t, l 1“ I‘M ‘ w M] ‘I I I 1 El‘ I H J I W 1 m' lil 11‘ M u ‘ ! { l ‘4 lh i! A STUDY OF THE RELATIVE VITAMIN COMPLEX CONTENT OF THE ' R AND S FORMS OF SACCHAROMYCES CEREVISIAE HANSEN. SAAZ ' THESIS FUR THE DEGREE OF M. E. Harold Buskirk I934 . ' I I . . . I I .n . .. I o no , . y .l . u,. .1! n. r In.a .. v. s 4'.4. . . . ~ - A!..Vu...sn‘.. 5.. ‘ . . . . E . p .. , . .. Q . l .. . . . . _ . l. UY.~»\I\.IWO.. .91? 9|).-2 r.. .urb:.. . r E l‘ . . L . .- . ,,. ‘_ ‘ ru-.. . . . . r . a . ... u E I 0 vlo. . C . . ‘ . . ? . .. .I I 9‘ . n . . 4.) P14. I . . I . . n . I . . . L... . .. -.. r. . .‘ #4:...a . . .ol, \ 44.1 2 o . . . . c . I o .. ...... r. . 7.. .»H¢¢.'l :. u If. v Ail! Ara . . I ' y . .. .0 a . _ . y I A 1 7.4 .\ v I «I ..J . c. ' A; n . ‘._. . . . . . I. .15 .I v r k F v. . . V. , I p . . . . . .1. .. 11%" o .0 I ”V . ”\IV: I .f .rfibof (v , .1. . ., .4 . ‘ ..! . . . ~ t . y p . ‘ E. r/z-vvotr/d f! _. ..-...‘. (I A STUDY OF THE RELATIVE VITAMIN B COMPLEX CONTENT OF THE R AND S FORMS OF SACCHAROMYOES CEREVISIAE HANSEfiy‘SAAZ \ - ‘ “MWedr THESIS Respectfully Submitted to the Graduate School of Michigan State College in Partial Fulfillment of the Requirements for the Degree of Master of Science BY 1'. ' Harold Buekirk 1954 , ('3') 1., t r‘J-J The author wishes to express his indebtedness and gratitude to Dr. C. A. Hoppert, Associate Professor of Chemistry, for his advice and supervision in the planning of this project and his assistance in carrying out the vitamin assays, and to Dr. F. W. Fabian, Associate Professor of Bacteriology, for his invaluable assistance and supervision in growing the yeast. This opportunity is taken to express my thanks to Dr. H. Halliday of the Home Economics Department for her advice in conducting vitamin B assays. *3 "JJ \W 1"“ ..JJ hp: INTRODUCTION In recent years a great deal of emphasis has been placed on vitamins and their role in nutrition. As a result, extensive studies have been made of the distribution. of vitamins. Yeast has long been recognized as one of the best sources of vitamins B and G and recently has been used extensively in the manufacture of ergosterol which can be converted into an antirachitic substance by treatment with ultra violet light. Extracts of yeast containing the vitamins as well as other nutrient parts have been prepared; These extracts are added to foods, medicines etc. Dried yeast has also been irradiated, thereby producing a material of high antirachitic potency. A milk of high vitamin D content is produced by feeding this yeast to milch cows. Studies have been made by various workers on the variation of vitamins E and G in different species of yeast and in different strains of the same species. A quantitative study has also been made of the amount of ergosterol produced in different species of yeast. In these studies no attention was given to the dissociated variant or form of the yeast which was used. In view of more recent work it is evident that yeasts, like bacteria, undergo dissociation. It has been shown that the normal smooth,S form, may be induced by various stimuli to dissociate into a rough, R form, and a gonidial, G form, and also various transitional forms. These three types are distinctly different from eachargoth culturally and morphologically, although they are derived from the same cell of the S form. Since the previous studies were made with yeast in which the forms were not known or recognized, they did not take into consideration the possibilityr that there might be a variation in the vitamin content of the dissociants of the same strain. Accordingly it was considered feasable to make an investigation of this phase of the subject. HISTORICAL Little work has been done on the variation of vitamins B and G in different species of yeast. Quinn, Whalen and Hartley (1930) however, studied ‘five samples of American and Canadian yeast for their vitamin B and G content. Amoung them were two samples of brewers yeast, two samples of dry powdered yeast, and a sample of fresh starch-free baker's yeast. One of the samples of brewers yeast was obtained from Canada while the other was from New York. The Canadian was "a top yeast" that had been dried at the plant in steam heated drums and then ground to a coarse powder. The New York sample was a "bottom yeast" that was obtainadin‘a moist condition and dried in the laboratory. They found that the vitamin B content of the two samples of brewers yeast was definitely greater than any of the other samples, the difference being at least ten fold. The Canadian ”top yeast” was slightly more potent than the New York ”bottom yeast", but the difference was very slight. The vitamin G content of all the samples was very uniform. Bills, Massengale and Prickett (1930) made an investigation of the ergosterol content of twenty-nine cultures of yeast. Eighteen different species and eleven different strains were used. They found that different species of yeast similarly cultured, may differ enormously in ergosterol content. Saccharomyces logos contained but a trace, while Saccharomyces oarlsbergensis yielded 2.0 per cent. Different strains of one species, Saccharomyces cerevisiae, ranged from 0.2 to 1.4 per cent. They Observed that runs giving heavier yields of yeast also showed a higher percentage of ergosterol. The fact that aeration during cultivation greatly influences the yield may have some bearing on the ergosterol content. Previously, the difference in * the cultural conditions had been used in explaining the variation in ergosterol content, however these workers found that different yeasts have different capacities for the production of ergosterol. These capacities may vary favorably or unfavorably according to the cultural conditions. In view of the fact that Bills gt El! found that the ergosterol content could be increased or repressed by manipulating the conditions under which the organisms were grown and also in the light of the recent work of Fabian and McCullough (1934) on dissociation in yeasts, it was suggested that there might be considerable variation in the vitamin content of the different dissociated forms of the same yeast. Since this work has been completed, one of Dr. Fabian's students, L. J. Wickerham, has found that one of the distinguishing characteristics of the R form of all yeasts studied so far is the presence of highly refractile bodies which he has demonstrated to be fat glabules. DESCRIPTION OF CULTURES USED The cultures used were: Saccharomyces cerevisiae Hansen, Saaz strain, American type culture No. 2552, an industrial beer yeast, producing a bottom alcoholic fermentation. No scum is produced on beer wort. The temperature limits for budding in beer wort are from 5° to 40°C. The cells are spherical and produce from two to four ascospores. Agar slant cultures have a smooth, white glistening appearance. For morphological and cultural characteristi O U) U] \D (D figures 3 and 4. t O O -— Sacchromyces cerevms1ae Saaz rough (d). This form was produced by Fabian and McCullough (1934) by converting single cell isolations of the normal S form by means of chemicals such as lithium chloride and brilliant green added to suitable media and also by nhySiCal influences such as desiccation and by aonornal temperatures. The R, form produces. rugose, dull, wrinkled colonies with filamentous projections extending from one to two mm. from the \J a colonial mass. The cells are elongrted averagins 14 to 20 microns in length. Highly refractile bodies were observed in the h cells and tests with Sudan 3 and osmic acid showed them to he fat. However, an extensive analysis has not yet been made. See figure 5. 1a " 7 Saccnromycer ellipsoidus Lansen is a yeast "g a bottow fermentation. In beer wort either round or elliptical cells are wroduced. The temper- :. .. .LHO - .p , . ",7; °., M 3 o . , 4.__ , O ature llh.ts ior bflflLlfl in weer wort are iron 0.5 ,o‘ - D . . -.‘ to 40 0. scum l rmation may occur on l1 uid media. {file cells ixi'hhe ecu 1rn¢rlw3 greatlfi'eilonfiwted. nscos ore formation occurs; the usci .re usually small and ellipsoidal and enclose from one to four ascospores. Agar slant cultures annear dull, white and Often slightly wrinkled. C TIL-T IV ATI 03" It is a rather simple task to grow small amounts of yeast in the laboratory, but when H larger ouuntit 4L es are needed the problem becomes more difficult and cosmercial methods must be adapted to a laboratory scale in so far as oossible. ‘Pbdéo‘rvtz tFwe (Bowrrtrésir of? a (30’T’Tilcix l frezlst producer a formula for a culture medium end sore helofu l instructions Here O‘tnined. The formula recommended for the *edium was as follows: 100 “arts -------------- sugar beet nolssses 2 H —————————————— mono basic annonium yfliosrru te 2 -------------- urea This naterial was diluted with tan water to five brix which is equivalent to five per cent sucrose by weight. The acidity Was adjusted to pH 5.0. As alternatives, three parts of ammonium sulfate may be used in place of urea, but this necessitates considerable more neutralizing. Aqua ammonia may also be used by adding it each tour and ad ustiug l._Jo the acidity. However, urea is recommended as it gives the least trouble. other formulae were used but the above gave-good yields and proved very satisfactory in all other respects. sl‘lkh’ hTTTS To insure greater “izlds, it was necessary to aerate the growing yeast. Therefore an annaratus had to be construcsed for this nurnose. An eight liter 1 Hide-n uth bottle v: .11 .s chosen for the recentacle. The aerating devise consisted of first;- a ten inch cotton filter thick filtered the air from the condressed air line renderinfi it sterile. "he filter was node by sacking cotton into a Tyrex tube one inch in diameter by ten inches lone. One-holed rubber stcvnirs, with % inch tubing inserted, were fitted into each end. (See Fig. 5) The filter Was sterilized for 50 minutes at 15 lbs. . , .. 1 ‘ .- 1 ' ('- r ~~r o .- 1 r' .' - . 4... 1,,.\ v. t ' ‘ ‘I .~., pressure. second,- a closed “frex UJUC, a ihcn b; A - ’ 1" - ' ‘. F‘- ‘Y . " I. ‘ ‘. 1 . .,.‘ ‘ 4- - ‘ . [I ' lij irlnies lxvi,, Rwltfll fetus-4 ixicn_xnihes ISOulffil on 4. . . .. "* n ~ ' ,.. s. . * J- 7‘ LflflJQS are”? Cit CL”: lCUVlur' tn.r1z.bout ; inches long. - - .: I. °- «A .-. .- '°. 1. - '33 these ;imr,nltions :Lflfl3.,ttached.~vilnni ”yrex . ‘u . jets two inches in length containing U~i (:9 E I...) ...—4 I)" O H CD CD on the usher side. The :ets were Connected with gum rubber tubing which made them flexible enough to be placed into the bottle through the four inch mouth. This Dart of the apnaratus was used to bubble air ue through the medium. It was sterilized by autoclaving for 50 minutes at 15 lbs. pressure. An incubator was constructed to accomodate the apparatus. This Was regulated t maintain a temper- o ature of 50 C. C3 GROVIEG OF 8160”” VEiST The medium Was nre ared according to the formula given previously. Usually from 500 to 800 grams of molasses was made un at one time yielding from six to ten liters of liquid. The acidity was adjusted with sulfuric acid to slightly below the desired pH to nrevent the nrecinitation of nhosnhates during the sterilization nrocess. Sulfuric acid was used in order ttct sulfates nijht be sur*li d to the medium. The material was nlaced in two 0_ "I“ liter I ask {.0 and autoclaved at 12 lbs. nressure for 50 minutes. The reaction was readjusted to OH 5 just before it Was used. All nH determinations were made electonetrically with a quinhydrone electrode. The bottle wa UJ sterilized chemical y with cleaning solution, rinsed with hot water and with the aerating device inserted, was nlaced in a steamer and heated. The hot medium was noured into the bottle filling it about 2. This was again heated to make sure that the bottle and contents were sterile. After cooling, the bottle was inoculated with afiout 100 cc. of yeast culture grown in molasses medium for two or three days or until a good growth was wroduced. Each inoculum was nrenared by washing a 24 hour growth from an agar slant into 100 cc. of molasses medium. Before transfering into ‘LO the bottle, however, each inoculum was examined micro- scopically for freedom from bacterial contamination. A few drons of corn oil was added to nrevent foaming. The aerating device was connected and air was allowed to bubble moderately through the liquid. The temner- ature was kept between 280 and 3000. Although the directions called for a DH determination at hourly intervals, it was found unnecessary from experience because no change was observed. Accordingly the pH was adjusted to the nroner noint at the beginning and checked at the close of each run. HAH‘ 'ESTI'I~JG AT‘ID DRYING When a sufficient amount of yeast was produced which was indicated by the turbidity of the medium, the air was turned off and the yeast was allowed to settle for several hours. At the end of this neriod the yeast had completely settled out leaving a clear supernatant liquid which could be easily synhoned off. The remaining liquid was removed by placing the material in centrifuge bottles ard centrifuging. The yeast was washed twice with nhysioloqical salt solution and removed by centrifuging. Refore harvesting, the yeart was again examined micro- scopically for bacterial contamination. All con- taminated yields were discarded. The yeast was nlaced in an evaoorating dish in which it was dried at 3700. This was accomnlished in about 24 hours. The dried yeast was ground in a hand mill and finally in a ball will which puliverized it to a fine powder. One hundred or more grams of each kind was necessary for the vitamin assays. GROWING OF hOWGW YEAST An attempt was made to grow rough yeast in the same manner as the smooth, but so much difficulty was experienced that the method had to be discarded. The rough type of yeast grows on the surface, where as the smooth type grows near the bottom. In aerating the rough forms a foam was nroduced which could not be checked nermanently with corn oil as was none in gnowing the smooth. This foam kept risins ntil it nushed the cork from the bottle and ran over the side. The roughs were, however, sucessfully grown in two liter flasks containing from one to one and one-half inches of molasses mediuw. The flasks were inocultted, shaken occasionally and allowed to grow for two or three days or until sufficient growth was obtained. Then the contents of all the flasks was poured into one and allowed to settle. Some of the yeast remained on too so that not as clear a separation could be made by syohoninq as was obtained with the smooth. 'Therefore, more of the yeast had to be separated by centifuqinq. In all other details the process of harvesting and drying was the sam as for the S form previouslv described. 11 ASSAYS FOR.VITAHIN B COMPLEX The purpose of this investigation was to make a comparison of the vitamin B complex in the S and R forms of Saccharomyces cerevisiae, Hansen, Saaz strain with that found in other species of yeast. For this purpose, they were compared with Saccharomyces ellipsoidus S and a commercial dried yeast. The method, in brief, involves feeding young healthy rats a diet complete in all vitamins except the ones to be studied. The material to be tested supplements the diet and is evaluated according to its ability to induce certain rates of growth in the animal. Since both vitamins E and G are required for growth, rate confined to a diet lacking of either vitamin in a short time start losing weight, but when the deficient diets are supplemented with materials containing these vitamins, the rats respond by gaining weight in proportion to the amount of vitamin given. The change in weight of the experimental animal is therefore taken as the chief criterion for the measurement of vitamin potency. Rats weighing from 60 to 70 grams were selected for the assay. Great care was taken in choosing animals that were uniform in weight, as this is a very important factor in the animal's response toward various amounts of vitamin B comnlex. The animals were fed a basal ration essentially sinilar to that devised by Barnett Sure (1955) which follows: Purified casein............. 20 “gal" a;>;a1‘.............oo.... 2 MaCollums salt #185......... 4 Filtered butter............. 10 DextriHCOOCOOOOOOIO000...... 6A.; A modification of Steenbock's salt #42 was used «7” P. CO 0": e in place of UaCollums s*l 9: as some vitamin B conslex is stored in the bodies of the rate, it was necessary to deplete the animals of these vitamins before starting the assay. This WLS accomplished bv fe ding the basal ration unsunnlemented. The point of depletion was recognized when the animals failed to gain weight. This required from two to three weeks. at the end of this *orioo, the aniMals were placed in individual cages with raised screen bottoms. Litter mates were used on corresponding levels of the R and 8 forms, but due to some irregularities in some of tne liters, this plan could not be followed throughout the exoeriment. Various samples of yeast were supplemented in the diet. A week's supply was given at one time. The yeast was put in an earthenware cup, mixed with a little basal ration and placed in the cage. The cup containing the basal ration was not removed. This was kept well filled, so that the animals could eat from this at will. Some animals from each groun were selected for controls. They received the basal ration only. The animals were weighed once a week on the day that they received their supply of yeast. A preliminary experiment was run on Saaz R to determine what levels to feed the yeast. The levels selected proved to be correct but as there was such a wide variation in the rats, it was thought advisable to discontinue this series and select a more uniform group of animals. (See table 1) The animals chosen proved to be very satisfactory. DISCUSSION As the vitamin B unit, defined by (Sherman), is given in terms of a daily dose, all levels of yeast fed were listed as grams per day, although it was actually administered in the form of a week's supply. For Saaz R and S, the levels of 0.25, 0.50 and 0.75 grams per day were found to be in the correct range. This also served for Sacch. ellipsoidus but with the commercial dried yeast, lower levels were necessary. Levels were chosen that were multiples of each other, in order to simplify calculations. Thus the levels were 0.0835, 0.1667 and 0.25 grams per day. 14 By inspection of the tables the results can be readily visualized. In table 1 a comnarison of the vitamin B complex in Saaz R and S was made. Some irregularities were noted among the data, but they seem to be minor and most of them can be accounted for. In the case of the 0.50 and 0.75 gram levels of Saaz S, one animal on each refused to eat the yeast in the alloted time, however, the animals finally became accustomed to it. Upon close observation of table 1 and 4, it can be easily seen that there is nractically no difference between the Saaz R and S, however, the data seems to indicate 9 the Saaz S to be slightly more note t. The difference however is not great enough to be significant and could easily be in the range of experimental error. In taolel2 and 5 a comnarison of the vitamin B complex was made with a commercial dried yeast (Northwestern brand) and Saacharomyces ellipsoidus. In the case of the commercial yeast, the levels were considerahry lower. The 0.0835 gram level gave as good a rrowth as the 0.25 gram level of Saaz R and S and much better than the Saccharomyces ellipsoidns S,. Thi indicates that the commercial 0) dried yeast has a potency three times grea,e than Saaz R and S. It also indicates that its rotency is greater than Saccharomyces ellipsoidus. The tests show that the R and S types of Saccharomyces 15 cerevisiae are slightly more potent than Saccharomyces ollipsoidus. When the tests were completed, the animals on the lower levels, at which they did not gain very readily, or were losing weight, were fed 0.25 grams per day of the Northwestern brand of dried yeast to determine if they had lost the ability to respond to the vitamin B complex. All of the animals gained from 10 to 20 grams per week. This indicated that the rats were normal in their response to the vitamin B complex, and their failure to grow was due entirely to a low content of this factor in the yeasts fed during the first part of the experimental period. STILZlinl‘L" all) COHCLETSI )1? As the result of these experiments, the followinv observations seem warranted: ) 'W 1. There is no sixniiicant difference in the .5 vitamin 3 complex coats t of the S and R _orms '40 of Saccharomyces cerevisire Nansen, Saaz stra n. 2. The corners al dried yeast contained appreciahly more of the vitawin B comilex than the S and R forms of Saccharongces cerevisiae and Saccharomyces ellipsoidus 8 form. 5- T33 GLVLI ir which the various veasts arranied tbemselves in their “otrncv of tbe vitamin B complex, is as follows: Sacch. ellipsoidus, the least potent; Sacch. cerevisiae, Saaz R and S forms the next most potent; Commercial form of dried yeast the most rotent of all. TABLE I Comparison of the Vitamin B complex of R and S Forms Saccharomyces cerevisiae Hansen, Saaz as Shown by Growth Records of White Rats. Ir Saccharomyces cerevisiae Saaz R Rat Gms.Yst Initial lst 2nd 5rd 4th 5th . Total Av.Gain No. per day weight ieek week week week week' Gain per wk. 2 0.25g 95g 955 955* 0 0 4 0.50 98 108 118 * 20 10 5 0.75 79 87 95 * 16 8 10 0.25 91 . 89 83 * -8 -4 15 0.50 75 78 75 * 0 0 11 0.75 94 104 116 * 22r 11 5 0 91 86 79 died -12' -6 9 0 82' 72 54 died -26 -15 15 0.25 72 79 85 84 81 as 15‘ 2.6 16 0.50 67 75 75 77 79 - 12 5.0 21 0.25 61 66 71 75 ' 72 - 11 2.75 22 0.50 60 71 ‘77 76 ‘ 79 - 19 4.75 Saccharomyces cerevisiae Saaz S 18 0.25 70 71 79 77 80 87 17 3.4 19 0.50 72 74 92 102 106 112 40 8.0 ' 20 0.75 75 90 109- 121 150 141 68 15.6 24 .25 59 65 7O 7O 7O 7O 11 2.2 25*“ 0.50 61 61 65 65 72 77 16 5.2 26*“ 0.75 56 60 66 70 80 85 27 5.4 * Animals were discontinued because they were not uniform. ** Animals did not eat all of their yeast supply in a week. The Vitamin B 00mplex in the commercial Dried least as Shown by Growth Records of White Rats. TABLE II Commercial Dried Yeast (Northwestern Brand) Rat Gms.Yst Initial lst 2nd 5rd 4th 5th lo. per day weight. week week week week week Total Av.Gain Gain per wk. 27 0.25g 68g 85g 91g 983 108g 115g . 475 9.4g 31 0.50 82 . 90 100 a ' 18 9.0 33 0.75 80 102 123 * 43 21.5 34 0.25 76 94 103 107 127 142 66 13.2 35 0.50 67 87 98 a 31 15.5 38 0.75 72 100 117 a . 45 22.5 28 0 84 77 66 died -18 -9.0 36 0 83 78 66 died -17 -8.5 40 0.0833 63 64 70 73 75 78 15' 3.0 41 0.1667 67 70 78 90 93 110 43 8.6 43 0.0833 81 78 87 88 89 94 13 2.6 44 0.1667 65 70 82 90 95 100 35 7.0___ * Animals were discontinued because the levels were too high. 18 19 TABLE III The Vitamin B Complex Content of Saccharomyces ellipsoidus as Shown by Growth Records of White Rats Saccharomyces ellipsoidus Hansen S Rat Gms.Yst Initial lst 2nd 5rd 4th 5th Total Av.Gain lo. per day weight week week week week week Gain per wk. 59 0 59g 45 died -l4g -l4.0g 45 0.25 64 60 57 51 58 61 -5 -O.6 46 0.50 50 58 65 75 80 80 50 6.0 47 0.0855 75 60 55 55 (67) (77)(82) 48 0.25 77 72 75 72 74 77 0 0 49 0.50 61 67 74 82 100 105 42 8.4 50 0.0855 62 57 57 57 (75) (90)(98) ( ) 0.25 grams of the Northwestern was given per day in place of the one indicated. TABLE Iv SUMMARY or DATA Showing the Daily Yeast Consumption and the Average Weekly Gains of Each Series of Rats Fed) on the Different Types of Yeast Yeast Gms. Yeast Average gain per day per week Sacch. cerevisiae 0.25gms. 2.67gms. Hansen, Saaz R 0.50 ~5.87 ' 0.75 9.50 Sacch. cerevisiae 0.25 2.80 Hansen, Saaz S 0.50 5.60 0.75 9.50 Sacch. ellipsoidus 0.25 -.50 Hansen 8 0.50 7.20 Commercial Dried Yeast 0.0833 2.80 (Northwestern Brand) 0.1667 7.80 0.25 11.50 0.50 . 12.10 0.75 22.00 ‘n” .. | 1' 91"- . ....r 'C 1113 lulu. 44.x-) ,._11 Quinn, Whalen and Hartley (1950) J. Nutrition 5, 257-65 Bills, Hassengele and Prickett (1950) J. Biol. Chem. 87, 259-64 Fabian and luCCullousjh (1954) J. Rest. 27, 585-625 Sure (1955) Proc. Exptl. Viol. Red. 50, 779-80 21 Fig. l Photomicrograph of 8. cerevisiae Hansen, Saaz showing typical 8 forms of the yeast. ( 560x ) Fig. 2 Photograph of a typical 8 colony of g. cerevisiae Hansen, Saaz. ( 8.5x ) Fig. 4 Photograph of a typical R colony of §. cerevisiae, Hansen, Saaz. ( 15x ) 25 Fig. 5 Photomicrograph ofl§.198revisiae Hansen, Saaz showing typical R forms of the yeast. ( 560x ) Fig. 5 ¥ 1 Compressed air line ‘--"-"I’A .- 'I’I'Il' Zjl71‘ — _ (N ‘ . _. ‘. . “. ‘1‘. ocniermitxic Lira vii ; 8 1“ . 1"]:1“. -_ 'w l. . f‘.’ k .L ‘J A - - hwr Var; '- .'e.atus large quantities of yeast. 7 17 Cotton filter I— 4 "7 I _ I usrxl tr) APPARATUS FOR GROWING YE%ST grow Thermometer Battery r ‘ --—-—b—Q—..' _._._...,. 1m 1(7-o-00tton Y7flz/[r/z'l b" - --I gfi \ \ \m\\\\x\\ \ixi \\ \ \ \\\\\\ a X \ \\X\\\\ \ \\\\ T Thermostat-- U plug ~~~Glflna , H tube H Large bottle-- Aerating device-—g;:1;:-;:- __ __ — — — _ — _ 3--.-_—.J Lea-..-: V '_C.'f\ 0‘,- [‘14: 1.... . \ -"‘-L.LI: ”‘4 \j T. --v-tl \i\\\\\\\\\\gh;nr. 1L ’(,/://;//J////II_////////////y/[l/I/lJ/l /// k r .1. k 8 u B lllllllllllINllll”INN”lllllillHIH’HIIUIIIIIIIHIIIWI 31293 02446 6645