Under microscope edges are fringed by branching chains of very long cells. Growth in Liquid Media. — Growth in all media tested consists first of powdery white colonies. These coalesce to form a resinous heavy film resembling a growth of vinegar bacteria. The growth sinks after several weeks and is replaced by a second film. No visible fermenta- tion in any liquids tested. Organism 78. Mycoderma from Contra Costa County Grapes Morphology. — Microscopical Appearance. — Sausage-shaped cells predominate. Very long filamentous cells not uncommon (fig. 4) . Sige. — Average, 10.5/x x 3/x. Maximum, 37.5/a x 3/x. Minimum, 4.5/x x 2{x. Spore Formation.— No spores at 22° C and 28° C. Colonies on Agar Agar. — Large and flat. Centers slightly elevated. Waxy luster. Edges show hazy growth of mycelium. Under micro- scope colonies are seen to be surrounded by radiating mycelial threads. Colonies on Gelatin — Large. Flat. Smooth. Translucent. Rapidly cover the entire surface of the gelatin. Gelatin rapidly softened to a syrupy consistency (see plate 2). Growth in Liquid Media. — In grape must, beer wort, and dextrose, solutions, growth is rapid and fermentation vigorous; in saccharose growth vigorous and fermentation slow ; little growth in lactose. Con- sists chiefly of surface growth. Small sediment. 26 University of California Publications in Agricultural Sciences [Vol. 4 Fermentation Tests of Mycoderma Forms from California Grapes. The data obtained by noting the loss in weight at 33° C of flasks containing 100 c.c. of grape must 23.01° Balling, grape must 29.8° Balling, beer wort 14° Balling, dextrose 15° Balling, saccharose 15° Balling, and lactose 15° Balling solutions, respectively, after inocula- tion with pure cultures of the above yeasts appear in table 3. The attenuation, representing the sugar destroyed, together with the yields of alcohol in the various sugar solutions tested are given in table 3. After each table the results of the tests are discussed. The nutrient solutions used were the same as those described for yeasts 35 and 72. See description of apiculatus yeasts. TABLE 3 Fermentation Tests of Mycoderma Forms from California Grapes Dextrose Saccharose Lactose Yeast number Time in hours Must of 23° Balling, loss in grams Must of 29° Balling, loss, in grams Wort loss, in grams 14 per cent solution loss, in grams 15 per cent solution loss, in grams 15 per cent solution loss, in grams 15 per cent solution 68 18 .00 .00 .04 .07 .00 .00 68 42 .70 .01 .21 .31 .10 .00 68 66 1.51 .24 .38 .93 .10 .00 68 92 2.45 .62 .37 2.10 .10 .00 68 123 3.35 1.32 .38 2.90 .10 .00 68 165 5.80 2.00 .40 5.89 .10 .03 68 236 8.45 3.30 .42 6.40 .10 .03 68 284 8.48 3.80 .50 6.40 .10 .03 68 314 8.48 3.80 .51 .10 .03 65 18 .00 .00 .00 .00 .00 .00 65 42 .03 .00 .00 .00 .01 .00 65 66 .03 .00 .03 .00 .01 .00 65 92 .03 .00 .08 .04 .01 .00 65 123 .03 .04 .08 .09 .07 .03 65 165 .03 .04 .12 .09 .07 .03 65 236 .03 .04 .17 .09 .07 .03 70 18 .02 .03 .00 .10 .00 .00 70 42 .10 .05 .06 .18 .20 .00 70 66 .38 .08 .33 .41 .20 .06 70 92 .80 .38 .42 .25 .05 70 123 1.90 .08 .80 1.30 .60 .20 70 165 2.47 .83 1.00 1.65 .90 .20 70 236 2.57 .95 1.00 1.65 .90 .30 71 46 .85 .35 .25 .00 .00 71 73 1.85 1.17 .34 .46 .00 71 96 2.70 2.19 .41 .00 71 118 3.74 .■',.19 .42 2.31 .01 71 167 5.47 3.89 .42 4.20 .00 .00 71 215 6.40 5.90 .44 .04 71 239 6.85 6.40 .45 .04 1918] Cruess: Fermentation Organisms of California Grapes 27 TABLE 3- -(Continued) Yeast lumber Time in hours Must of 23° Ballin loss in grams Must of g, 29° Balling loss, in grams Wort loss, in grams 14 per cent solution Dextrose loss, in grams 15 per cent solution Saccharose loss, in grams 15 per cent solution Lactose loss, in grams 15 per cent solution 73 27 .20 .06 .18 .66 .10 .00 73 52 1.09 .61 .34 1.79 .28 .00 73 77 2.26 1.56 .49 3.21 .58 .00 73 125 4.14 3.34 .59 4.11 .93 .00 73 167 5.69 4.59 .61 5.72 1.19 .00 73 244 9.25 6.62 .72 7.07 1.41 .00 73 341 9.32 8.24 .87 7.35 1.52 .00 73 421 9.52 9.65 .92 .00 76 27 .00 .04 .00 .00 .00 .00 76 52 .04 .08 .04 .07 .00 76 77 .00 76 125 .00 76 167 .31 .13 .24 .20 .00 76 244 .42 .39 .46 .41 .00 76 341 .62 .69 .62 .50 .00 76 421 .64 .69 .70 .54 .00 78 27 .23 .16 .23 .38 .23 .00 78 52 1.12 .44 .54 .80 .54 .00 78 77 2.37 1.64 1.75 2.05 1.37 .00 78 125 4.34 3.29 3.24 3.38 1.74 .00 78 167 6.05 4.60 4.52 1.95 .00 78 244 7.55 6.20 5.25 6.16 2.26 .00 78 341 9.50 7.50 6.98 2.50 .00 78 421 9.85 8.05 7.29 2.70 .00 The seven different varieties of mycoderma yeasts, whose rates of fermentation in normal must, concentrated must, beer wort, dextrose, saccharose and lactose solutions were studied gave great variation in the rate of fermentation and the amounts of sugar fermented. Organ- ism 65 did not cause fermentation in any of the liquids tested, though it gave a vigorous growth. On the other hand, yeasts 68, 73, and 78, developed fairly strong fermentations in must and beer wort, destroy- ing nearly as much sugar as the true wine yeasts. Organism 76, caused a very feeble fermentation in grape must but did not ferment any of the other liquids tested. The mycoderma forms did not ferment so rapidly in dextrose solution as in grape must. Contrasted with the mycoderma forms the true wine yeasts did not exhibit this difference. Lactose was not fermented by any of the mycoderma forms. Sac- charose was attacked to a slight degree by forms 70, 73, and 78, the amount of sugar actually fermented in each case being small. In general, the fermentation curves of the forms shown in the diagrams 28 University of California Publications in Agricultural Sciences [Vol. 4 differ from those of the true wine yeasts shown in figures 12 to 15, by their low and gentle slope throughout. In most cases they show a slower start of fermentation than do the 8. ellipsoideus yeasts. Be- 4 n fl rt /, ?<0 /< 5G_^20O 2-TO 230 j'vSO 3 zn Ana ^o q T7A/y "/A/ / /o/y^f <5 >»— -> - 75 - ---73 ■ —78 / 63 A 7 y ^- fy o \y 7/ f) (V- 'V vy 4 CD ^•' r rr FfA/r/ JT&T 10N?> /?T 3 ■~». // ^Tr / A/ A7/ f$T j ZTWo / 5/?/ 2 ,••> 4r V 1 o „> ^,*' " — — '- r' 76 q .. --" 75 fi ^- ^."* ..~-7tf 7 ^ ^ -—'" ~"~"' • yy -7 A <7/ r rr ^MFt 1TR-T /OA/^ RT "> > ,--" ty srr: IN F rrFx WOf it 3 R x*^ ^" / 8 ys ^r ...... • j n .y£ ^^ -"'" r/ *•£"// / A/Oi y/=?s ^ tf fc K) 1 «# / £0 £ bo 4 70 A 50 3 o znn 2.-40 2.?>n ^zo ?*>f>c> ioo 4*0 -s =?/^fa ITHTI ONS j IT TbWc n c IN ?S RCCh IHRC ^F .«*)/ in ION ? 76 • i 3 ^..—- - . ■ — ■ 75 s' — — timl 70 IN h OURZ \ *— » ^ itr~h I bo h bO ft 00 z- W X vo o> ZO 3 bO *l 00 -5^C Fig. 9. — Fermentation curves of Mycoderma forms in dextrose and saccha- rose solutions. Organism 77. Torula from Contra Costa County Grapes Morphology. — Microscopical Appearance. — Vary from ellipsoidal to sausage-shaped with ellipsoidal form predominating (see fig. 3). Tends to form short chains. Size. — Average, 4/x, x 2.1 fx. Maximum, 8p x 4.5^. Minimum, 2.2/*xl.5/x. Colonies on Agar Agar. — Small. White. Convex. Waxy luster. Spherical colonies in the agar. Under the microscope colonies are found to be surrounded by straight chains of long cells. Most other yeasts examined that formed side chains in this manner produced branching cells. Colonies on Gelatin. — Contoured surface with highest elevation at centers of colonies. Centers of colonies colorless. Chalklike at edges. 32 University of California Publications in Agricultural Sciences [Vol. 4 Undermicroscope, colonies are fringed with branching chains of long cells. Growth in Liquid Media. — Growth in all liquids slow. Feeble fer- mentation in grape must, beer wort, and dextose. No surface growth. No fermentation in lactose and saccharose. Rates of Fermentation of Torulas 37 and 77 in Various Media. Kates of fermentation in grape must of 23° Balling, and 29.8° Balling, beer wort of 14° Balling, and 15% solutions of dextrose, saccharose, and lactose were tested as previously described for apiculatus and mycoderma forms. The results are given in table 5 and figure 10. TABLE 5 Fermentation Tests at 33° C op Torula Forms from California Yfasts Must of Dextrose Saccharose Lactose Must of of29°8 Wort loss, loss in loss, in loss, in 13° Balling, Balling, in grams grams grams grams Yeast number Time in hours loss in grams loss, in grams 14 per cent solution 15 per cent solution 15 per cent solution 15 per ci solutio 37 18 .01 ,00 .04 .12 .07 .02 37 42 .19 .07 .31 .54 .29 .07 37 66 .51 .45 .98 .38 37 92 .63 .50 1.40 .43 .08 37 165 .87 .51 2.74 .54 .08 37 236 .87 .13 .51 3.64 .54 .09 77 46 .17 .01 .23 .23 .01 .03 77 73 .84 .15 .48 .48 77 96 1.14 .42 .58 .58 .10 77 118 1.30 .81 .68 .68 77 167 1.37 .86 .78 .78 .10 77 215 1.44 1.03 .83 .83 77 239 1.48 1.08 .95 .95 .03 Torulas 37 and 77 were able to produce only very feeble fermenta- tions in the liquids tested. Torula yeast 77 was not able to ferment must of 30° Balling, or saccharose solution. In must of 23° Balling, and in beer wort the fermentations of the two yeasts were about equal. In must, beer wort, and saccharose the fermentations were completed in a short time, about 120 hours. In comparison with the fermenta- tion curves of the 8. elUpsoideus yeasts (figs. 12 to 15), the yeasts above gave very weak and incomplete fermentations. The flavors of the fermented liquids were disagreeable. Therefore, the yeasts seen i to have no practical value but may cause defective flavors in wine where their growth is not prevented. Attenuation and Yields of Alcohol. — The fermented liquids were analyzed for alcohol with the results reported in table 6. 1918] Cruess: Fermentation Organisms of California Grapes 33 40 A o /^o /< 60 2 on &io 2,no 7>2,o 7SJ ^^^z. $ 4 i TFFFF?MFNT/?V ON AT. 57TC 3 c<5 IN MUST £9 %PAI / ING #, ^ i § 77 o vi ^ -.**""" $ * 1 JIT FFFfMFNT FIT ION FIT 3 i- 3=s o r //V /=3/ Tff W OF?T ^ / § ,1 ^i * •= J7 77 ? -f I 75 > "57 ft WFFFF MFNTft von m -7\rc l o IN DF> TffOSt •LSQLL TION o 77 5- 4 fZ TZFFF? WFNTR VON Ft r7F?C 7J $ IN SRC mHRR OSF ^ >OI . 2 < 1 5$ vl 57 77/ 1E IN HOUF\ \3 *— ► -7 E" 5 o /a! o n >0 £ 20 & ?0 ^ 80 &, KO &t bO-9&. \ Fig. 10. — Fermentation curves of yeasts 37 and 77 in grape must, beer wort, dextrose and saccharose solutions. 34 University of California Publications in Agricultural Sciences [Vol.4 TABLE 6 Yeasts 37 and 77. Attenuation and Yields of Alcohol in Must, Beer Wort, Dextrose, Saccharose, and Lactose Solutions Attenuation Per cent of in grams Theoretical Observed theoretical Yeast per 100 yield of yield of yield No. Medium c.c. alcohol alcohol obtained 37 Must of 23° Balling 5.21 3.42 .60 17.6 37 Must of 29?8 Balling 05 .03 .00 .0 37 Beer wort of 14° Balling .20 37 Dextrose solution, 15% 7.96 5.22 3.50 67.3 37 Saccharose solution, 15% 64 .43 .40 93.02 37 Lactose solution, 15% 06 .04 .00 .0 77 Must of 23° Balling 4.58 2.99 1.31 43.7 77 Must of 29?8 Balling 3.85 2.53 .65 25.7 77 Beer wort of 14° Balling .... 2.91 2.00 .30 15.0 77 Dextrose solution, 15% 4.55 2.99 .65 21.7 77 Saccharose solution, 15% .00 .0 77 Lactose solution, 15% 06 .04 .00 .0 Analyses of the fermented liquids from fermentation of Torulas 37 and 77 in grape must, etc., showed that the absolute amounts of alcohol formed were small and that the yields for each per cent of sugar destroyed were in all cases, except for yeast 37 in dextrose, very low. In the majority of the fermentations, the yields were less than 50 per cent of the theoretical. These yeasts have the defects of giving slow incomplete fermentations, low yields of alcohol for sugar fer- mented and of producing liquids of disagreeable odor and flavor. PASTORIANUS AND WILLI A YEAST FROM CALIFORNIA GRAPES One S. pastorianus and one Willia yeast were obtained from grapes in 1911. Both are true yeasts and form spores. Organism 36. S. pastorianus Yeast from Grapes Grown at Davis Morphology. — Microscopical Appearance. — Form varies from spherical to sausage-shaped. The ellipsoidal form predominating. Much smaller than true ellipsoideus yeast (fig. 3). Size. — Average, 4.5/* x 3/x. Maximum, 7.5w x 3/x. Minimum, 1.5/x x 1.5ft. Spore. — Produced in great abundance in 24 hours on gypsum spore blocks. Spores also form in grape must cultures. Spores spherical in form. Number per cell, 2 to 4. Size of spores, 2/x x 2/x, (fig. 7) . Colonies on Agar Agar. — Round. Slightly convex. White. "Waxy Luster. Colonies in agar spherical. Under the low power, the colonies 1918] Cruess : Fermentation Organisms of California Grapes 35 are surrounded by chains of cells made up of long central cells with pairs of short ellipsoidal cells at the junctures of the larger cells. Colonies on Gelatin. — Flat. Concentric rings of growth. Outer edges of colonies sharply indented. Gelatin slowly softened but not completely liquefied. Growth in Liquid Media. — Heavy wrinkled surface growth which sinks to bottom frequently. No visible fermentation. Taste of liquids bitter and disagreeable. Organism 69. Willia Species (Saccharomyces anomalus) from Grapes Grown at Acampo, California Morphology. — Microscopical Appearance. — Size and shape very irregular. The larger cells usually sausage-shaped or ellipsoidal and the smaller ones spherical. Larger than typical S. anomalus in the University collection (fig. 3). Size. — Average, 6/a x 3^. Maximum, 14^ x 2.5//,. Minimum, 3/x x 3/x. Spore Formation. — Spores are formed abundantly on gysum blocks at 28° C and 22° C, in three days. Spores are of typical Willia form, i.e., hat-shaped (fig. 7). Number of spores per cell varies from 2 to 8. Size of spores, 4//, x 1.5/x. Colonies on Agar Agar. — Medium size. Flat. White. Waxy luster. Edges entire. Colonies on the agar are spherical. LTnder the microscope the edges of the colonies are entire. Colonies on Gelatin. — Large. Flat. White. Chalklike. Colonies spread rapidly and soon cover entire surface and the gelatin is rapidly liquefied. Growth in Liquid Media. — A chalky white film develops on all liquids tested, with flocculent growth at bottom of flask. Prolonged top fermentation takes place in all liquids except lactose. Strong odor of ethyl acetate is developed. The fermented liquids possess a burning taste. Rates of Fermentation of Yeasts 36 and 69. — These yeasts were tested for their rates of fermentation in various media as previously described for other organisms. The results are given in table 7 and figure 11. From the data given in table 7, yeast 36 is seen to give scarcely any fermentation in any of the liquids tested, the maximum loss in weight per 100 c.c. being .68 grams in must of 29.8° Balling. On the other hand, yeast 69 gave fairly strong fermentations in must, beer wort, dextrose, and saccharose solutions. Neither yeast fermented lactose, 36 University of California Publications in Agricultural Sciences ' [Vol. 4 TABLE 7 Data of Eates of Fermentation of Yeasts Numbers 36 and 69 in Various Sugar Solutions Yeast number Time in hours Must of 23° Balling, loss in grams Concentrated Wort loss, must, loss in grams in grams 14 per cent 29? 8 Ball. Balling. loss, in grams 15 per cent solution loss, in grams 15 per cent solution loss, in grams 15 per cer solution 36 46 .03 .03 .03 .03 .00 .00 36 73 .04 .04 .03 .06 .00 .00 36 96 .09 .11 .03 .08 .00 .01 36 118 .24 .16 .13 .13 .00 .01 36 167 .33 .43 .13 .19 .00 .02 36 215 .45 .63 .13 .26 .00 .02 36 239 .46 .68 .28 .00 .02 69 18 .01 .10 .13 .01 .10 .03 69 42 .58 .18 .46 .13 .28 .14 69 66 1.10 .23 .56 .77 .86 .14 69 92 1.79 .80 1.71 1.77 1.90 69 123 2.49 2.20 1.75 2.87 2.88 69 165 3.22 2.50 1.81 3.99 3.78 69 236 4.02 4.88 1.92 5.31 4.62 .14 69 283 4.49 5.55 1.93 5.77 4.98 69 314 4.62 5.75 5.79 5.03 while yeast 36 did not attack saccharose. Although yeast 69 gives more vigorous fermentations than does yeast 36, still it does not ap- proach the S. ellipsoideus yeasts from California grapes in rate of fermentation or fermentative power. Attenuation and Alcohol Yields of Yeasts 36 and 69. — The fer- mented liquids were analyzed with the results given in the accom- panying table. TABLE 8 Yeasts Numbers 36 and 69. Attenuation and Yields of Alcohol in Must, Beer Wort, Dextrose, Saccharose, and Lactose Solutions Attenuation Per cent of in grams Theoretical Ons€rved theoretical Yeast per 100 yield of yield of yield No. Medium c.c alcohol alcohol obtained 36 Must of 23° Balling 2.49 1.95 .30 15.3 36 Must of 29?8 Balling 1.65 1.08 .00 .0 36 Beer wort of 14° Balling 31 .20 .00 .0 36 Dextrose solution, 15% 1.95 1.28 .24 18.9 36 Saccharose solution, 15% 02 .67 .00 36 Lactose solution, 15% 11 .07 .00 69 Must of 23° Balling 11.12 7.30 1.25 17.1 69 Must of 29?8 Balling 2.92 1.90 1.00 52.6 69 Beer wort of 14° Balling .... 1.40 .92 .50 54.3 69 Dextrose solution, 15% 11.03 7.25 3.10 42.6 69 Saccharose solution, 15% 7.73 5.09 2.60 51.2 69 Lactose solution, 15% 1.25 .80 .00 .0 1918] Crusss: Fermentation Organisms of California Grapes 37 4Q SO 120 I6Q 2QQ 2*Q 280 7*>20 ^0^00 tim e: >N HOURfS -4>9 FEFi' M EN iriT/O h 1AL MU ST,37> ?q , QBLUfSlB. RT7 > *£*. tb9 4 T TF F RM fNTFI T IQN I N MU S T 3 0% tffiU.ING AT 3 :m 36 to 4 W FFF?MFN TRTinN IN BF.FR W Q&T RT s:tc 69 6 — 36 W s W F rRMFA/TrtT/ON IN OEX TF? OSE SOLU T ION TT m- al 3L'FC R KCNT ftT/Q N JJSL U n zoo £40 % no i*&.n 75&n <+nn ^o ft Til* F IN Hoim •5 ti- ff . __ — — " — 66 ID n __-- " Q P * i _^ -t n- H 7 i i r FFR >MFN1 V? T7n. Vv-5 /?7 7i3°C 6 s t / IN A U/ST / ?,?)% t =*/?/_/ Il IG. S § i / -? * t if fl 8 / — 67 £ 8/ r .^- — - — — / // / / ' . — .- *^" ** ' /*> P a a it CD .~J± 10 g a^^' ~ =r ~ 9 C * 19 ff **? 7 k i/ 1 *r IN i VUS T tt% F^HLI in a. -* O li a ft ij ^ / 7 ij TIM £" IN A fouFt: > J 4 3 Ik \o h bd % OO A iv & bO 3 £0 3 60 4 CO -Mb Fig. 12. — Fermentation curves of true wine yeasts from California grapes. reason that some wines will ferment completely even at temperatures above 38° C, and others stick with unfermented sugar is because of the difference in the character of the natural wine yeast present. The possibility of a yeast similar to yeast 67 being the only wine yeast present in a natural wine fermentation makes it very desirable to 44 University of California Publications in Agricultural Sciences [Vol. 4 insure good fermentations by the use of pure and selected varieties of wine yeast. Lactose supported the growth of the 8. ellipsoideus yeasts discussed above, but was not fermented by any of them. Attenuation and Alcohol Formation at 33° C. — The fermented liquids were analyzed with the results in table 10. TABLE 10 Yeasts Numbers 64, 66, 67, 74, 75, 79. Attenuation and Yields of Alcohol in Must, Beer Wort, Dextrose, Saccharose, and Lactose Solutions Sfeasl No. Medium Attenuation in grams per 100 c.c Theoretical yield of alcohol Observed yield of alcohol Per cent of theoretical yield obtained 64 Must of 23° Balling ... 19.70 12.80 11.75 93.4 64 Must of 29?8 Balling ... 21.40 13.90 12.80 92.1 64 Beer wort of 14° Balling ... ... 6.16 4.39 3.40 77.4 64 Dextrose solution, 15% ... 11.69 7.60 6.00 79.0 64 Saccharose solution, 15% ... ... 12.23 8.80 7.15 81.4 64 Lactose solution, 15% ... .00 .0 66 Must of 23° Balling ... 19.41 12.70 12.90 11.40 11.40 89.7 66 Must of 29?8 Balling ... 19.75 88.4 66 Beer wort, of 14° Balling . 5.70 66 Dextrose solution, 15% ... 13.20 8.60 7.95 92.4 66 Saccharose solution, 15% ... ... 12.03 8.30 7.55 90.9 66 Lactose solution, 15% .00 67 Must of 23° Balling ... 5.41 3.55 2.00 56.4 67 Must of 29?8 Balling ... 3.85 2.53 1.30 51.3 67 Beer wort of 14° Balling . ... 1.61 1.11 .65 58.6 67 Dextrose solution, 15% ... 12.75 8.38 7.95 94.7 67 Saccharose solution, 15% ... ... 2.82 1.94 1.00 51.5 67 Lactose solution, 15% ... .36 .24 .00 .0 74 Must of 23° Balling ... 19.31 12.70 11.40 89.7 74 Must of 29?8 Balling ... 20.95 13.70 11.40 83.2 74 Beer wort, of 14° Balling . ... 7.96 5.30 4.10 75.3 74 Dextrose solution, 15% ... 11.64 7.60 5.70 75.0 74 Saccharose solution, 15% ... ... 11.83 8.18 7.95 97.2 74 Lactose solution, 15% .00 75 Must of 23° Balling ... 18.31 12.03 8.01 66.6 75 Must of 29?8 Balling ... 22.10 14.53 75 Beer wort of 14° Balling . ... 6.07 4.17 2.34 57.3 75 Dextrose solution, 15% ... 12.55 8.60 6.40 74.4 75 Saccharose solution, 15% ... ... 12.62 8.70 8.60 98.8 75 Lactose solution, 15% ... .36 .25 .00 .0 79 Must of 23° Balling ... 18.51 12.17 79 Must of 29?8 Balling ... 18.54 12.20 11.45 93.8 79 Beer wort of 14° Balling . ... 6.36 4.38 2.35 53.7 79 Dextrose solution, 15% ... 12.60 8.20 6.00 72.9 79 Saccharose solution, 15% ... ... 11.43 7.87 6.00 76.2 79 Lactose solution, 15% ... .06 .04 .00 .0 1918] Cruess : Fermentation Organisms of California Grapes 45 ^ ^0 im tm *o n z^n &m 3$a -y? ^pn ^y ^F7D Fig. 13. — Fermentation curves of true wine yeasts from California grapes. The effect of a moderately high temperature on yeast 67 is shown by the analyses of the fermented liquids, from which it is seen that the absolute amounts of alcohol formed by this yeast are small and that the per cents of the theoretical yields obtained were very small. Thus at 33° C it not only gives very incomplete fermentations, but is also very wasteful of the sugar that it ferments. Yeast 75 gave the highest yield of alcohol, 14.53 per cent. None of the yeasts gave as much alcohol in must of 30° Balling, at 33° C as they did at 24° C. Contrasted with the yeasts discussed in the previous pages the S. ellipsoideus yeasts gave larger yields of alcohol and in general a larger per cent of the sugar destroyed was transformed into alcohol. 46 University of California Publications in Agricultural Sciences [Vol.4 Comparison of Fermentations at 33° C and 24° C (all Varieties) The nineteen fermentation organisms discussed in the preceding pages were transferred to grape must of 29.8° Balling, and the pro- ducts of fermentation at 24° C and 33° C studied and compared. The results appear in table 11. TABLE 11 Comparison of Products of Fermentation of California Yeasts in Grape Must of 30° Balling Fermented at 24° C and 33° C Attenuation in grams per 100 Organism c.c. 35. S. apiculatus 8.31 72. S. apiculatus 6.80 65. Mycoderma species .... 2.70 68. Mycoderma species ... . 4.20 70. Mycoderma species .... 3.40 71. Mycoderma species .... 8.20 73. Mycoderma species .... 23.90 76. Mycoderma species .... 3.20 78. Mycoderma species .... 10.10 37. Torula species 3.70 77. Torula species 6.47 36. S. pastorianus 2.80 69. Willia anomala 5.11 64. S. ellipsoideus 24.60 66. S. ellipsoideus 22.00 67. S. ellipsoideus 24.01 74. S. ellipsoideus 22.50 75. S. ellipsoideus 22.70 79. S. ellipsoideus 25.30 35. S. apiculatus 6.15 72. S. apiculatus 1.20 65. Mycoderma vini 0.00 68. Mycoderma vini 7.84 70. Mycoderma vini 2.05 71. Mycoderma vini 73. Mycoderma vini 14.75 76. Mycoderma vini 00.00 78. Mycoderma vini 14.13 37. Torula species 05 77. Torula species 3.85 36. S. pastorianus 1.65 69. Willia Anomala 2.92 64. S. ellipsoideus 21.40 00. S. ellipsoideus 19.75 07. S. ellipsoideus 3.85 74. S. ellipsoideus 20.95 7;1. S. Hlipsoi /< bn &no z^n & HO .T. ?.n ?% «=>0 <400 <44Ck 9 r//v/r / 'V AYO/ JFf^ » fl V / 1 -66 7 & £ / / s r /*/■> WtZNTt 1T/ONZ /IT 75. 5°C f> , / //V /5/ r F /=? W rOFfT. *> / / / ^ / / 1 k / / 6-f 2 p * / / / 8/ / / /Lr-^ / e>/ 9 fi «6 7 n rrF 'MFNT ATI ON. 5 FIT 75 =5°C 6 IN F EELR ^ VOFfT 5 ^. -f 1 3 2 s -—75 — . to J /^ __ "■"* -J.---« 39^ — / 53 / ^' fe' ' 9 a T" 7 __ __ _--^6 67 6 g • ^^* — --' — •* 5 y 4 m FEt ^Mdd rnnot VS FIT 33° C 3 // 7 /' IN DL XTFfC ■)^E 5 ILIITI ON % $ / // 1 O y ^ TIM tit N HOC r* ^ 10 s a /} W 1 bO % A3 ^ 40 X 30 3 SO 3 60 -4C <*H0 Fig. 14. — Fermentation curves of true wine yeasts from California grapes. that are suitable for the production of wine. Although several of the wild yeasts produce fairly large amounts of alcohol and low volatile acid during fermentation, they can not be used in wine making, be- cause they produce undesirable flavors and odors. An objection to the California 8. ellipsoideus yeasts is the fine grained character of their sediments, giving slow clearing after fermentation and difficulty in racking. Another defect is that some of them seem to be weakened very perceptibily by such a moderate temperature as 90° F (32° C). 50 University of California Publications in Agricultural Sciences [Vol. 4 2Q 12Q. IW. &na &3Q. ZiW TifiQ 7>W dQQ "HP 366) 1UO 44b Fig. 15. — Fermentation curves of true wine yeasts from California grapes. Several varieties of 8. ellipsoideus isolated in France and cultures of which are in the University collection give much better results than any of the yeasts isolated from California grapes in this laboratory. Consequently i1 seems desirable to use one of the French varieties rather than any of the above California yeasts for commercial fer- mentation of grapes for wine. 1918] Cruess : Fermentation Organisms of California Grapes 51 IV. INFLUENCE OF LOCALITY ON THE CHARACTER OF THE MICRO-ORGANSIMS ON GRAPES Methods of Taking Samples 6 Samples of grapes were obtained from Davis, Fresno, and El Centro, all localities in California. Two Mason fruit jars of one quart capacity each were plugged with cotton wool, packed into a small box with excelsior, and the box and contents were sterilized at 150° C in a dry heat sterilizer. Two such jars were sent to each of the above localities. Several small bunches of grapes were cut and allowed to fall into the open jars. The cotton plugs were then replaced. The grapes were sent to Berkeley where the numbers of the different groups of yeasts present were determined. Samples from Contra Costa County, California, were taken in sterile paper bags. The organ- isms on grapes from Ripon, San Joaquin County, California, were determined at the vineyard. Method of Counting Living Cells on Grapes The same sort of grape must agar, and sterile petri dishes pre- viously described were employed in the counting tests. In addition to the above apparatus a number of sterile one c.c. pipettes, and test tubes, each containing 9 c.c. of sterile water were necessary. On arrival at the laboratory, the grapes were crushed in the jars by means of a sterile pestle. One cubic centimeter of the fresh juice was re- moved by means of a sterile 1 c.c. pipette and transferred to a tube of 9 c.c. sterile water. With another sterile 1 c.c. pipette one cubic centimeter of the liquid in the first tube of water was transferred to a second tube. This process was carried progressively to a third, fourth, and occasionally a fifth tube, depending on the condition of the grapes. This gave a dilution of 1 :10 in the first tube, 1 :100 in the second tube, 1 :1000 in the third, and 1 :10,000 in the fourth. From each of these tubes one cubic centimeter of the liquid was removed to separate petri dishes. Melted agar must at 40° C to 45° C was poured into each plate and the plates set away to allow development of the individual cells into colonies. Counts of the number of colonies and notes of the numbers of each type of organism present were made. From the dilutions used it was possible to calculate the number of active cells of each kind of micro-organism present in a unit volume of the original liquid (see plate 1). 6 The writer wishes to express his appreciation of the aid given him by Messrs. Flossfeder, Way, and Packard in taking samples. 52 University of California Publications in Agricultural Sciences [Vol. 4 Results op Counting The results of the countings made, appear in the following tables TABLE 12 Micro-Organisims on Grapes from Davis Sample number 1, Sample number 2, first crop, second crop, Muscat grapes, Muscat grapes, Organism cells per c.c. cells per c.c. Penicillium, green mold (probably glaucum none none Aspergillus mold (probably niger) none 1,000 Mucor mold 20,000 none Dematium mold 130,000 105,000 Powdery white mold none 3,000 S. apiculatus (a wild yeast) less. than 100* less than 100 Other wild yeasts 200,000 True wine yeast (S. ellipsoideus) none none * The dilutions below 1:100 were so badly overgrown with the dematium mold that it was not possible to differentiate the yeasts present, so that "less than 100 per c.c." may mean from none to 100 per c.c. It signifies that no S. apiculatus or true wine yeasts appeared on the dilution of 1:100. There were no true wine yeast cells present, however, because all of the samples were plated out again during fermentation and in this way true wine yeast cells were proven to be absent. Added proof of the absence of true wine yeast is given by the fact that the crushed grapes did not undergo true wine yeast fermentation but after a feeble wild yeast fermentation became moldy and rotted. TABLE 13 Micro-Organisms on Grapes from El Centro Sample number 1, Sample number 2, Organism cells per c.c. cells per c.c. Penicillium (olive green mold) 2,400,000 20,000,000 Penicillium (blue green mold) none 50,000 Fusarium-like mold 30,000 none Aspergillus mold 40,000 100,000 S. apiculatus less than 100 less than 100 Wild yeasts, Mycoderma vini type .... 400,000 8,000,000 True wine yeasts (S. ellipsoideus) none none There were no true wine yeasts present because the grapes did not undergo a wine yeast fermentation but simply became very moldy and finally rotted. TABLE 14 Micro-Organisms from Grapes Grown at Fresno White grapes, Red grapes, Organism cells per c.c. cells per c.c. Penicillium (olive green mold) 10,000 15,000 Aspergillus mold : 10,000 1,000 Fusarium-like mold 10,000 4,000 Dematium mold 7,500 560,000 Mucor mold 200 less than 100 S. apiculatus less than 100 less than 100 True wine yeast (S. ellipsoideus less than 100 less than 100 These grapes underwent true wine yeast fermentation, proving the presence of a few 8. ellipsoideus. 1918] Cruess: Fermentation Organisms of California Grapes 53 TABLE 15 Micro-Organisms on Grapes from Vineyard of J. Swett & Son, Martinez Alicante Sauvignon Isabella Bouschet grapes, verte grapes, grapes, Organism cells per c.c. cells per c.c. cells per c.c. Penicillium (green mold) 5,000 3,000 15 Mucor (gray mold) 2,000 none none Dematium (tree mold) 17,000 22,000 18,000 Aspergillus mold none none 200 Monilia mold 12,000 30,000 10,000 Wild yeasts 14,000 36,000 100 True wine yeast (S. ellipsoideus a few a few a few These grapes underwent normal alcoholic fermentation, proving the presence of S. ellipsoideus. TABLE 16 Micro-Organisms from Grapes Grown at Kipon* Organism Cells per c.c. Penicillium (blue green mold) 1,700 Mucor mold 100 Aspergillus mold less than 100 Wild yeasts 2,600 True wine yeasts less than 100 but more than * The above counts were made by plating the grapes in the vineyard. Yeasts From the Tulare Experiment Station In 1906 grapes were gathered at the Tulare substation by Pro- fessor Bioletti 7 under conditions that precluded contamination by organisms not present on the grapes. The yeasts present on them were isolated by H. C. Holm and their properties studied. All of them were found to be wild yeasts of very low fermenting power and all gave undrinkable fermented musts. No true wine yeast was found. Discussion of Tables 12 to 16, Inclusive The grapes from Davis, Fresno, El Centro, Ripon, and Martinez were all allowed to ferment after crushing and plating tests were made during fermentation in order to obtain the true wine yeast if present. The Davis grapes and the El Centro samples did not undergo true yeast fermentations, but after a short wild-yeast fermentation, became very moldy and finally rotted. The grapes from the other localities underwent normal true wine yeast fermentations and the yeasts of this type present were found on dilutions in agar must made during fermentation. 7 Bioletti, F. T., and Holm, H. C, Calif. Exp. Sta. Bull. 197, pp. 169, 175, 1908. 54 University of California Publications in Agricultural Sciences [Vol. 4 Wine is not made in the regions in the immediate vicinity of Tulare and El Centro, and none is made at the University Farm, Davis. The grapes from Fresno, Ripon, and Martinez came from places near wineries and on all of these samples true wine yeasts were found. These tests indicate that grapes from regions remote from wineries have smaller numbers of true wine yeast cells on their surfaces than grapes from wine making regions. Reasoning from these facts it is probable that the grapes gathered during the beginning of the season will have fewer true wine yeast cells than those picked later in the crushing season. Thus the use of pure yeast is more necessary on grapes from regions distant from wineries and on grapes gathered during the forepart of the season that it is on those picked in wine making regions after the season has progressed for several weeks. The figures given in Part IV indicate the influence of the time of picking on the numbers of yeasts, etc., present. All the samples tested above gave a great preponderance of molds and wild yeasts over the true wine yeasts. It can easily be seen that if there are several hundred thousand wild yeasts and a million or two mold cells for each one to a hundred true yeast cells, as was actually the case in several instances, the true wine yeasts will have overwhelming odds against which to develop. Where such a condi- tion of affairs exists the wild yeasts must of necessity cause consider- able damage. It is not probable that the molds do a great deal of damage after crushing because the conditions are not very favorable for their growth in the crushed grapes, but they undoubtedly de- preciate the value of grapes for wine making during long shipments. In summarizing the above data it may be stated that grapes, both from wine making regions and from those in which wine is not made, are deficient in true wine yeasts and carry a large number of molds and wild yeasts prejudicial to the making of the best wines. Grapes from regions remote from wineries seem to bear smaller numbers of the true wine yeasts than those from wine making localities. V. INFLUENCE OF THE STAGE OF RIPENESS ON THE CHARACTER OF THE MICRO-ORGANISMS ON GRAPES Sampling Grapes were gathered in sterile paper bags from a Zinfandel vine in a large vineyard at Muir Station in Contra Costa County, Cali- fornia, in 1912. One sample was taken when the grapes were green, a second when they had begun to turn, and the third in the middle of 1918] Cruess : Fermentation Organisms of California Grapes 55 September when the grapes were ripe. Since the samples were all from the same vine, they may be taken as being more or less com- parative. A sample was taken from an Alicante Bouschet vine in the same vineyard as the Zinfandel when the grapes were green and a second sample in the middle of September from another vine of the same variety in the vicinity of the first one. The grapes were crushed in the laboratory, using precautions against outside infection, and counts of the active cells present were made as in the tests recorded in Part III. Eesults The results of the tests appear in the accompanying tables . TABLE 17 Numbers of Micro-Organisms on Grapes at Different Stages of Ripeness Ripeness of grapes at sampling Type of organisms found Cells per c.c. of must, Zinfandel grapes Cells per c.c. of must, Alicante Bouschet grapes r Molds 1,040,000 100 Hard, greenJ "Wild yeasts less than 10 less than 10 I S. ellipsoideus none none r Molds 1,000,000 ginning to eolorJ Wild yeasts 175,000 1 S. ellipsoideus none f Molds 190 22,000 RipeJ Wild yeasts 3,360 26,000 1 S. ellipsoideus less than l s but present a few The crushed samples were allowed to stand in sterile jars plugged with cotton. Samples taken when the grapes were hard green and beginning to color, molded and putrefied, but did not ferment, indi- cating absence of 8. ellipsoideus. The samples of ripe grapes both fermented, showing presence of 8. ellipsoideus. On the green grapes molds were about the only kind of organisms present. When the grapes began to turn in color the wild yeasts had made their appearance in large numbers, but no true yeasts were in evidence. On the ripe grapes were found chiefly wild yeasts and molds and in addition to these a few 8. ellipsoideus cells, not enough to develop on the plates of dilution 1 :10, but still enough to cause the grapes to undergo a true yeast fermentation after standing several days in a sterile flask plugged with cotton wool. 56 University of California Publications in Agricultural Sciences [Vol. 4 The surfaces of the hard green grapes are very poor places for the development of micro-organisms; and molds predominate probably because they are merely present in the dust on the surface of the grapes. As the grapes soften during ripening some of them are broken by birds and insects and the yeasts, etc., develop in the broken berries. From these the cells are carried to the surfaces of other grapes by insects, etc., so that as the season advances the numbers of yeasts increase. Even on the ripe grapes, however, the numbers of the true wine yeast cells, 8. ellipsoideus, were small and were greatly exceeded by those of the molds and wild yeasts. The data of this table do not confirm the statement often made that the numbers of micro-organisms increase during ripening. VI. CHANGES IN THE NUMBERS AND CHARACTER OF THE MICRO-ORGANISMS ON GRAPES DURING SHIPMENT FROM VINEYARD TO CELLAR Burger grapes were picked at the vineyard of J. Swett & Son, Ripon, California, and dilution plates on agar were made at the vine- yard on the freshly picked grapes. Counts were also made on grapes from the same block of the vineyard after they had stood about eigh- teen hours in boxes. The sample in this latter case was taken from a lot of two boxes that were crushed into a sterile barrel. After the grapes arrived at the winery in Martinez three days later an average sample was taken and counts of the micro-organisms present again made. Alicante Bouschet grapes were plated at the vineyard and on arrival three days later at the winery. The results of the above tests appear in tables 18 and 19. TABLE 18 Change in Micro-Organisms on Burger Grapes After Picking Grapes after Grapes after Grapes in 18 hours 3 days on vineyard, in boxes, board cars, cells per c.c. cells per c.c. cells per c.c. Organism of must of must of must 1. Penicillium mold 1,700 145,000 ] 2. Mucor mold 100 50,000 ( 3. Aspergillus mold none none ^9,200,000 4. Dematium mold less than 1 72,000 5. Myeodera forms and Torula yeasts 2,500 160,000 4,700,000 0. S. apiculatus yeast less than 1 less than 10 less than 1,000 7. True wine yeast (S. ellipsoideus) less than 1 less than 100 500,000 8. Vinegar bacteria none none 800,000 1918] Cruess: Fermentation Organisms of California Grapes 57 TABLE 19 Change in Micro-Organisms on Alicante Bouschet Grapes During Shipment Grapes three days Grrapes in vineyard, later at cellar, cells per c.c. cells per c.c. Organism of must of must 1. Penicillium mold 320,000 2,500,000 2. Mycoderma forms 125 5,400,000 3. Other wild yeasts none less than 1,000 4. True wine yeast (S. ellipsoideus) 25 440,000 5. Vinegar bacteria none less than 100 The effect of standing over night in the boxes was to greatly increase the numbers of wild yeasts and molds present without mate- rially affecting the quantity of true wine yeast present. No doubt a great deal of the mold and wild yeast cells came from the sides and bottoms of the boxes, where they had collected from broken and moldy berries during previous shipments. Thus, it may be seen how grapes almost free from micro-organisms could acquire large numbers by standing in boxes a few hours. Some of the increase is, of course, due to normal increase of the cells present on the surface of the grapes and to increase of organisms in the grapes broken during picking. The grapes were three days on the road from the vineyard to the winery and arrived in a moldy condition, but in no worse state than other Burger grapes shipped under like conditions. The plating tests bore out what the appearance indicated, namely, the presence of great numbers of molds, 9,000,000 per cubic centimeter; wild yeasts, 4,700,000, and even large numbers of vinegar bacteria, 800,000 per cubic centimeter. The wine yeast was present in less numbers than any of the other organisms, there being only 500,000 of them per cubic centimeter. The Alicante Bouschet grapes gave similar results, but being firmer grapes they arrived in better condition than the Burgers, as was shown by their appearance and by counting tests. A method of controlling the micro-organisms on grapes during shipment is discussed under Part VII. VII. CHARACTER AND NUMBERS OF MICRO-ORGANISMS ON GRAPES AS RECEIVED AT THE WINERY DURING THE SEASONS OF 1911 AND 1912 Samples of the freshly crushed grapes from two wineries in Contra Costa County were taken in 1911 and 1912 and counts of the active cells present were made with the results given in the following table : 58 University of California Publications in Agricultural Sciences [Vol. 4 CD o 1 "* * ft" 2 52 s-i 22 (B OS S ^ ££ T fa ^ h fa C fa W ft 03 P3 W CD CD O ^ 'ft ft "o S l 03 .e8 03 o P- i= "" o co a oqcor^io m to s oo o 1918] Cruess: Fermentation Organisms of California Grapes 59 These tests justify the statement that grapes ordinarily arrive at the winery with a preponderance of the undesirable organisms as com- pared with the desirable true wine yeast, 8. ellipsoideus. The actual numbers of all molds, wild yeasts, etc., are less on grapes gathered near the winery than on those shipped considerable distances before crushing; but the ratio of molds and wild yeasts to true yeasts is greater on clean grapes than on moldy ones judging from the above tests. Therefore, there is just as much, or even a greater need for the use of pure yeast with clean grapes than with grapes in poor condition, although it is extremely desirable to check the growth of the undesirable and promote the development of the desirable types in both cases. VIII. EXPERIMENTS UPON THE CONTROL OF MICRO- ORGANISMS ON GRAPES FOR WINE MAKING Before Shipment Burger and Alicante Bouschet grapes from the vineyard of J. Swett & Son were used in the tests discussed below. Several boxes of Burgers were crushed into a barrel that had been sterilized with strong potassium metabisulfite solution and rinsed out with clean water to remove the metabisulfite. To the crushed grapes was added potassium metabisulfite at the rate of 12 ounces 8 per ton of grapes. This was added in the form of a 10 per cent solution. Four clean Mason fruit jars were filled with the crushed grapes and sul- fited at the rates of 6, 8, 9, and 14 ounces per ton. The metabisulfite is decomposed by the tartaric acid of the grapes to give sulfur dioxide and cream of tartar. Several bunches of Alicante Bouschet grapes were crushed into a Mason jar and were treated at the rate of about eight ounces of meta- bisulfite per ton. The treated Burgers and Alicante Bouschet were shipped from Ripon to Martinez in the same car with untreated grapes. Counts of the micro-organisms on the grapes before treatment and on the treated and untreated grapes at the winery upon their arrival three days later were made. s Twelve ounces of K 2 S 2 3 (potassium metabisulfite) per ton corresponds to approximately 190 milligrams S0 2 (sulfur dioxide) per kilogram of grapes. 60 University of California Publications in Agricultural Sciences [Vol. 4 3 c o .a o o^ «« A O o o 00 cd o ^ a o c o o ■£ s u OS o c c o o o o £ «w o o in -, ° ° 2 o3 Fn £ ee c * IC " R] w c- « •r< ^_ cfi -»- fc ci P 'a R p a > w ES 03 ft 5, > a p 8 S Eh . rt 6 C R 4- O 'r P C - 5 ° ■+- T a to a> Eh P E 'a p A ftl u CL r f- c o