.it.-<, the A.-i>iiini-itf.'«, must not, however, be tjilcen to imply that the fungi of this class are ex- clusively reprotlucetl by means of ascospores. On the contrary, emphasis must be given to the fact that the prof the Phi/romi/ret'it, it must, for this purpose, prcxluce internally as many tninsverse septa as will corresjx)nd with the number of resting cells to be formed. This constitutes the exceptional ca.se referred to in § 218. where the occurrence of septa in the unicellular my- celium of a Phi/com ycetes can be observed under normal cir- cumstiinces. Special partition walls ai-e also forme«l in such cases in the alre;idy septiitvd mycelimn of the Mt.C"inijctt'f. The formation of germinative resting cells by the breaking up of the mvcelium, was first observed in the cjise of a funmis known as Oidiinii / ^-mi.-, in.- ile nutrient solution) were ex- posed to an atmosphere containing 60 per cent, of oxygen and 40 per cent. of C4irl)on dioxidi'. Es('11K.\ha(;ex (I.) obsei'ved siuiilai- malformations in cul- tures grown in excessively rich nutrient solutions, /'.>/. a 60 p*'r cent, sugar solu- tion. According to M. U. UEi.\H.\HnT(I.; they also occur in mixed cultures, as u result of the injurious elTect, on one of the symbiotic organisms, of the meta- bolic products of tlie other. A fungus is s.iid to be monomor- phous when the s;ime is only known to fructify in one single maiinei-; whereas fungi exhibiting two or more methoil> of fi i»cti(j«-ation are termed pleomor- phous. The Mu<-i>riuf^a (J5 235) form a go(Ml example of pleomorphisuj, and the .s;ime occurs in the, to us, still more in- teresting Stirr/Kinniii/'-, f,.t, which latter always exiiibit three dilVertMit methods of fructitication.viz. l>v conidia, gemnne. and endospores Owing to their Pro- tean chaiacter, these organisms (xvupv u peculiar position in the genenil mor- phology of the fungi ; and in them we see, more than elsewhere, liow the same organ can change its nature and undergo miMlilicji- tion from one to anothei-. .According to the comprehensive researches of H.vxsen (X.\ \ 1 I 1.). the yea.st cell may .serve (i) as a coniiliiun, for veget^ilive reprcxluction ; (j) as jvut of a mycelium ; or (3) jis an ascus and therefore jtrtnlucing internal spores. Finally (4), the spore is capable oi not only acting vegetatively (germinating), but also, under certain conrv|>araUon o( the «;i-lmcn. )lni;n. S^). (A itfr 26 GENERAL MORPHOLOGY. Among the Eumycetes, the oidia and gemmas are the most frequent varieties of fruit ; but this cu-cumstance, coupled with the simplicity and uniformity of their structure, renders these characteristics almost entirely valueless as a basis of classifica- tion for the fvmgi, since this classification is mainly founded on differences in the occui-rence, mode of production, and develop- ment of one or more of the other three reproductive organs (endospores, conidia, zygospores), in the species to be differen- tiated. Fungi wherein these organs have not yet been observed, or wherein the latter appear in a form which does not permit their inclusion in the existing system, are set apart in a special class as " Fungi imperfecti," a term, however, expressing not incompleteness in the fungi themselves, but only in our know- ledge concerning them. A few of these species, e.g. the so-called Saccharomyces ajjiculatus, certain Mycodermas, the Moniiice, the Toruhe, kc, fall within the province of the present work, and will be dealt with fully in the final Section. § 224— The Germination of Spores. Their Tenacity of Life. The spore is ripe when it has acquired the capacity of de- veloping into a new individual of the species from which it originated. The first stage of this development is termed ger- mination, and, in the case of endospores and conidia, is a com- paratively simple process. It has already been fully discussed in § 218, and illustrated in Figs. 92 and 93. Deviations from the main lines theie laid down, however, sometimes occur ; and with one of these we shall later on become acquainted in the case of Saccharoniyces Ludwigii. While previous separation from the parent plant is unnecessary for the commencement of germination in the conidia, the endospores must have been set free therefrom by the decomposition or breaking down of the wall of the sporangium or ascus. More will be said on this point in § 235. Special experiments by P. Lesage (III.) have placed beyond doubt that the development to mycelium of a spore geiminating on a solid nutrient substratum proceeds the more rapidly and luxuriantly the higher the water-vapour ten- sion of the superincumbent air. The germination of the zygospores commences by the burst- ing of the episporium, and the extrusion of the endosporium, in one or more places, by the pressure of the swelling cell contents. When the zygospore is submerged in a liquid, the development is of a vegetative character, a mycelium being produced ; but when, on the other hand, the spore is exposed to the air, it puts forth (fructificatively) a fiuit-bearing stem, which then produces spores in its turn. An example of both instances, in zygospores THE OKIIMINATIOX OF SpOKhX 27 of one ami tlie s;inie species, is given in i' i;,'. 107. See ali>o division 5 in Fig. loi. The Slime also applies, in many instiinces, to chlam\'Josj>ores, i.e. these also may germinate in two ways : either vegetativelv 4^4 Vh.. 107.— Spdrodlnla Aspergillus S<-hrmnk. FriKtifli«tivelyRem)tiiatinc np. -i-Tf. .Magn- ^«:v. (AOtr Brt/eld.) V«T;i-UtiVfly Koniiiiintiiif: ■ ,, ., Two ripe si>onintdii of ihi .ring stem. Magn. iso. (AJXer tK Barv.) Azyguapore. Mapi. i,^. \Ajt, , 1. rvti Tar*V, or fructitiftitively. An example of a fungus |X)ssessing these l>ri)perties is afTordeil by Chlatnijdoinwor n The fructi- fii'utive germination of its chlamydosjx>ri - ..- i«pri*senteesfris, and others wherein A. de Bary had observed the assumed fungus cellulose — this worker, in 1881, proved that when such membranes did not immediately give the reactions in question (especially with iodine), they could be induced to do so by steeping them for not less than a fortnight in a 7 /to 8 per cent, solution of caustic potash. The elementary composition of the membrane so treated, he found to correspond to the formula 7^{C^'H.y^^0r). Even this observation did not remain unopposed, the same hypothesis being urged against it that every investigation into the nature of the cell membrane has had to contend with ever since the days of Pay en, namely, the question whether this preliminary treatment of the membrane merely results in the ex- traction of the extraneous admixtures, or whether it is not rather that the fungus cellulose is converted into true cellulose. How- ever, even apart from this doubt — which will be further con- sidered in subsequent paragraphs — Richter's observations do not disprove the assumption that a substance, differing from pure cellulose, occurs in the membrane of fungi, since, in some of his experiments, the cellulose reaction could not be observed in the preparations employed. Nevertheless, although, in view of the last-named circumstance, it cannot be admitted that, strictly speaking, Richter's experiments actually identified fungus cellu- CELLULOSK. v> lose witii tiuf cellulose, it is j(i8tifiiil)ie to nssuiiio from his ri-.-,uiu tlmt j>uii« eellulosM iimHv does occur iu the c«*ll lueiuljntue of fungi. An utteinpt Ui supiiort this assuiuptiuu wus uiade by I. Ukeyfl'SS (I.) in 1S93. For the purpose of extnictiuf; the cell contents jintie sotlii, and then heating to iSo" C with concentrated c;iu>tic potiish, whereby — a.s was then .sup{>oseJ — everything except pure cellulo.se was decomj)oseeiuj,', moreover, ascertiiiuoil to cont'Spoiid witli tlie foniiula CnIl.,^N'.,0,,j. This substance is ii Imse, the chloriile of which is thi*o\vn lf in 2 t<» 3 per cent, hydrocliloric acid or in very dihite acetic acid. A sohiti(jn of iotline in |x>tassiuui icxlide, conUiining a trace of free aci,' a hlue to blue- violet coloration. These last reactions closely resemble those of cellulose, and mi^'ht easily be mistaken, by an inexperienced ob.server, as indicatinjj^ the pre.sence of that sukstiince. This possibility will be referred to later on. A closer indication of the position to be allocited to mycosin in the immense lieM (if organic nitro-^en compounds wasatforded in a .second investigation of Wixtehsteix's (II.) by the discovery that this substance is decomposed, on hydrolysis with 3 per cent, sulphuric acid, into: «/-f;lucose as the chief product; then probal)ly other hexoses as well, and then into acetic acid and an undetermineil nitroj^enous organic sub.stance. Deci.sive conclu- sion as to the method of combin.ition of the nitrogen in this cell-membrane substance was .soon afterwards furnished by WiN- TEUSTEIK (III.) through the discovery that, when heated with concentrated hydrochloric acid, it yielded a ciyst:illis:ible fission product, which proved identical with the hydrochloride of chito- .samine, C.11,,0,.X11.,.11C1 — at that time erroneouslv ts;in is basic in character, and is precipi- tated from its acetic solution by an excess of caustic jHita-sh. The chloride is insoluble in .strong hvtlrochloric acid. A similar dissoci^ation pnxluct is already known in the mycosin prejvireil 36 THE CELL MEMBRANE OF EUMYCETES. by Gilson in 1894; and as a matter of fact, chitosan and mycosin are identical, and can be isolated by the same method, both from animal chitin and the cell membrane of fungi. In this manner it was proved that chitin is also present in the fungoid kingdom, and is not, as was formerly supposed, of ex- clusively animal origin ; and indeed the proof was strengthened J by E. GiLSON (III.) and E. Winterstein (IV.) isolating chitin, as such, from Agaricus campestris. The nitrogen content of this substance was determined by these workers as 6.24 per cent., a value agreeing fairly well with the 6.01 per cent, calcu- lated from the formula (OisH3oN20^2) ^^t up for animal chitin by G. Stjcdeler (I.) and confirmed by T. Araki (I.) in 1895. On the basis of this formula the two hydrolytic reactions above referred to may be expressed by the equations : — C18H30N2O12 + 2H0O = C14H06N2O10 + 2C2H4O2 Chitin in the Chitosan Acetic acid potash C14H26N2O10 + 2H2O = 2C6H13NO5 + C2H4O2 Chitosan in the Chitosamine Acetic cone. HCl acid It may be mentioned that chitosamine, and even chitin, can be split up by the prolonged action of concentrated hydrochloric acid into ammonia and a sugar : — CeHiiOo. N H2 + H2O = NH3 + CsHiaOs to which Berthelot has given the name chitose. The formation of this sugar by the action of concentrated hydrochloric acid on preparations of the cell membrane of fungi has already been observed by St.edeler (I.) without, however, the nature of the reaction having been recognised. Bearing in mind the fact that Dreyfuss employed concen- trated potash at 180° C., tnider which circumstances the chitin present would be converted into chitosan, the latter — according to Gilson — then behaving like cellulose in presence of certain reagents, it will be readily understood why Dreyfuss arrived at the conclusion that he had to deal with pure cellulose. Accord- ing to the results obtained by Wisselingh (L), the same con- version, and consequently the same liability to deception, also occurs when chitin is exposed for a considerable time to the- action of dilute (7 to 8 per cent.) caustic potash at the ordinary temperature, i.e. the same treatment as employed by Richter in purifying the cell membrane preparations. Finally, it is not inappropriate to recall that the formula given above for chitin is not yet entirely beyond dispute. It cannot at present be definitely stated whether the relative- « values of the bodies taking part in the foregoing reactions are 1 accurately represented by the equations laid down by Araki ; in || fact we have still to face the possibility of there being a whole ("HITIN. 37 series of isomeric or nearly allied substances, aiul therefore of the necessity of rej.'anlin'; the name cliitin as a collective ttMm. The extent to which chitin is jiresent in revious jiaraf^raiths. Its absence was confirmed by this worker in the case of liaderia, Ounn/cetejt {S(tpruleSac''Jian/iiii/relen examined by him. In all the other species of finifji examined, however (about a liundred), the presence of chilin was invariably detected, e.'j. in MuL'ur muceilo, M. rucemosm*, Rhizoinu* nii/rirann, Penicil- h'utn ij/itnruiii, Tn'rothecinm rosruin, in the sclerotia of fiutnjtis rini'rea and C/arire/ix jntrjiurra, itc. At j)resent no inst;ince of the simidtuneous occurrence of cellulose and chitiu in the cell membi-ane of a funjrus is known. In many cji-ses, e.if. the jterithecial wall and the a.sci of A/i/xri/il/iin i/luui-if^, the membrane is partly conj[)osed of other substjinces. With the assistance of im})roved microchemical tests \Vi.sselin;,'h has also studie-ling. Fouh- ling. and Pe-fuh ling, this worker i.solated a carluihydnite of the formula ^^'ooH^s^.j^' which, on being treated with dilute acid, is converted into a licjuid capable of nvlucing Fehling's .solution. To this carbohydrate^ he gave the name pachyman. l-'iter, E. WlXTEUSTElN (II.). isolate.l from the edible boletus (/hit'cipitated by alcolud. This substance 1ms re*^'eiveideriible (piantities of other compounds, poorer in or altogether devoid of nitrogen, were also pi-esent. At tiio jtresent time there is very little that is reliable known with regartain information on this point; on the contrai-y, these date as far back as the efforts made by W. FiKi.sTiNC! (I.) in 1S68. At a later period the matter was energetically taken up by L. M.\N<:i.\ (11.), who repoited that, in the Miirorintd examinetl by iiim, the inner layer of the .septii and aerial hy[tha' consists of cellulose, whilst the outer layer is compost'(lof pectin bodies. I'nfortunatfly the above-mentioned obseivations of \\ i>S(lingh have seriously called in (piestion the reliability of the microchemical reactions on which Mangin based his assumptions. The reader will not expici to tind here any genenil lepoits on the thickness ot the cell membrane in fungi. Nevertheless, mention m.-iy be made, in this connection, of a fact determined by Fk. KscMKNH.vtiKN" (I.), namely, that the concentnition of the nutrient .S()lution luis a direct inlluence on the thickness of the cell membrane of the organism grt)wn therein. The cell membrane of fungi also often exhibits in a liigh . . . degree the capacity of swelling ; which, indeed, is fre4puMitlv an indispi-nsable faculty, especially in ajKO'cnii/ia and (/.red one may r(>a.sonably a.ssume the pre.sence of pectin substances in the cell or menilirane. According to the concordant results obtained by A. lJriu;KK- STEIN (I.), M. NiGGL (I.), and C. C). Hauz (II.), lignitication of the nuMubraiie doe.s not seem to occur in Mucur miiccdo, Peiiicil- liutii i//(inrii)ii, Asj>er(;tllus ij/nurui^, and Sorrfiaromi/riii rrrtvisict, or, according to the ob.servations of the la.st-named worker, in j\Iucor nicfrirati^. Aspen/ill ii,t ro>ioih(ilothe<'ium rosteufii, Tuhrr rifni- riuni, T. ifstivum, f'^'v''-'>p.'i jiur/nirea, and Torula. On the other 40 THE CELL MEMBRANE OF EUMYCETES. hand, the lignin reaction (with sulphuric aniline sulphate) was furnished by the large pileated fungi, though whether the pre- sence of lignin is thereby proved must remain an open question. Not infrequently a deposition of colouring matter is found in the cell membrane, usually in such a condition as to be incap- able of extraction by any known solvent ; this is the case with the conidia of AspergiUns and Penicillium. In other coloured fungi the colouring matter is embedded in the plasma ; of this a fine and technically important example has already been given in § 219. Whether the waterproof character of some cell membranes, e.fi. in the conidia of Penirillivvi and AfpergtUus, should be attri- buted to the deposition of excreted fatty or waxy substances, must be left undetermined. Biologically this phenomenon is important since it prevents the penetration of toxic substances from the surrounding aqueous medium, and thereby also opposes the attempts of the mycologist to kill such fungi by means of aqueous toxic solutions. Deposits and incrustations of calcium oxalate crystals are of very frequent occurrence in the membrane of fungi, especially on the surface of the spores. In many cases their presence and appearance afford characteristic indications valuable for the jiurposes of classification. ( iiArrKiJ XT.r. .MlNKKAl- XT'THIKNT MATEIUALS. J5 228. Alkalis. A GLANCE tliroii^'li the existing unalvti-iil will sodii ii-Vf.il tliiit thechief of these eoiistitvients Hie phosphoric acid mul potash. The latter sely I^iKitic (II.). contains 54.5 per cent, of K.,0 an ..f the.se three must invariaMy be present. With regard to ca-sium, all sub.sef|uent investigators, how- ever, agree that this met-al is unsuitable for replacing pot;issium for the purpose in (piestion. InstJiiices of this are furnishetl bv S. W'lMHiUAnsKY (XI.) in his culture experiments with Mi/ru- (I'-niiii rini : W. Hkxecke (II.) in the case of I'' itirilltum ijhiurnm and Aspenjillus ///_//'•;•; and sub.setptently by E. (Jue.nthek (I.) for Miicor cori/mhifW, Jihi^ojnis ui'ji iraiiJ^, and Ii(>fri/fi.< riturea. Opinions are divided as to the suitability of rubi experiments of W. Henej'KE (II.) potassium was found replaceable by tiiis allied met^il. but only in cases where mt>rely vegetative development was in (pies- tion. Finally, the experiments of E. (irEXTUEii (1.) furnished no uniform results: the cultivation in solutions containing rubi- dium, but nt> potassium, being succe>sful in the avse of liotriiti^ cinerea, but not so with I{/ii:.'>})us ui(fnraiitt. Now, in oitK'r to rightly appreciate these results it will be neces,s;iry to b»'ar in mind the great dilliculty experienced in completelv freeing the 4* 42 MINERAL NUTRIENT MATERIALS. rubidium salt from the accompanying potassium salts. This difficulty is, moreover, accentviated by the comparatively ready solubility (even at simple boiling temperature) of the glass of the culture vessels, the consequence being that a little alkali finds its way into the nutiient solution from the glass during the sterilising process. In order to eliminate this soui'ce of error several workers have already recommended the use of metallic culture vessels for the purpose in question, but soon had to abandon same on account of the toxic action and conse- quent retardation of development produced on the sowing. This was observed, in the case of silver, by Raulin (III.), whose similar observation in the case of tin was confirmed by W. Benecke (III.). This last-named worker also excluded aluminium from the list of sviitable metals, for the reason that it sustained corx-osion and therefore caused an alteration of the nutrient solution, although Th. Bokorny (I.) employed it, apparently with good results, in his experiments on the nutrition of aJgce. Finally platinum, which, according to Bokorny, has a poisonous effect on the green thallophytes in question, was found by Benecke to be innocuous in the case of Aqjeryillus viijer ; it is, however, too expensive to use for large series of experiments. The difficulties encountered in purifying the nutrient salts, in order that the experiments conducted therewith may be perfectly reliable, can best be appreciated by the aid of the following data, for which we are indebted to W. Benecke (II.) and E. Guenther (I.). The figuies relate to the minimum quantity of KCl which will enable the development of the sowing to proceed when added to loo c.c. of a nutrient solution previously free from potash. Aspergillus niger is sensitive to 0.02 mg., Rhizopns nigricans to 0.0 1 mg,, Mucor corymbifer to 0.02 mg., and Botrytis cinerea to 0.0 1 mg. of KCl. The essential requirements of the fungi in respect of potash are therefore very moderate ; and, in fact, if the necessary quantity be exceeded, to the extent of several units per cent., the growth may be injured. The maximum amount of potash salts which Rhizopus nigricans will stand, and still continue not only to grow but also to fructify, has been determined by E. Guentuer (I.) as follows: KCl, about 7.5 per cent. ; KNO3, a,bout 7 percent. ; whilst in the case of K.,SO^ the organism will still bear up to 10 per cent, (concentrated solu- tion) very well. In opposition to the concordant results obtained by earlier workers, Carl Wehmer (IV.) assumed that sodium is able to replace potassium as a nutrient material for fungi. This view was, however, disproved by the experiments of W. Benecke (III.) on Asjiergillus itiger, an undescribed species of PeniciUium, Mucw stolonifer, Botrytis cinerea, and a pure-culture wine yeast from Winningen ; these results being also strengthened by E. Guenxher's (I.) culture experiments with Mucor corymbifer , ALKALIS. 43 ]{hi::(ijii(s iiirucia1ilL' utility as ivpirJ* tilt' nutrition of funj^i, and run be entirely ilisjienseil with. The UiaxiuiuMi (juantities of the salts of this metal that can he pre- sent in the nutrient solution without injury, have been deter- mined hy E. (luenthei-, in the case of Hliizoj'us nit/nram, as follows: NaCl. 12 per cent.; Na.,SO^+io iup, 26 per cent.; uiul NaNf)^, 6 per ci-uf. With re^'anl to lithiiun, W . IUcnecke (111.) has shown — in refut^ition of the contrary assunijition hy Nae^eli — that this metal is not a foodstutf for fun<.M, althouju'li a strong stimulant. When lithium sjilts were piest-nt in the nutrient solution it was found that the conidia of Axj eriji/liix imji r did not {,'erminate, and that no conidia were formed in the case of an uns|iecified species of PfniriUiiiiii. The extent to which the various species ure M-nsitive to the action of this metal must Uuctuate consider- ably, since, whilst E. (SfEXTiiEU (L) found 0.05 per cent to be the largest addition of lithium nitrate that Hhtiitjmt; nitjricans coultl stmd and still continue to thrive, 11. M. KiriiAUDS (I.) was able to observe that A}'/>'i-ium has, however, Ihh'U disproved by WlNo»;u.\USKV (Xi.). who showed that the latti'r is iiulispen.sjible for the development of Mi/ro- ■/■ III, I riiii. The siime results were obtained bv Adolf Maver in culture experinu-nts with beer yeast; by 11. Moi>iscii (11.) ami W. iU:.N'EC'KE (1.) with PenirU/ium tjlawnin aiul As]terijilliis tiiijrr ; and by E. (!t EXTllEK (1.) with Mnrur ron/iiihi/er, lihizopua niijri- tins, and Jinfri/iis ciinnn. Mow .sensitive and responsive the tungi are to a .small adtlition of magnesium is evident from the ()bservation rect)r«led by Henecke (1 1 1.) as to the consiilei-able dilTerence in tlevidopment exhibited bv two, otherwise «'i.>iarv to induce a sowin;: of I{/ii:opus ni(jrit'ans to grow at all. In refutation of an e.ulier assumption bv Sestini, it has been shown by II. Mol.iscii (II.) and W. Hknecke ( I.), and after- 44 MINERAL NUTRIENT MATERIALS. wards confirmed by E. Guenther (I.), that not only calcium, barium, and strontium, but also the closely allied metals beryl- lium, zinc, and cadmium are unsuitable for replacing magnesium ; and that, in fact, they behave as poisons when added in slightly larger amount to the nutrient medium. An addition of 0.02 2)er mil of cadmium sulphate or cadmium chloride is sufficient for Aspergillus and Penicillium ; whilst for Rhizopus nigricans 0.00 1 per mil is enough. According to Guenther, an addition of 0.2 per cent, of beryllium chloride is necessary to restrict the development of the last-named Phyromijces. With regard to zinc, it was observed by J. Raulin (I. and III.) before 1870, in his experiments with Aspergillus niger, that the mycelial development of this fungus could be consider- ably facilitated by a small addition of zinc sulphate to the nutrient medium. The conclusion drawn therefrom that, in contradistinction to the earlier discoveries of the same worker, zinc is indispensable to the structure of the fungus in question was, however, unable to stand subsequent investigation. Both in this fungus and in the case of Penicillium glaucum and Botrytis cinerea it was found by W. Pfeffer (II.), H. M. Richards (I.), and Ono (I.), that the action of zinc is stimulative (§81) in the sense of H. Schulz's law (I.). Even an addition of 0.0005 per cent, of zinc sulphate to a nutrient solution of, e.g. saccharose and mineral salts, resulted in a considerable increase in cropping. This attained double the yield (furnished in the absence of zinc) when the addition reached the optimum amount of about 0.003 per cent, of zinc sulphate ; but, on raising the addition to 0.05 per cent., a poisonous action was observed. Rhizopus nigricans seems to be still more sensitive, since, according to E. Guexther (I.), it will not stand more than o.oi gram of zinc sulphate in 100 c.c. of nutrient solution. A noteworthy observation made by several workers is that this stimulation is really a kind of fattening process, the stimulative influence being confined to the develop- ment of the mycelium, that is, to the vegetative portion of the thallus ; whilst the production of conidia, or organs of fructifi- cation, is retarded, and even entirely suppressed. Supported by other experimental results (relating to copper as well as zinc), Andr. Richter (I.) has pointed out that, in such a state of dilu- tion, the salts {e.g. zinc sulphate) are no longer capable of acting as such, but — in accordance with the theory of dissociation — are more or less separated into their components, the ions Zn and SO^, which are therefore the real stimulants. Moreover, because the extent of the dissociation is also dependent upon the nature of the solvent — in this case the nutrient solution — the action exerted b}' an addition of such saline stimulants is also determined thereby. The present is a suitable occasion for referring to the labours of Th. Paul and B. Kkoenig (I. and II.), SCHEURLEN and Spiro (I.), and others, to whom we are indebted MKTALS OF THE ALKALINK EAIlTHS. 45 — since the puMimtiijii (jf \u\. i. — -for the application of the dii»- sooiutioii thi'Oi-v to the stiuly of the action of |KjisonK on micro- organisms (^sj 79 un will thrive in the absence of CJilcium. c.7. Stichoroccuji bat-dlaris Na*geli, Uldhi'ix snbtilis Kuetzing, but not Vaucheria or Spiro'iijra. For the future, therefore, the axiom must be changed, and calcium regarded as indispen.sjible for the higher green plants, but not essential to the fungi and to certtiin algie. 80 far as Ivirium and strontium are concerned, it has been placetl lieyond doubt that tho.se two metals are not only u.seless, but also injurious, and act as poi.sons toward the fungi. Thus, for example, in the experimentHS of E. tiuenther, the develo|>- ment of sowings of liliiiujiUK HKjririuin cea.sed in presence of 1.0 per cent, of barium nitnite, or 1.5 per cent, of strontium nitnite, in the nutrient .solution. Even calcium, it may bo remarked in passing, is capable of acting injuriously when present in larger tpi;intities, the last-named worker having found the limit of safety to be 4 per cent, of cjilcium nitrate in the case of the sivme J'Jn/coniifces. i 230.- Elements of the Iron Group. On the fact that iron had liei'u shown uiiii>pen.-cerUiiuili{» the iinpoi-tiilit fact that the plasmic acid (|^ 252) isohiteil from the* lutrKMu of wtist contiins about i per cent, of (uuisketl) iron, whicli is |)rol>aljly attfiched direct to tin* |»ho.s|)hoiu.> .itoiu. Attempts have iih-eadv been made to utilise this fact in pliarinacy and meion pnxluct of nucleiii, to wliich the name ferratogea has l)een ^'iven, .md which contjiins alxjut I per cent, of iron in or^'anic c(anbination and readily absorbed in the intestines. According to G. Maki'Mann (II.), the iron in fungi (and especially in I'eiiirilliuvi) is usually in the ferrous state, and oidy exceptitnially present in a higher stiige of oxidation ; this has been deinon.stiiited by treatment with hydrochloric .solutions of potjissium ferro- and ferri-cyanide. Au exception is itlVorded — at least according to \\. KfssEuuw (II.) — by yeast (pres.sed yeast in jiarticular), the staltilitv of which is .saifl tt) be inJluenced by its abundant content of ferric phosphate. In addition to its part as an indispensjible foodstutf, iron also .seems to act as a .stimulant : according to the indica- tions alVorded by compai-.itive experiments undertaken bv 11. M. KiCU.VUDS (I.). Tlie nu»t;ils allied to iron, namely nickel, cobalt, and man- ganese, have Iteen tested on Asjwri/i//u.-< ni5 J 29). The optimum ipiantity of the .sidt of the tir.st-nameij>t'ier cent, of manganese. Subseipu-nt experiment,s have led this worker to conclude — though this h.is not vet been contirmed — th.»t nianiranoe is the real active aijent in the 48 MINERAL NUTRIENT MATERIALS. oxidases, by reason of the convertibility of its protoxide, this being readily oxidised to peroxide, which in turn as readily parts with oxygen and is reduced to the protoxide. Consequently, in Bertraxd's (VI.) opinion, the organic constituents of the oxidases merely play the part of carriers of manganese. For the purpose in view, this latter cannot be replaced by any allied or other metal. According to the results obtained by Richards (I.) in cul- ture experiments with Asjjenjillu.^ m'ger, aluminium is not only non-essential as a foodstuff, but has no appreciable action as a stimulant. § 231.— Sulphur, Selenium, Silicon, Phosphorus, Arsenic. Strictly speaking, it has not yet been proved that sulphur is essential to the growth of fungi ; that it is so having been con- cluded from the (still disputed) assumption that this element forms an important constituent of the albuminoids. The attempts hitherto made to carry out perfectly convincing ex- periments, in nutrient media, positively free from sulphur, have proved futile. Thus, neither Adolf Mayer nor E. GuENTUER (I.) succeeded in fully eliminating this element from the saccharose vised in the preparation of nutrient solutions, a few thousandths of a per cent, remaining in combination as an ineradicable impurity. According to the investigations of Nsegeli, sulphates as well as sulphites and hyposulphites may serve as a source of sulphur ; but ammonium thiocyanate and sulphurea are unsuitable. A careful confirmation of this report is the more desirable because Adolf Mayer, in his cultures of beer yeast, found sulphates unsuitable for this purpose. Selenium appears to be incapable of replacing its near ally, sulphur, as a nutrient material for fungi. At any rate, the experiments of E. Guenthek (I.) with Rhizopus nigricans have shown that an addition of even 0.0005 P®^' cent, of sodium selenate will suffice to prevent the germination of spores of this fungus in a nutrient solution of glycerin and mineral salts. Silicon also, according to J. Raulix (III.), must be included in the list of foodstuffs essential to fungi, though no support to this view is afforded by the later cultivation expei-iments con- ducted on this point by H. M. Richards (I.). Nevertheless — in view of the observation (unfoi'tunately not followed up) of E. Wixtersteix (I.) that the ash of his so-called fungocellulose (§ 225) consisted almost exclusively of silica — it may be regarded as not impossible that silica (as is undoubtedly the case in the higher plants), while not e^senfial to the structure of fungi is very useful for strengthening their membranes. The present SL'LPHUlt, SKLENU'M. SILICON, ETC. 49 is a suitiilile i)|ij)ortuiiity fur laentioiiin^ a ntitrient solution {Litjuiile Jiuii/iii) still used in Fifucli hiliorut^jiifs, fur lx»th moulds iind fission fungi. It was i-oiii|Miund<'d l»v UaL'LIX (I. and III.) on thf basis of his observations, which we now know to liave been si>iiiiuli:it imperfect, and is com{jo.sed of: — (iruriis. Gramt. Water . . . . 15000 I'utas.iiuni carbonate 060 Saccharose 700 Tartaric aciling analyst's place the figures at between 15 and 60 per cent.; though it should be mentioned that not all the reports on this point are of efpial value, sonie of them relating to ca.ses where insufficient regard was j>aid to the volatility of phosphoric aciletely extracted the phosphoric iu-id from decayed oak woixl. With a view toascertiiin- ing tlie local distribution of phosphoric acid among the in method, PinirilUinn hreviraulf has proved r'(i«*i7<' pniirtjui. On the other hand, A.fjierijilliis jl(iru.g — which, according to II. ScuMiuT (I.), has a very powerful reducing action — ..4. iiiijtr, A. iiuhfmcKit, A. funu'(/afii.<, I'enicilliuni ijlauruin, Mwor mucalo, ami others, have been found iinsuiti\ble, the odtnn- of garlic being either entirely ab.sent, or else njaske ajiparent on even merely superticial observation, namely phototropism. or the influence exerted by light on the direction of growth. At the outset research was conflned to the narrower tield of the form of ilhnnination mo>t common under natunil conditions, viz. by the sun's rays (heliotropism) : and, according as this influence proved .stimulative, rctiirdative, or inert^ the fungi alYectt'd thereby were classed as jmsitively heliotropic, negatively heliotro])ic, or aheliotropic. An example of each of the two lattt'r possibilities was furnished by ,1. SriiMiTZ (I.) in iS|; and l>y KiiAls (I.) in 1876, the latter of whom founcl Ji/ii:.o)itis ///;/r/i(j//.-' {Mit'-iir fttn/oni/t'r) presumably aheliotropic. Sehmitz observed that the mycelial threads at that time chi^sed 53 54 STIMULATIVE INFLUENCES. independently as Rhizomorpha, but subsequently assigned to the cycle of development of Aijaiicus meJhus, turned away from the light, though Brefeld (III.) was unable to confirm this behaviour. E. Chr. Hansen (XXII.) made us acquainted, in 1897, with three new examples of negative heliotropism, in species of the families Coprinus and Atjaricus. With regard to all the remaining fungi examined for their sensitiveness to light and found to be exclusively positive in their heliotropism — such, for example, as the conidiophores of Pezi'^M FurJieliana examined by G. Winter (I.), the sporangial hyphte of Muror mucedo, Phycomyces nitens, and a species of Pilobolus, examined by Kraus (I.) and Vines (I.), and the stalk of CopriJius lapojms examined by Brefeld (III.) — an advance was then made by separately examining the influence of the different colours of the spectrum on growth. No uniform results, however, were obtained ; for, whereas Fischer von Waldheim (I. ) found that Pilobohis cridaUinus was only heliotropic under blue light, both its congener, PliiluhoJus micwsporiis, and Muror mucedo are also sensitive to yellow light, according to Brefeld (IY.) and Regel (I.). The further researches of Wiesner (I.), in contradiction to those of Fischer von Waldheim, show that PiJobolux cridal- lirms and Coprinus niveus still continue to turn helioti'opically, even in the ultra-red rays. We are indebted to Friedr. Olt- MANNS (I.) for the settlement of this discrepancy, and also for raising the considei-ation of this phenomenon to a higher plane than before. His researches were performed on Phijcomyces nitens ; that is to say, the very fungus that had hitherto been regarded as decidedly positive in its heliotropism, and the one chiefly used in lecture demonstrations on account of the unusual sensitiveness of its long sporangial hyphse to light. By using a very powerful electric arc light (up to 5300 Hefner units), Oltmanns found that the fungus in question behaved posi- tively phototropie under weak illumination, but negatively so under a powerful light, whilst at an intermediate stage of illumination it remained aphototropic. The universal law of stimulants thus applies also to the phototropy of fungi, the sign — to speak mathematically — of the stimulative effect being determined by the strength of the infliience. However, the degree of stimulation necessary to the production of a given effect is also dependent on the actual condition of vitality of the individual under examination, age, in particular, being an important factor. Thus, in the case of Phyromijces nitens, a given degree of illumination causes attraction in the young sporangial hyphre (with just grey sporangia), whereas in the older ones (with already blackened sporangia) it induces repulsion. The applicability, to the phototropism of fungi, of Weber's law (§ 233) of the I'atio between the degree of stimulation and the effect, has been demonstrated by J. Massart (III.) in the case INl'LUKXCE OK LKiHT ON EUMYc KTES. 55 of Phycornijces nitt^ii^. Tlie existence of after elTet-ts of htiniula- tion WHS first reimirketl, in this comiection, by Wiekxeb (I.). Although the iiillueHfe of light on all the other vit:il manifestations of fungi has been the subject of numerous observations and experiments, no unifornt results have been secured, nor has the stime elevatexi>oriuiii, or the endospores of Rhizcjms ni-jrirauK {Muror sti)tiini/er). Contrary results were afterwards ol)tiiined by vox Weitsteix (I.), who found germination retarded by light in the case of spores of RhMlomycex KorJiii ; and by F. Klkvixg (I.), who proved that intense sunlight entirely prevents germination in the conidia of Asi/rriji/hts i//aitruis. The reports of experimentei's al.so differ with regard to the influence of light on the vegetative development — increase in the size oi tiie clIIs, and the power of growth. Thus, wherea.*!, according to a report by J. Schmitz (I.), SjiJu'rid ctiijiuphila grows more strongly in the dark than in daylight, I^eziza Furkeliaiin — according to (5. Wixtek (I.) — ceases to grow in the dark, and perishes entirely if the exclusion of light be prolonged. Vax Tieghem (IX.) and Oaillaud (I.) found illumination exercise a favourable influence on the development of Ptnifiltiuiu and certain yeasts respectively. KuAl's (I.), on the other han fihahi attiiin their greatest length in the dark, rather than in red, yellow, or blue light. Brefeld al.so made the sjime observation with regard to the stalks of cert;iin species of Ci>j,riiiuf. (\. H. VlXES (I.) found the growth of the sporangial hyplue of I'liijcoinijo-^s niteriK prejudicially affected by light, and traced this action to the influence of the blue rays. Tiie first to investigate the influence of light on cell fission, and therefore on cell reproduction, w.is L. Knv (1.), in the case of i)re.ssed veast. He failed to di.scover anv ditVerence in the rate of reproduction in the ilark and under minlerate illumina- tion by gaslight. This is, however, altered in tlu> ca.se of strong insolation, as will be referreil to later on. Al.so in respect of fructification the individual speiies of fungi seem to dilVer with regard to the strength of illumination neces.sjvry to the production of a given result ; such at least is the conclusion furnished by compiring the reports of variouu observations conducted on this point, the original probability, based on considerations of a general physiological character, 56 STIMULATIVE INFLUENCES. being heightened by the determinations of Oltmanns. Accord- ing to H. Hoffmann (II.), E. Loew (III.)) ^^^ A.. Lendner (I.), the formation of sporangia or conidia in Rliizopus nvjricans, lliamnidium eler/ans, and Mucor vnicedo, or PpniciUium gJaucum and Trichuthecmm ro^eum respectively, proceeds just as well by daylight as in the dark. C. Werner (I.) also failed to detect any influence of light on the formation of conidia in two of the higher Ascomycetes. In the case of Botrytis ciiierea — according to RiNDFLEisCH (I.) — this formation occurs solely by night ; and in this instance, as was determined by L. Klein (III.), the retardation is attributable mainly to the blue-violet rays of the spectrum. The converse has been observed in the case of Rhizopus nigricans, which, according to A. Lendner, puts forth its sporangia two days later in the dark, or in red and yellow light, than in white, blue, or violet light. In the dark, Mucor raremosus produces merely barren sporangia ; whilst, in the case of the Thamnidium aurantiacuvi described by Rochard (I.), this fructification is said to proceed most favourably in twilight, and to be prejudiced and retarded by strong light as well as by an absence of illumination. This report, however, has been contra- dicted both by Payen (I.) and Poggiale (L). A similar observation to that of Pochard was made by A. Lendner with regard to the formation of conidia in Aspergilhis luteus, Asp. niger, and Bofri/fis (cinerea ?). Elfving (I.) states that the formation of perithecia in Aspergillus glaucus is entirely or to a large extent suppressed by light ; and the same applies to the formation of the pileus in certain members of the genus Coprinus — congeners of the mushroom — observed by Brefeld (IIL). Nevertheless, the question of the influence of light on the fructification of any given fungus cannot be answered off-hand. On the contrary, it has been established beyond doubt that the nature of the action exerted by light depends on the other conditions of vitality, Brefeld (III.) having shown, in the case of Coprinus stercorarius, that the formation of the pileus ceases in the dark when the temperature remains below 15° C. This discovery that light rays can be replaced by heat rays becomes of greater interest when it is borne in mind that — as was de- termined by Brefeld — the only light rays having any influence on that development are the blue-violet ones, and not the less refractive (e.g. the yellow) luys. A detei'minative influence is also exercised by the composition of the nutrient substratum. Thus, A. Lendner made the obsei'vation — which deserves follow- ing up — that spoi'angiation in Mucor Ji avid us occurs in white light, bvit ceases in yellow or red light or in the dark, when the oi'ganism is grown in Raulin's nutrient solution (§231); but that the converse is the case when van Tieghem's nutrient solution is used. INFLUENCE OF LIGHT OX El'MYCETES. 57 (Innvn on solid media this fungus seems to develop sponnigia under Jinv kind of illuiiiiiiution. A fact of not less inii>nriif, a fun;,Mis of the Mihorarea family (.^ 235), namely, the after effect of illumination. When kej)t in the dark from the bejiinnin'', the mveelium of this fun-nis remains entiiely and permanently barren ; but if exjtosed to the light foi- a eouple of hours, and then plaeed in the darkl)ef()re any signs of fruetifica- tion appear, the sporangia will develop, though ordinarily they oidy do .so in the light. Still more scanty is our knowledge of the influence of light on the internal life, /.<. the chemieo-physiological ea}>aeity, df the Eumycetes. In fact it i.s almost entirely confined to a series of ns made on respiration (^i 23.S), /.<. the exhalation of carbon dioxide. The lir.st experiments were tomlucted by Wilson (I.), who failed to discover any influence due to light; and also by Bonnieh and Mancjix (I.), and Plhiewitsch (II.), who experimented with various pileated fungi (iiittr alia, Aif'iriciis cat/iji'fiti.-') and J'liijrtDiii/ff-s iiifi'ih<, and in all cases found respiration hindered by dilfused daylight, the .strongest elTect being produced by the least lefrangible rays. The.se experiments, however, were of a merely preliminary character, no regard having been paid to the circum.stiince that the ex- haled carbon dioxide may originate from fm) distinct vital processes ; viz. either from the conversion of matter in the course of Ijuilding up the cell, or from the prtK-ess of combustion within the full-grown cell for the purpo.se of replacing di."riskly. All the above-mentioned workers regarded the total amount of car\>on dioxide, liberated l)V the cultinvs under observation, as a mea.sure of the respiration ; but H. Koi.kwitz (I.) took into oonsideratitui. as a by no means negligible .<(auve of error in prolonged experinu>nts, the gradual decompo.»itii>n sustained by oxalic acid under the influence of light (>5 21), since this acid is of fretpuMit occurrence among the metabolic prinluctij of fungi. On excluding this source of inaccin-;u'v, by examining 58 STIMULATIVE INFLUENCES. the cultures under a brief illumination by a powerful electx-ic arc, this worker ascertained that considerable acceleration of respiration is experienced in the case of Oidiuvi ladis, Asper- gillus nhjf'Y, and one species each of Muc.or and Penicillium. The action of sunlight has been the object of a number of observations, which merit our attention the more in that they were chiefly made with yeasts, and partly relate to the biology of the fungoid flora of the vine. Of the other fungi, Asjienjillus glaucus was examined by Elfvixg (I.) in this connection, whereby it appears that the conidia when ripe will stand insolation for several weeks in succession at the latitude of Helsingfors, with- out injury, though they are killed in a few days when in a young, immature condition. Far inferior powei's of resistance were presented by the yeasts subjected to insolation in the south of France by V. Mautinaxd (II.), whose results were afterwards confirmed by G. Tolomei (YIL). It was found that both sporogenic and sporeless cells in two races of the group Saccharomyces eUipsoideus, and also cells of S. apiculatus, perished after four hours' exposure to the sun's rays at an atmospheric temperature of 41° to 45° C. A similar result followed insolation for three days at 36° to 37' C, whereas other specimens of the same species remained alive when kept in the dark under otherwise equal conditions. This coincides with the results of the experiments made by W. Lohmanx (I.) with Sarcliaromyces Pastovianus I. Hansen, two species of I'orula, two film-yeasts {Mijcoderma), and a distillery yeast (Race II. of the Berlin Experimental Station), these organisms being killed in a few hours by insolation, as well as by illumination with an electric arc lamp of 8000 to 11,540 metric candle power. The last-named culture yeast succumbed first, whereas the first of the wild yeasts proved the most resistant. The unfavourable conditions artificially produced by Martinand are experienced in piuctical viticulture by the yeast cells that make their habitat on the grapes most fully exposed to the sun, i.e. particularly those on the upper part of the vine-stocks. Consequently we should expect to find in that position a smaller number of living cells than on the grapes lower down. This observation of Martinand's also leads to the conclusion that the grapes from southei-n countries — which often give very poor fermentation — are less al)undantly inliabited by yeast cells, and are chiefly infested by such races as are less sensitive to insolation. It is therefore probable, as a result of this factor of natural selection, that the wine yeasts of southein latitudes are physiologically different from those whose progenitors have lived for centuries under a cooler sky. An assertion deserving closer investigation is that of Ward (VII.), to the effect that the colouiing matters of chromatic fungi iifford protection against the injurious influence of light. INFLUEXCE OF LIOHT oX KL'MVCETES. 59 Ward >m)j)urt.s this view \t\ the peiKOiial observation that the colourless spores of Oiiliuiii lurfin, Clialara iiiyoMlenna, and SfW' channnijceK pyrijumtix were killed by insolation, whereas this was not the case with those of AKjmyillu/' ifhttirw, J'fiii»'illiuiii rnigfuituhi {!'. i/ltiitrum), Mucor ract'inoKux, ami Uutrijtin chitiiu. It must not, however, be forgotten that the greater j)ower of resistance oiTered by tliese spores may well be tlue to the con- siderable tiiicUness of the meml)rane. Nevertheless, as tho foregoing discoveries by Eifving have shown, all means of pro- tection fail in presence of prolonged insolation. With regard to the influence of the Roentgen rays on the vitality of tho Eumyeeto, an expei iiiHiit was idiiducted on Phycomyceti niferijt by L. Eiiuaha (I.), but furnished no definite results. Further research on this point is therefore desinible. ^ 233. Chemotropism General Remarks on the Enzymes of Eumycetes. Chemotaxis has already been explained (>; 41 of vol. i.)as the attraction or repulsion of motile microorganisms by chemical stimulants. Motile cells, which are therefore capaVile of being similarly inlluenced, are al.so found among the Kuinycetei!, namely, the zoospores (J5 220) of the Oouiycefef! And Clit/triiliacea; i.e. fungi that do not come within the scope of the present work. In the case of the other higher Kunniciti.K, the elVects of such a stimulus are manifested by the atTected individual either develoi)ing with particular strength towards the seat of the stimulus (inclining thereto) or turning in the oi)posite direction. These phenomena are termed respectively positive and negative chemotropism. The earliest statements and ob.servation.s on this point were made l)y W. Pkkkkkii (IV.), then by J. WuUT- MAXX(Vlll.), y\. Wauu(V1.). M. (K KEixnAKUT(I.). and others. More thorough investigations were conducted by ^1. Mivosni (I.) with Miiror muodo, J'fiyrdiiiyri's jtiten.<, liJiizopua m'/jricaiif, l\'nirilliu)u ijlauriDn, and A.ositive chemoti-ojiism begins to turn into negative in consequence of a strongly in- creased concentration of the stimulant. Further investigation is merited by the question of how far chemotropic action is concerned in the phenomenon known as rheotropism, the outward sign of which is the adoption by fungoid hyphfe of a definite position with respect to the direc- tion of flow of the surrounding liquid. Jcexsson (I), to whom we are indebted for the first observation on this point, found that the mycelial hyphre of BotrytU grow against the current, but those of Mucor and Phycomyces with the stream. The first- named fungus he termed positively rheotropic, the other two negatively so. The tendency of fungoid hyph* to gi'ow towards a positively chemotropic stimulus is manifested not merely when the latter is freely accessible, but also when it is separated from the in- fluenced fungus by a partition. In some species and under special CHEMOTKOPISM. 6i conclitiuiLS, tliis tendfiicy is expressed by the excretion of sub- Htances capable of attacking and jn-rforating the siiid paititious. Among such sul)stjinces may be mentioned carbon dioxide and oxalic aciil, which come into action niort' especially when the fungus is in conUict with a calcareous substnitum, sueh as an egg-shell or a bone. On this point a num)>er of experiments were conductdl by K. Lind (1.) with Asjteiyillus uiijei; I'tnicil- liuin (jlawuiii, and JiufryttK nttt-mi. Of the organic substunces naturally forming such a ]Mirtitioii wall, viz. either chitiu or cellulose, together with allied carbo- hydrates, the first comes under consitleration in all cases where the fungus endeavours t*j perforate the shell of an insect. In order to elTect this it has need of an enzyme cajtable of dis- solving chitin, on whith point several reports have been jnade by Zoi'F (X.). However, even when the object infested with the i«irasitic fungus is a fungus itself, the collaboration of a similar enzyme is necessary, since chitin (.^ 226) forms ai principal constituent of the membrane of many members of this class. The faculty of excreting an enzyme capable of dissulving cellulose and allied carbohydmtes liecomes maiidy apparent when higher plants are infested by a fungus. The fijst notice of the occurrence of such an enzyme in KiDinjii^tes was that of DE Baky (II.) in 1SS6, in the case of Si-lnutiuia {I'l^iza) Libert iana ; and a similar one was recorded in 1S8S by !M. Wauu (I), in an allied species injurious to lilie.s. A year later, E. Kissling found the same faculty in S<-lei<)/iuiu Fui-Lxliuna, and this was confirmed by J. Behkexs (IV.), M. MiYosui (II.), and M. NouuiiAisKN (I.). Subseijuently, on the basis of his own experinu'iits, ^1. O. Ueixhakut (I.) came to the conclusion that the mend)rane-di.s.solving enzyme secreted by the lir>t-named iSrlerotinia is different from that of the second species. This is by no means .surprising, in view of the great vaiiety of carbo- hydrates (J^ 227) taking part in the stjucture of the vegetable cell wall. The ability of liotri/iis cinena to secrete an enzyme ciipable of dissolving true cellulose has been demonstiiited by an experiment, carried out by J. Beuhexs (IX.) in a manner to which no objection can l>e niised. On the other hand, J'vuiril- liuin (jluuciim, 1\ luteuin, Jihir.ujnis ni'jru'aii.f, and probably also Monilia fniclii/ena, have been proved incapable, though, with the exception of the last-named species, they can attack the so-called centnil lamella (.^ 119) and therefore .secrete a ptHrtin- dissolving enzyme. Another organism capable of dissolving true cellulose is the vine rt)ot mould, examined by J. Behkexs (Xll.), and termed by him P.o.udoJhniatoj/iora. This is a nou-^withogenic mould fungus occupying an unknown po.sitiuu in the botanical .system; it grows generally on wood, and has a particular atlinity for vine-wood, which it rots and destroys. Accordini: to C/atek (II.), the lignilied cell walls of higher 62 STIMULATIVE INFLUENCES. plants consist of an ethereal combination of cellulose with a substance to which he gives the name liadromal. The dissocia- tion of this compound and the liberation of cellulose, are probably effected by an enzyme which this worker has dis- covered in certain fungi, e.g. dry-rot (§ 80), and which he terms hadromase. Fungi that have invaded trees and timber find, in the bark and cambium layer of same, oftentimes large amounts of glucosides, such as salicin, populin, amygdalin, coniferin, &c. Thanks to the action of certain excreted enzymes, such as emulsin, they are able to utilise for their own nutrition the carbohydrates separable from these substances. A number of these parasites were examined in this I'espect, with affirmative results, by E. Bourquelot (III. and IV.). Further mention of Eumycetes enzymes capable of decomposing glucosides will be made, with reference to certain special examples, in a later section. . However useful to parasites may be the faculty of secreting an enzyme capable of dissolving the cell walls of the infested host, there are certain cases where the parasites can dis- pense with this faculty and still attain the end in view. This is effected by the purely mechanical pressure exerted by the apex of the hypha on the cell wall of the host, when the hypha itself has formed an appres- sorium (§ 237) and thus pro- vided an abutment. In this manner it succeeds in per- forating the cell wall. This faculty has been confirmed by M. MiYOSHi (II) in the case of Penicillium glaucum and Botrytls cinerea, both of which proved capable of penetrating thin gold leaf quite free from holes. In this case chemical action was entirely precluded. When fungoid hyphse obtain a footing on the epidermis of such parts of plants as exhibit stomata, they often prefer to obey the chemotropic attraction exercised by the cell contents, in a peculiar manner, by growing towards the nearest stoma, passing, by this means, into the interior of the plant tissue, and then penetrating the cell membranes, which are far thiniier than that of the epidermis. An example of this is shown in Fig. no. The successful entrance of the hyphai in this manner is not, however, invariably followed by the further development of the parasite, the sequel depending on the con- stitution of the cell contents. The latter, without prejudice Fig. 1 10. — Khizopus nigricans. Hyphfe from Ave spores, which, twenty- seven hours previously, were sown on tlie under surface of a leaf of Ti-adescantia dincolur that had been injected witli a 2 per cent, solution of ammonium chloride, making their way to the stoma, and passing there through into tlie in- terior of the leaf tissue. Magn. 100. {After Miyoshl.) I ClIEM(JTnui'lSM. 63 to their piuijorty uf uttnicting the invader, may he of such a character as to jtreclui()logic-alIy interesting and jinieticallv important question may In- fuund in handbooks on jdant diseaHos ; and a few relevant remarks will also he made later on in connection with the rotting of fruit and " sweet-rot " iu grapes. In the absence of a jnore favourable opportunity, a few geuei-ul remarks on the secretion of proteolytic enzymes (^ 170) by Kuuiycetes may be made here. The first record of a gelatin- u)it, liofn/fis rinen'd, and Cejihaloihi'i-ium rut^'.'uin will litjuefy about one half the medium within ten days, and produce complete lique- faction in two to three weeks. The result,s were indefinite in the case of AKpn-'/i//uit i/lancit.<, A. /iniiii/a/i<.<, and A. rariatin. .Vccording to later report-s by the same worker, a slight liijuefac- tion of the gelatin is also elTected by Murur Itouj-ii and .1/. javaiiinu<. In the course of a comprehensive investigation on the occmience and activity of a gelatin-dissolving enzvme in various members of the vegetable kingdom — including a nund)er of fungi — Fkk.mi anil BrscACi.io.M (I.) obtiiinivl atlirmativo results with sundiy edible fungi, as also with ''Inricejts 2fit>Tii''''^} Aaji't'i/illiut jiaru.'i, itc. The occurrence of enzymes capable of dissolving casein was investigated by E. liofugrEl.oT and H. IIkkissev (I.), who lound such a one present in about 20 out of 12b species of fungi examined, cj. in Amanita muscaria, Boletus rtluiis, Jcc. Botli worki'rs lu)ld this enzyme to bo identical, or at least certainlv very nearly allied to, trypsin, since, like the latter, it furnishes tjnrosin. J. IIjokt (l.> succeeiled in detecting the pre.-ence of similar enzymes, capabU- of digesting fibrin, in various higher fungi; they afterwards completely degrade the peptone fornuxl, trom the above substmce, along with leucin and tvrosin. E*'^ albumin is also attacked at the ordinary temperature. ZorK 64 STIMULATIVE INFLUENCES. (X.) has published an observation on fungi capable of dissolving horny matter (keratin). Diastatic enzymas, i.e. such as are capable of converting starch into sugar, are also very often (§ 117) found in Eumyretes. Many species of the latter are specially productive in this respect, and are employed on this account in the fermentation industries ; this is particularly the case with the species of Mucor that will be described in § 240, and the Koji fungus {Aspergillus ory?:ie) dealt with in the last section but one. At present we shall not treat of the technico-mycological side of this faculty, but merely make a remark of a geneial physio- logical character, namely with regard to the dependence of the formation of this enzyme on the external conditions. In this connection W. Pfeffer (V.) and Jul. Katz (I.) examined PeniciUiiuii (jJaucu/n, Aspenjilhis 7iujei; and Bacillus meijatheriu7n, and found that these two Eumycetes produce diastase, even in the absence of starch, provided no adverse influence comes into play. Such an influence has been traced to the presence of various sugars, in the case of Penicillimn t/Iaucuiti, the formation of diastase at medium temperatures ceasing when the (otherwise identical) nutrient media received an addition of either 1.5 per cent, of saccharose, 2 per cent, of grape sugar, or 10 per cent. of galactose. The secretion was also retarded by 5 per cent, of maltose. Enriching the nutrient properties of the medium is succeeded by an increase in the limit of the foregoing additions. In the case of Aspertjillus ni(jer, an addition of even 30 per cent. of saccharose merely restricts the formation of diastase (at 31.5° C.) without suppressing it altogether. The amount of diastase secreted per vxnit of time by this fungus was found to be greater (other conditions being equal) in the case of cultiu-es in which provision had been made for the immediate separation of the resulting diastase by additions of tannin. Enzymes (lipases — Hanriot (I.)— or steapsines — W. Bieder- MANN (I.)) capable of decomposing fats, are found not only in the pancreas, blood, and other corporeal fluids of all animals hitherto examined, but also in the vegetable kingdom. Their occurrence in Enmijcetes was first detected in the case of Penicilliarii gJaucum, the discovery being made by E. Gerard (II.) and L. Camus (I.). They were then found by the last named (II.) in Aspergillus niger, and by R. H. Biffen (I.) in an unidentified fungus infesting the cocoanut. J. Hanus and A. Stocky (I.) repoi'ted having observed the production of a similar enzyme by a number of mould fungi in the course of their investigations — referred to in a later section — on mouldi- ness in butter. It is not improbable that several vai-ieties of lipases exist in the fungoid kingdom. The faculty of producing such an enzyme is of particular importance when the nutrient medium is rich in fat, e.g. in the case of fungoid parasites CHEMOTROPIS.M. 65 infesting the fatty matter of insects. This faculty uiay be regarded as non-essential to such fungi as make their habitiit on oilcake (§ 2^5) and consume thn fat therein, since — as has het-n shown, particularly V>y W. 8it;Mi nd (I ) — the oil .seeds freijiit-ntly contain lipase, and it is very probable that some portion of this is left behind in the oilcake. It is. however, doubtful whether this residue has n«jt alieady lo.st its jKjwer, and is consequently incapable of being utili.sed by the infesting orgauism.s, the lipases being very sen.sitive to injurious intiuences (acids, salts, iVrc.) For this rea.son, it may be remarked in pa.ssing, all attempts at their i.solation in a pure state have faile-ill therefore be dismissed with a few brief explanatory remarks. The family of the Entomophthorese forms a connecting link between the orders of Zygomycetes and Oomycetes. The species of this family are almost exclusively parasitic on living organisms (insects, fungi, ferns), and one of them will, from its efi'ects at any rate, be, superficially, known to the reader, namely, E)iipusa 66 SUHDIVISION OK THK (JllDKK OF ZYOOMVfETKS. 67 mutcit, the cause of a ilisesvse att^wking the house-fly in late suininer ami autumn. From this cause iiumherH of the.<>u iu- Hects are fouml to adhere, stnicldle lej'f^e*!, to walls ami wiudowrt, aij'l l)ei'oino surrouui^ting of unieellular eonitlia ilispersed by tlie c-onidiophores protruding from the IkmIv of the insect. The.se coniuh;i' of which they attjich themselves by means of special organs (haustoria), and then penetrate the interior for the puipo.se of ab.Ntnicting nourishment. This behaviour they exhibit in common with the species of the third family of conidiophorous Zt/'/omi/ct'te^f nanu'ly, the Chaetocladiaceae, whose conidia are unicellular and may be regarded a> sporangia, whilst the contents are united to form a single endospore, instead of being dividef the.se six f.imilies, only the first and la.st will be dealt with in the following ]>a nigra |>hs, chiefly from the .stJindpoint of Physiology and the technology of fermentation, lejiving out of considei-ation their morphology ami development except in so far as mention of these is al>.s<.duttdy essential to our purptx-e. Readers desirous of obtaining fuller information on the two latter points lU'e referred to Alkued Fiscmek's (III.) monogniph on the Phijroxnircfe.-i ami Zijijoinijote.*. 68 MORPHOLOGY OF THE MUCORS. § 235.— Subdivision of the Mucor family. From the explanations in the preceding paragraphs, the MucorarecB may be defined as Zygomijcetes which exhibit spor- angial, but not conidial, fructification, and produce naked zygospores. This family may be subdivided into three sub- families, of which, however, only one falls within the scope of the present work, whilst the others will merely be referred to in order to facilitate comprehension of the connection existing between them. The sub-family of Thamnidieae is characterised by the posses- sion of two kinds of sporangia : on the one hand, a large, normal, polysporous sporangium, on the crown of the sporangio- phore (terminal sporangium) ; and, on the other hand, certain far smaller sporangia, which are situated lower down and put forth by whorled lateral branches of the sporangiophore. This second kind of sporangia are destitute of columella, contain only a pair of endospores — sometimes only a single one — and are known as sporangioles. The most closely investigated member of this sub-family, Thamnidium elegam, is shown in Fig. in. Starting from observations conducted by Brefeld (IX.), the dependence of fructification, in this species, on the external conditions (chemical composition and concentration of the nutrient substratum ; temperature) was examined by J. Bach- MANN (I.). This worker demonstrated that, by controlling these conditions, it is possible to compel the fungus to produce either terminal sporangia or sporangioles exclusively, or both together, or again to remain barren of fruit. The second sub-family of the Mucoraeece, namely, the Pilobolese, is distinguished by the featiu-e that the ripe spor- angium— owing to its peculiar structure — is released, and even forcibly expelled from, the organ on which it has been developed. Of the species belonging to this group, mention may be made of Piloholus cri^stallinus, which is often found on horse droppings. Finally, tlie Mucorese constitute the third sub-family. They are distinguishable from the first by producing only a single kind of sporangia, which, unlike those of the second sub-family, do not separate from the sporangiophore before discharging their contents, but remain attached thereto after bursting. The discharge is effected in consequence of either the lique- faction or brittleness of the membrane of the ripe sporangium. One of the genera in this third family, viz. the genus Sporodinia, is characterised by the forked branchings of its spor- angiophores and the suspensores of the zygospores. This form is represented in Fig. 107. On the other hand, in all the re- maining Mucorece — which have been arranged into five genera by A. Fischer (III.) — these organs are either not branched at. all or at least not forked. SUBDIVISION OF THE MUCOll lAMILY. 69 (3> , Kio. III.— Thainiiiiliuni i'K-(jniis Link. I. Snoranfjiophore, slightly (6) niai:nilU'^' rf^^\ The genus Phycomyces is characterised by the possession of spinous prolongations on the suspensores and sporangiophores, which latter are unbranched, olive-green in colour, and possess metallic lustre. One species of this genus, PJiycomyces nifens, is plentifully met with in empty oil-casks, on oil-cakes, in concentrated fodder works, and similar places, and puts forth stiff, upright spor- angiophores 7 to 30 cm. long and 50 to 150 ju. in diameter. These become crowned with an initially orange, but finally black, globular sporangium, 0.25 to i.o m.m. across, exhibiting a cylindrical columella and filled with ellipsoidal, yellow-brown, thick- walled endospores, ib to 30 /x long and 8 to 15 /x broad. It has not yet been ascertained whether this species feeds on the fats present in the aforesaid medium ; in fact, the whole ques- tion of the decomposition of fats by moidd fungi (i:^ 233), on which sundry observations have been communicated by R. H. Schmidt I.) and by Ritthausen and Bau- MANN (II), is a matter still requiring closer attention. The genus Mucor is distin- guished by the absence of spiny branches on the suspensores, by the silky gloss of the sporangio- phores, and by the liquefaction of the ripe sporangium membrane. The contents of the ripe sporangium are only partly consumed in building up the spores, the remainder serving as a matrix wherein the individual ripe spores are embedded and separated from each other. Now this matrix is capable of distension, and holds the crowd of spores together, even in microscopical preparations ; whereas on the other hand the sporangial membrane, being liquefiable, is either quite invisible in such (aqueous) preparations, or at most is only seen as a residual trace at the point of attachment to the sporan- giophore. An attempt is made to portray this in Fig. 112. The outside of the sporangial membrane is found to be more or less closely set with crystals of calcium oxalate. A few of the species composing this genus are pathogenic, and therefore interesting to the pathologist, since they are able to set up mycosis (in this case Mucor-mycosis) or fungification of the body they have infested, or into which they have been artificially inoculated. Of these pathogenic species of Mucor— some of which have not yet been properly examined and classified in a botanical sense — mention may be made of the following : Mucor pusilhis, discovered by Fig. -Mucor mucilagineus Brefeld. Newly burst sporangium, m is the membrane, z the matrix, sp the spores. Several of the latter have been squeezed out of the sporangium. Magn. 300. (After Brefeld.) THK (J EX US MUCUH. 71 Limit; Miicor roripnhih I , i-.\;miiiied by Lichtheim. and wliicli '\n pioliiiltly iiltMiticjil with Limit's Mitnn' rurtfinnxun, siiliMMjiU'iitlv also imiiifd Ji'/ti.:oputi raniutiiin ; liiially Siehemnaim'K Mwor itepta- ttts, which is jHM-haps the sjiine iis Miimr rai'ttinmuf. Thes*^ patlio- geiiic sjn'cifs have all het*n tried on wariii-blofMled uniiiialK, and therefore thrive at iiu-ultatiuii temperature. § 236." The Genus Mucor was estjxblished by Mieheli as far baek as ijjc). l)urinj; the suceeefliiig 140 years it received attention at the hands of a larj^'e niunber of workers, and a considerable nundter of specie.s have been described foi- the nio.st j>ait ini|terfectlv. The species of this genus may be divideil into three groups. One of tln'iii comprises all the species with unbranched spor- angiophores, the cinef representjitive ijciiig Mtfn man do. with which is associated .1/. iiiifi7(('/i7ieuf, itc. The species of the .second group may exhil)it clustered branchings of tlie sporangio- phores ; they inchule Mucor raciiiiosiDf, M. luertiia, M. tenuis, M. fiaiiilix, M. rori/iii/>i/i'i% and M. j'iixi//ii.<. Finally, the char- acteri.stic featiu-e of the third group is a more or le.ss decided S3rmpodial branching of the sporangiophore. This group com- prises Miiiur tipiiiu.een diagrammatii-ally illustrated on p. 2. From tlu* mycelimn itself arise stilV sporangiophores, 30 to 40 /* thick and (according tt) the conditions of growth) 2 to 15 cm. in height The apt'X of eacli supports a single globular sporangium, which is closely coveied with tine needles of calcium oxalate, and usually measures 100 to 200 /» in dia- meter, though, as shown in Fig. 113, it maybe much smaller under unfavourable comlitions The spores, which are in the shajie of an elon;.'ated ellipsoid, and about twice as Ion*; as thev are broail, may dilYer in size in one and the .siiuu* sjM»ningiuni. the usual measurements, however, being 6 to 1 2 /i and 3 to 6 /» respectively. The cell contents are faint yellow in colour, the nuMubrjine colouile.ss and smooth. The /vgosjKires, the gradual development of whidi is represented in Fig. 101. genninjite bv putting t\)rth direct an uidu-ancheil sporangiophore with an attached spomngium. This species does not produce gemnue. A s]H>rangivnu of Mucor mucilatjiueus — which was first men- tioned by linKi Ki.i) (TV.) is .shown in Fig. 112. which alsIiort*s. Tlie !izy;ro.s|M>iM>s are forini'il, sinfjly iiiul indo- peiiiKMitly, un the iipirt«.s of hnuiclies uf jin cioct 8|*oriiiif.'io|>li(jie, arising' fioiii tlie nutrient substiatuui (or tlie iiiyceliuui), and wliicli tliLMefore an-sinnt-s a clusteriMl appwiranc-e. It is repre- seiiteil in Fi-;. 115. On tlu* utln-r liiiiul, noriniil zygospores have n(»t liitlierto bei-n discovereil on this fun^'us. Mucor /rui/iliit liainier resemhhjs the foregoinj^ species as regards the structure of the sporangium ; except that the hitter is smaller. The siime applies al>o to the spores, which are oval in shape, about 4 /t in length, and 2 fi in breadth. Mu'ur s/>itiinfitj< run 'I'ii'ijhetii owes its sj)ecitic name to the spiny projections, exhibited, to the num- ber of about a dozen, on the crown of the cylinilrical or pearshajieil columella. In preparations sub- merged in water, the membrane quickly dissolves and vanishes from sight, leaving a picture recalling that of the head of a conidiophore of any A.\ > in-d i^on r>>tii'ii apples) nnil (Kvseribed bv .Vi-KKKO Fisi hkk(I 1 1.), was .so called on .account of the pear-like shape of the ctdumella, the V«i«ider (alnnit 140 to 2 So /t) upper end of which extends a dist^mce of about 200 to 300 /t within the sporangium. When light is admitted to cultures of this fungus, grown in a suitable nutrient solution, it elabonites compamtively huge tpiantities of citric acid fj-oin Kiu. 1 14.— Chtin>yiI>«iiiucor race- iiinsiis HrvfoUI. I. Branclietl Mntni. fv>. .. 0|itii'nl Kectioii of s|Hiniiii;iuni. more hJKlilv niii^iiitltKi ( uhuhh' from tlie x-eht, there is developed l)etween these two organs, in the Mortier- fllu and Jtlii:.i>ji'',i, an inteiiiiediate or^'an which determines the h)cation »jf the sporan^jiophore. This is effected in the folhjwinjj manner: a hypha, whicli, from its peculiarities, is called a stolon, be«;ins to project outwaid from the mycelium ^(rtiwiii'r in and upon the nutrient suh^tiatum. I)escriliin}'U,'< >iii/n'iHt/ij<. 0\i comiiif,' into contact with the desired sub- stratum, the st<)lon first endeavours to attach itself thereto by puttinj; forth a system of short hypha-, which fit close a;,'ain.st the substratum, and on account of theii- branched and root-like form arc termed rhizoids. In their entirety they const it uic ;iii adhesive organ, called an appres- sorinm, which is coj>iously su]>plied by the stolon with nutrient materials frtnii the distant mycelium, aiul in tin-n puts forth stolons of the second order. These now extend to farther distances and i>roduce aitpressoria of I'M-ha- (A), which h«vi. i.r.H-,f.icd , , • , 1 • 1 r 1 '""" ""^ «toh>ii (»!>. Magn. loa the second order, wluih form the {,A/trr Bif/flJ.)^ starting point for stolons of the third or/>ii,<, the sporangiophores take their rise exclu- sively from the appres.soria, from each of which spring usually three to five, more nirely tip to jvs many as ten. In the genus Mf ■>•■., -■^nr fm. ii6.— Mortirrrlla lUxtathiskll Bnfeld. The lower ciul i>i tlie »tM>ranf;io- Iili^-rt' (f) is I'liVfloiHil tiy a |i|f\iii of 76 MORPHOLOGY OF THE MUCORS. Ahddia, on the other hand, the sporangiophores spring from the crown of the arch formed by the curved stolon. The development of appressoria is a consequence of the mechanical attraction exerted on the tip of the stolon by the objects with which it comes in contact. If means be taken Fig. 117. — Rhizopus nigricans Ehrenberg. (a) is the extremity of a stolon, which has developed into the appressorium {h). This latter is the starting point of the sporangiophores (0. four of which are shown with the sporangia (■<) unbroken, whilst the columella (e) is all that remains of the fifth. Magn. 30. (After Brefeld.) to prevent the occurrence of such contact, for example by compelling the stolon to grow vertically downwards, the stolon itself will develop into a sporangiophore direct Instead of forming an appressorium, the apex — as J. Wortmann (XIY.) has shown in the case of Rhuopus nigricam — develops into a normal sporangium. Another instance, in the fungoid king- dom, of the effect of such contact attraction — which is also frequently observed in the higher plants, especially in the curvature of tendrils — is met with in Phijcomyces nitens, and was first noticed by Errera (VI II.), but afterwards more fully KIIIZOI'E.K 77 investigattil \>\ W outmann (XIII.), The sporaiipiophoreH of this Mucui'ii when {gently touched at the hide, ciu ve in Mich a manner that the phiee touchecl aiihunies a concave form. This phenoniemm is termed haptotroplsm. We are indebted! to A. DK l>AUV (II.) for the e;irlie>t t'l)ser\iitiiin« on tlie rehitiou between contact atti-action and the formation of ajtjiressoria in fungi. The matter afterwards enpi«;ed tlie attention of M. Blesoen (II.) — in tlie case of liutiytis ciiierea — and other.s. Ji/ii::i'/>us nii/riranx is the hest and longest known member of tliis family. In 1818 it was described by Euhexuekg under the name Muror Htolonihr, which is still ii.sed bv several workeis ; and the .same authority afterwards conferred on it the new appellation. This sajirophyte readily and almost invariably infests vegetable substratii — e.y. fruit — when the latter have been exposed to the air for some time in a damp condition. The stolons often attjiin a length of 3 cm. : the sporangiophores may grow to a height of 4 m.m. (Fig. 117). The .sporangium is pure white at first, afterwards yellow, and finally almost black. When rij>e, the membrane licjuefies on coming in contact with water, and therefore cea.ses to be visible in ordinary mici-oscopical {(reparations. In such case, beginners generally mistake the large columella for the wall of the sporangium. The spores are of varied form, mostly oval, and their longitudinal measurement is 6 to 1 7 /i. The name lUtizojmf^ onjza: has been given by Went and PiaxsEN CJeekligs (I.) to a fungus discovered by theui in Ragi (241), the .sponxngia and spore of which organism axe con- siderably snialler than those of li. niyriram. Nevertheless, fluctuations in .size are met with in both cases, according to the conditions of nutrition and deve]t)pment. This species was examined by C Wkilmkii (XIV.), who failed, however, to find any fundamentjil tlitVerence between the same and li. rtii/riconti ; and it is therefore probably nothing more than a variety of this last fungu.s. CHAPTER XLIV. FERMENTATIOX BY MUCORS. § 238.— IntPamolecular Respiration and Alcoholic Fermentation. The exhalation of carbon dioxide, to which the name respiration has been given, may occur, in both animal and vegetable cells, under two widely different sets of conditions : either in the presence and unrestricted access of oxygen, or in the entire absence of this gas. In the former case the process is termed oxygen respii-ation ; but only a few remarks can here be made in connection therewith, for the most part to supplement the thorough description given in Pfeffer's (III.) Handhool; of Verje- tahJe PhydrAogij. The volumetric ratio between the absorbed oxygen and the evolved carbon dioxide, or, as it is termed, the quotient of respiration (CO., : O.,), was found by Saussure, as long ago as 1833, to vary with the nature and actual condition of the plant under examination, as well as with the conditions of nutrition. With regard to this latter factor, it was shown by DiAKOxow (II.), in the case of Penicillium ijlaucum, that — other conditions being equal — the quotient of respiration is equal to unity when the food.stuff is sugar, but 2.9 when tartaric acid forms the nutrient substance. Puriewitsch (III.) then demon- strated, in the case of Aspi'ir/iUus niger, that an increase in the concentration of the nutrient solution causes the quotient to rise at first, but afterwards to recede. The influence of temperatui-e on the quotient of respiration was investigated by C. Gerber (I.) in cultures of the last-named fungus grown in Raulin's nutrient solution, with an addition of tartaric (malic or citric) acid, either alone or in conjunction with saccharose, as the organic foodstuff. Mention has already been made in |:^ 233 of the influence of the degree of illumination on the quotient of respiration, as well as on a few other points in connection therewith. We shall have to deal in a more thoroughgoing manner with the second kind of respiration, to which the name of intra- molecular respiration has been given in consequence of a pro- posal made by Pflueger in 1875. This phenomenon consists of the persistent exhalation of carbon dioxide by cells excluded from a supply of free oxvgen. The sole materials for this 78 INTIIAMOLECULAH KKSPIKATION. 79 respinitioii are the eurbohydi-iites, tmd in fact, priiiiurily aud diiertlv, oiil\ reitiiiii kinds of Kugiir, wliich are deeOKed in siu'li a iJiaiiiiiM- as to fiiinish, in addition to cailMin ilioxide, a residual product poorer in oxygen. This product, however, is 1)V no means a uniform entitv, hut consists of a mixture of several suhstances, alcohol beinj; an invariable constituent. In lii^rher plants the enerj^y with which carbon dioxide is liberated during intnunolecular respiration is not infreubject has been collected in a meritorious treatise by C. CJekuek (1.). It is not within the province of the piesent work to go more closely into the botanici>-physiological aspect of this phenomenon ; and besides it will be found thoroughly leported in the above- mentioned Ihindliooli of Pfelfer's, by all who are interested in the matter. The tirst observation of iutiamolecular respinition in fungi was that of Bail (1.), who, in 1857. .showed that the mycelium of certain (unspecified) Muciirf, when kept submerged in a sjicchariiie licpjid ami out of contact with air, a-s-sumes an unusual foiMi (si'f ,^ 219), and in.stead of consuming the sugar to carbon dit)xide, as it does in presence of air. converts the same in such a manner as to produce alcohol. This new fact was .seized U|H)n by PastEIK (11.), who endeavoured to utili>e it as a pillar of support for his novel theory of fermentJition. This we have already referred to in ^ 16 and <$ 113, mentioning at the time that it is not universiilly applicable. In fact it is only uncon- ditionally true in the ca.se of a single group of phenomena, namely those produced by strictly ana-robic agents, the butyric- acid bacteria in particular. On the other hand, teur and his predecessors, concerning the prcnluction of alcohol by Miirontf under the specified conditions, remain unimpeached ; only, this formation of alcohol must not be regarded as perfectly identical in nature with the alcoholic fermentation excited bv veasts. 8o FERMENTATION BY MUCORS Mention will be made further on respecting the difference between the two processes. As already hinted, the presence and accessibility of certain kinds of sugar must be regarded as an indispensable preliminary condition for the occurrence of intramolecular respiration ; and this will be more fully elucidated in subsequent paragraphs. Even under otherwise equal conditions, differences exist among the various fungi in respect of the extent to which intra- molecular respiration may develop, and of the energy with which the resulting liberation of carbon dioxide proceeds, in some species the phenomenon seems almost entirely absent, and in others, e.ij. Rhizopus nigricans, it is very feeble. According to DiAKONOw (I.), Penirillium (jlaurum and Aspergillus iiiijer cease to give off carbon dioxide vei'y soon after tlie exclusion of air has been effected, and they perish within an hour, even when grown in nutrient solutions containing sugar. In this case, as was shown by Pasteur (III.) and Brefeld (XIII.), merely extremely minute traces of alcohol are formed. According to a private communication from Prof. Hansen, Schioenning has failed to confirm the contrary report of Elfving (I.) to the effect that he found considerable amounts (up to 4.2 per cent, by vol.) of alcohol in cultures of the first-named fungus ; consequently this report must undoubtedly be based on error. Apart from the true alcohol-yeasts, in the restricted sense of the term, the producers of the largest (percentage) quantities of alcohol are to be found among the Murorecv. The behaviour of these under the con- ditions inducing intramolecular respiration will be dealt with in the next paragraph. We will now tvirn to the difference between intramolecular respiration and alcoholic fermentation, and ask ourselves whether this difference is one of principle or merely of degree. Those who inclined to favour the latter hypothesis were able to advance many circumstances in its favour. The chief products (alcohol and carbon dioxide) and the subsidiary ones (glycerin and succinic acid) are the same in both decomposition processes. The objection raised by the opposite party, namely that the ratio of transposition is different in intramolecular respiration, and that a smaller quantity of alcohol is formed per unit weight of sugar consumed, was disposed of by the discovery of Godlewski and PoLZENiusz (1.), in 1897, to the effect that peas stored out of contact with air produce 1 01 to 103 parts of alcohol to 100 parts of carbon dioxide exhaled, i.e. nearly as much as the 104.5 P®^' cent, that should result from alcoholic fermentation according to Gay- Lussac's equation. Nevertheless, so far as we are able to judge in the present state of our knowledge, there exist two funda- mental differences between the processes in question. One of them has already been mentioned, namely the inception of alcoholic feimentation by yeasts in presence of air ; and the Ml ruli lEKMKNTATlu.N.^. 8i Kecoiul wjII benmie apiHtrent as tin* iiuH-lianics of the two pLeuo- uiena are exuiuiiu'd and foiiijs«.*rveil iti liifjliei- ]>]anti^ aiul Mucijrt'u, inti-jiiiiulfcular if.spimtion is muiouliteilh an intra- cellular process, a inanifestiition of vitality on the jjart of the cell as such, and thfrefuiv insejtarahle fniui the corpus of tin* cell. On the other haiiil such a local limitation cannot he assnnn-ti iu the case of alcoholic fermentation by yeast since it has become known that this phenomenon is not due to the action of the cell ]'er ^•«', but to a contained enzyme that is able to exert its powers outside, ami independent of. the cell in which it originated, and can incite the aforesaid decomposition of supir when placed in a test-tube. Throuf^h the discovery of this new fact, the pheno- menon of alcoholic fermentation by yeast has lost, at one blow, its character as a direct manifestiition of vitiility on the jwirt of the yeast cell, and has been transferred to tlie cjitegory of enzy- matic action. Consequently, those who defend the hypothesis that these two processes are identical in princij>le, and ditler merely in degree, will have to seek for the concomitiint action of a similar enzyme in intnimolecular respiration as well. So long, howevei", as this has not been i)roved we must still uphold the assumption that, on the basis of facts — so far as the.se are known — a di.stinction must be dniwu between the formation of alcohol by intraniolecidar respiration (pna-eeding entirely within the cells themselves) and the alcoholic feruienLition excitetl by a yeast enzyme (which may abso act outside the cells). Xeverthe- le.ss, for the sake of simplicity, we may still continue to speak in the following ]»nragraphs of alcoholic ferment-ition induced by Mu'-iir'n. provided the ilitlerence specitied be borne constiintly in mind. .^ 239. -Mucor Fermentations. Mail, who discovered that alcohol is foinied by intramolecular respiration in certain species of .!/»/•»//>, diy E. Ch. Hansen (VIll.), who in 18S7 reported on his experiments with Mwor iiiuct'do, M. rai'euKn'U-!', M. er'pinosu!!. His dis- VOL. II. ¥ 82 FERMENTATION BY MUCORS. coveries were afterwards supplemented by other workers who will be mentioned later on. The degree of susceptibility towards alcohol is an important factor in solving the question as to the suitability of these fungi for the purposes of the distiller, since it is this sensitivity which, other conditions being equal, determines the attainable produc- tion of alcohol. Another iniiuential factor is the fermentative activity, i.e. the duration of time in which a given quantity of alcohol is produced. A few reports on this point are collected below : — Mucor muredo has produced :— According to After In Pasteur (III) Brepeld (XV.) FiTZ (XI.) at 30° C. Hansen (VIII.) at 23° C. and at room temperature 14 months | wort 7 weeks 15 days 6 months must wort wort Alcohol 1.8 % bv weight 2.6% ■ „ 0.8 % „ 0.5 % by volume 3-0 % . Mnror racemosus has produced According to FiTZ (IX.) . Brefeld (XIII.) Brefeld(XIII.) Pasteur (III.) . Pasteur (III) • FiTz (XI.) . After f " many \ \ months " J 2 years 2^ years 1^ monthfi In At Hansen (VIII.) . > 12 months must f nutrient) \ soln. J wort must w'ort 25-26° C 15° C. f high I \temiJ.J f room \ \temp.J 25-30° C. / room\ \temp. j Alcohol 3.5-4.0% b weight 4-5% up to 5-5% 3.3% 3-4% 2.3-2.7% 7.0% by volume According to U. Gayon (V.) the production of alcohol by Mncor sidno-^us ceases as soon as the amount formed attains 1.5 to 2.0 per cent. ; but Hansen, on the other hand, succeedecl m de- tecting 5.4 per cent, by volume in wort cultures at 22 O., atter 61 months. Jhimv drcineVoides, according to Gayon (VI.), pro- duces 5.5 per cent, of alcohol. Mucor eredus, according to Hansen, when grown in wort cultures at room temperature, furnishes 8 per cent by volume in ih months, but only 7 per cent, at 25 G. It therefore probably forms an exception to the rule applying to all the other MtmorecB mentioned, namely, that they produce larger quantities of alcohol at higher temperatures than at lower. Thus, according to Fixz (IX.), Mucur racenwsus is very sluggish MUC'()1{ KKKMKXTATIUXS. 83 in this respect ut teiiipeiaUuiv, below 15° C. The weakest of all Beeuisto be /{/ii::<>j,Uf: ///<;ilts, at 25° C, air Ijeing excluded: alcohol, 1.46 percent.; succinic acid, 0*02 per cent. ; glycerin, 0.12 per cent. The mutual iiitio between these three products is 100 : i .4 : S.3. The amount of succinic acid seems to differ with different species; at least HitKKELD (XIII.) .states, on the basis of his experiments with Jilii.:ii/ius 7//«/r//-a«,<, Mu-nr iiucedo, and M. raceinom^, that cultures of the first of these organisms yield the relatively largest pro{>ortion of acid, and tho.se of the third tiie sm.illest, though still more than is furnished l)y beer yeast under similar conditions. The.so results pre- sumably relate solely to succinic acid ; at lea.st this is probably the case with .1/. ruri'iiiosit.t, since, according to Vu. HioiutiE (I.), this organism does not prcxluce any volatile acids. Pix>duct.s of the ester cla.ss have been often ob.servod to result from the intramolecular respinition of Mueontv ; e.g. an otlour i-esem- Iding that of pears, in cultures of .)f. ru'emosiw by Brefeld. and one recallim: that of plums, in .l/'/-./- ./r.-//;' //..//.> bv Gayon. We will now in.juire as to the kinds of carbohydrates which may be ilrawn upon by the Mm-oon for the purjK..-o of forming 84 FERMENTATION BY MUCOES. alcohol ; and we will divide these into sugars on the one hand and starch and dextrin on the other, so as to be able to classify the species of this genus on the basis of their practical applica- bility. Starch and dextrin do not yield alcohol direct, but only after they have been converted into sugar ; consequently they can only come into consideration in the case of such Mticors as are capable of producing saccharifying enzymes. These species will be fully described in the next chapter, and for the present we will deal solely with the formation of alcohol from sugars, and with the inversion of the latter. So far as is known, saccharose is split up by only two of the species hitherto men- tioned. One of these — according to Fitz (IX. and XL), Bre- FELD (XV.), and Haxsek (VIII.) — is Muror racemosus ; the other was observed by the last-named worker, and is probably nothing more than a sport of the same organism. These two species produce an inverting enzyme, which faculty constitutes a sharply defined distinction between them and the Mucor and Rhizopus species hitherto known. Hansen's comparative ex- periments revealed the importance of this physiological charac- teristic as a means of differentiating the individual species ; and its value for the purpose in view is so much the greater from the fact that its occurrence in these two species, and absence in all the rest, is quite independent of the envii'oument ; which is more than can be said of morphological characteristics. It is therefore advisable to point out at once that saccharose cannot be inverted or fermented by Mucor viucedo, M. erectus, M. spi?i- osus, M. alternans, M. circinelloides, M. Mouxii, or Rhhopus^ nigrica7is, though they can draw upon it as a source of carbon. On the other hand, with the exception of Rhizopus nigricaTiSy they can all attack maltose and convert it into alcohol. Whether the fermentation is preceded by a fission of this disaccharide into hexoses, as occurs in fermentation with true yeasts, has not yet been investigated. None of them is capable of convei-ting galactose into alcohol direct, though they can do so after inver- sion, as shown by Fitz (XI.) in the case of Mucor racemosug. This latter is therefore equally incapable with the other six species of pi'oducing an enzyme that can hydrolyse milk sugar. According to Dubourg (I.), trehalose can be fermented to alcohol by Mucor alternant, though raflOnose cannot. From dextrose and Isevulose each of the above-mentioned Mucorece can produce alcohol by intramolecidar respiration ; and the same- naturally also applies to the invert sugar formed from sac- charose, by chemical reagents or the preparatory activity of other organisms. Presuming the above-mentioned preliminary hydrolysis of maltose to be correct, the formation of alcohol by the intramolecular respiration of Alucorece will fall under the same law as has already been found to prevail, without excep- tion, in the alcoholic fermentation excited by Swrliaromycetea MrcOll FKltMKNTA'nuXS. - 85 and other l)u. Miliar Iuu^m, luniiely, that the hoxose« alone are cajHiltle (A fiiiiiisliiu^' ah-uhol diiect. HiiHin^ on tlie oni' Ijund 011 the iiuifregn.iliility of saecharose, aixl on the susceptihility to dissociation of tlie hexoses on the otlitT, (J.wox (IV.) has atU-nipted t<» (dal.urat.' a prcx-ess for extim-tin',' supu- from niohissi-s. The cliief feature of tlie pro- cess is the decomposition, by Miu-or cin-iric/foiihs, of the invert siipir, which, as is well kn(.wn, prevents the crystillisiition of the acconipanyin'.' saccharose in molasses; the resultin;; alcohol is then distilled off, and the s^iccharose is finally sepamtcHl from the residue by crystiillisiition. In the n)anufacture of snnfT, a certjiin part seems to be played by the intramolecular respinition of Mwui-k, especially in connection with the after-fernjentjition in the so-called " jiig- tails" to which reference has alreauro'iii'isu.<) both in tlie pig- tiil.s and on tobacco leaves, both jrreen and dry ; and he also observed that this latter organism acts therein lis a prcxlucer of alcohol, drawing upon the sugar contiiined in the " sjiuces " (Jj 1051. On the other hand, he rarely succeeded in finding Mucor species on tobaccos that weie not treated with "' s;iuce." With regard to the part played by Mucors in turning bread moulily and in the rotting of fruit, this and correlative questions will be dealt with in the final section. O. Johax-Olsen (II.) in dest-rilting the prepanition and com- position of (Jammelost (a Norwegian chee.se), states that certain species of '' -.l///<-.y forming pungent fission |>roducts. CHAPTER XLV. THE USE OF MUCOREvE IN THE SPIRIT INDUSTRY. § 240.— Mucor Rouxii and other species of Amylomycetes. The faculty of producing diastatic enzymes and thereby exei-ting a saccharifying action on both starch and dextrin, is possessed by a considerable number of the Mucors. For the eailiest informa- tion on this point we are indebted to U. Gayon and E. Dubouhg (II.), who observed this faculty in Alucor circineUoides in 1886, and also found that this organism was only capable of exerting the influence specified when it developed in aggregations of gemmte, but not when in the form of its normal mycelium. Shortly afterwards they reported (III.) on further similar experi- ments with Mucor alternans, and demonstrated that, under equal conditions, this organism converts starch into maltose and not into a hexose. The faculty is lacking in other species, e.g. Mucor racemosus, which, according to FiTZ (IX.) also leaves inulin unacted upon. There are other more powerful species of Mucors, which, on account of their pi-actical impoitance for the spirit industry, will now be fully described. For the preparation of rice spirit there is produced in China, Cochin China, and the neighbouring countries, an article known as Chinese Yeast, and put on the market in the form of flat mealy balls, about the size of a half-crown. Its preparation, composition, and application were first described in 1892 by E. Calmette (I.), whose reports were extended and supple- mented by C. EiJKMAN (II.) in 1894. According to these authorities this so-called yeast is prepared by mixing equal quantities of husked rice — steeped in cold water — or rice meal and water, and pulverised aromatic drugs. The number of the latter varies, in different recipes, up to 46, bvit always includes garlic and galanga root. The resulting stiff paste is divided into small cakes of the shape mentioned above. These are laid on mats, previously coated with a thin but close layer of moistened rice husks (paddy), or are covered over with a layer of rice straw, and are kept in the dark for two to three days, at an air temperature of about 30° C. The balls, which by this time smell like yeast and have become coated with a fine 86 ML'COU KOUXII. 87 white velvety covering, are next dried, either by exiK)Sure to the sun, or (in rainy weatiier^ liy Lr,^i,t|o liie heat, and are thi-n packed into bags for Siile to the distiUers. In Indiji and China these balls are employed in the following manner : about 1 .•, jtarts by weight of the finely crushed or ground mass are s|irinkle. (20 litres), so as to half fill the sjime, and are left covered up. The starcli gnulually saccharifies, and the mass con.se<|uently becomes partly liots are filled up with rivei- water, and the sweet licjuid is left to undergo alccdiolic fermentiition, which .sets in very .soon. This .stjige htsts two days, and the mash is then distilled in leaden retorts heated by direct fire. Each 100 kilos, of rice furnish about 60 litres of spirits, cou- Uiining about ^h jier cent, of alcohol. The flora of Chinese yeast consi.sts of bacteria, which, how- ever, are of no uioimMit ; then yeast cells, which must be regarded as the exciting agents of the alcoholic feiinentation ; and certain Mucors, which effect the .sacchariGcation of the stiirch. Of the la.st-named organisms, which alone concern us here, C'almette i.solated a species, which, in honoiu- of his teacher and colleague, E. Koux, he named Aun/lonii/res lioiwii. He did not, lu»wever, ascertain anything definite respecting the morphology of this fungus and its position in the botanical .system ; but it was afterwanls recognised as a Mucor Ijy Eijkman, and its name clianged to Mwor avi ijloiii i/rex lioiurii. The first thoroughgoing investigations regarding the progress of development of this organism were pulilished l>y (.". Weilmek (XI.) in 1900, who at the .sjime time propo.-^e /x in diameter) or oval. They are colourless, or pale yellow to light brown, and have a very thick, smooth, colourless mem- brane. It is solely in the form of these resting cells that the fungus appears in Chinese yeast, to which, as Calmette has shown, it gains access from the rice husks and rice straw. This explains why, in many places, the manufacturers of Chinese yeast consider it indispensable to press a few moistened rice husks into each of the fresh, pasty yeast cakes. As soon as the Fig. ii8. — ^lucor Ronxii. Sympodial branched sporangiophore, from a 7-days old culture on sugar-agar. ilagn. about 160. (After Wc/niier.) Fig 119. — Mucor Rouxii. Sporangium almost entirely empty. Part of the wall still hanging in large shreds on the sporangiophore. In front of the globular columella are still lying three spores. From a culture on rice. Magn. about 230. (After Wehmer.) gemmae arrive in a suitable nutrient medium they put forth germinating tubes and develop into mycelia. Up to the present no zygospores or yeast-like budding cells have been observed in this fungus. When grown on solid media (steamed rice in par- ticular) the mycelium gradually assumes a highly characteristic orange colour due to golden yellow drops (oil ?) appearing in the cell, but only at room tempei'ature, though not at 40° C. The optimum temperature for the development of the mycelium lies between 35° and 40° C. According to Calmette, a tempera- ture of 75° C. kills the organism within half-an-hour, and 80° C. is fatal in 1 5 minutes. The same observer states that the diastatic enzyme produced by Mucor Rouxii exerts its most powerful effects at 35 to 38° C, and is destroyed on being warmed to 72° C. According to BoiDiN and Rolants (I.), the sugar thus formed from the starch is glucose. Like Itevulose, saccharose, maltose, and lactose, this MUC'oij Korxn. 89 sugiir serves un u suurcnof carbon tul>stratiini. thf ;.'hic().>t« is also converted into alccihol. A. SirxiKuKK and W. HiiMMKi. (I.) state that the same also occurs in presence of '/-mannose, fruc- tose, ^^aliictose, trehalose, maltose, dextrin, and f<-meth\l trlu- coside, but not in presence of rathnose, hictose, saccharose, melibiose, xylose, arabino>e, rhamnose, tiigatose, /:f-meth\l gliico- fiide, inulin, A'c. The inability of this fungus to produce invertin Was recognised by J. SANciiNhmi (I ). This worker alst) per- fonned a series of comparative experiments on the elfective force and conditions of the diastatic enzymes produced by Munyr Huiwii, M. aJteniaiifi, and Asjttnfi/fuj^ <>ri/r.ii-, and found that the last-named is weaker than the other two, so far as regards the S;iccharilication of dextrin, the greatest power in this respect being exliibited by the second. The appeanince of free acids in the cultures of Miirnr liiinxii is also recorded by the same observer; Calmette supposes oxalic acid, Eijkman, on the con- trary, lactic acid. The amount of acid may be so abundant as to kill the culture. The imlustrial utili.sjition of this fungus in Europe will be dealt with in the next paragraph but one. At present we will conclude with the remark that Hoidin and Rolant.s' propositi to employ this fungus for converting into sugar, or .ilcohol, the dextrin left in the residues from distillei-ies operated on existing lines, would prol>;ibly be too expensive out- side the laboratory. fi-AmijIovryff^ and y-Ann/loniyn-is are the names of two species of Mifni- wliich liave recently found (-mjilovment in the so-called amylo process (,^ 2^2), inste;id of Munir liimwii (o-Ami/lomyceK). Colette and Moidin found the li species on Japanese rice, the otlu-r {y-Anii/fdiin/rf;^) on Tonkin rice; and we are indebteil to A. Sitmkokk and W. R0M.MEI. (I.) for fuller researches on their moiidiolo^v and phvsiolo^'v. Accoitlinir to these workers, cultures of the.se two species on a nutrient •solution contjiining glucose can be readily distingui.shed from those of Mitmr limuii^hy the circum>tance that, whereas the latter develops as a weakly, barren mycelium almost entirelv within the liipiitl, the ft and y specie.s, on the other hand, alst) git)w on the surface, forming well-develoju'd .terial mycelia, which pro- ject above the level of the liquid a ilistance of jis much as 3 cm., and bring forth sporangia. The latter often develop in a special manner. Instead of the tip of the sponingiophore gradu.allv enlarging to a sporangium, the swelling here constitutes merelv a preliminary st;ige. It does not itself develop into a sporangium, but puts forth one or nu)re threadlike pnK^esses which mav then become crowneil with sporangi.i (one each). This is illu.stn»t4Hl m Figs, ijo and i::i. In both species the ripe sponingia are 90 USE OF MUCOREvE IN THE SPIRIT INDUSTRY. black, and of slightly flattened globular form, the transverse diameter varying between 45 and 95 /ji, whilst the longitudinal diameter is about 15 yu, smaller. The spores are oval or elliptical, brown in colour, and attain the following dimensions, in the Fig. 120. — Sporangiation in ^-Amylomyces. The globular swelling at the apex of the hypha produces in the one case two branches, each carrying a sporangium, in the other case five branches, three of them exhibiting each a single sporangium in different stages of development. Magn. about 120. {After Rommel's original drawing.) Fig. 121.— Sporangiation in 7-Aniylomyces. In the one example the knohlike swelling has put forth three branches, each with a sporangium ; in the other, one of the branches is short and has no sporangmm, the other two being long, declinate, and carrying sporangia. Magn. about 80. (Ajter Rommel's original draiving.) dry state: ;8-species, length about 9 /x, breadth about 5.7 ^; y- species, 7 and 4 /x respectively. The formation of gemmaj on the niycelia is of very frequent occurrence, especially when air is excluded. The behaviour of these two fungi towards I!A(a AN J) TAPEJ. 91 KUgars Wiis exaiiiiiuil \>\ the Limliier mifi-o-ftMiiu'iitiition iiit'tlitKl. The results sliowed that ghu-nsf, fructose, (/-iiianuose, j;uhictot>e, maltiise, ami dextrin are feriiieiited hy both these speciei*, ak'ohdl beiii^' fornieil ; whilst, on tlie 5t and carefully examined by 'J'. CiiiiZASzrz (I.), will produce 1 06 per cent, of alcohol in twenty days in a nutrient .solution containing to per cent, of dextro.se. Sporan^iation proceeds in a similar manner to the two last-named species of Amylomyces; })ut the organism differs from these by forming rhizoid proce.s.ses, and therefore presumably belongs to the genus Uhizojius. % 241.— Ragi and Tapej. The raw material employeil in Java for the production of arrack consists of wa.ste molasses from sugar works, containing about 20 to 30 per cent, of glucose (or invert sugar) and 25 Ui 35 per cent, of .sjiccharose. This molas.ses is reduced to al)out 15" Be. (26"6' Pall.) by dilution with rivi-r watei-, and is then treated, ill oi'der to reliably accelerate alcoholic fermentJitioii, with an auxiliary, which is comjiarable with the leaven (!; 14S) u.sed in European distillerie.s, and is known as Tapej This is prepared from rice by the aid of a second aiixili;ir\ material, wliich the Malay natives of .Tav.i term Ragi or Eaggi, and the Chiuese settlers ftill Peh-Khak. According to A. (J. Vokukum.w (I.) this Ragi is prepart'd by crushing together ])ieces (rich in sugar) of the sugar-cane and the root.stock of galanga (A/jiinia ti(tlitnle fragnuMits are removed, and the pulpv residue is made up into llat, round cakes, about 4 cm in diameter, which are left to drv anil harden in the sun. t^enerallv. how- ever, they must first be laiil in rice .stniw for a couple of tituent of sUiivh j^iaiuih'S in the othrr kinne, aspara- gin,and ammoniinu .sjilts, but not from nitrates and nitrites. It is incapable of producing invertin. A new species, allied to the foregoing, has been de.scribeil, under the name Mnror javaiticuf, by C. Wkjimek (Xlll.). It occurs both in Kagi and Chine.se yea.st, mainly in the form of gemuuv, which retiin their geiniinativi' jiower for at least five yeai-s in these media. Moiphologically, they doselv resemble Muror alteruavK and .1/. rirritwl/nitftv. The mvcelial hyplue measure i 3 t from the mycelium a den.se herbage of sponingiophmes, which form sympoilial branches, attain a height of i cm. and over, and «levelop a globular sporangium on each of their six or more branches. The dimensions of the sporangiiun increase with the height of the support and vary between the limits of z,o and 20 a in diameter. The colour of the sponingium is vel- lowish-grey to brownish : and the membrane is ahnost Jilwavs smooth, as is also the globular (10 to 35 a) columella. The spores are globular to elliptical, colourless, .snjooth and thin in the nuMnbrane. and measure 5 to 6 a, or 5 to 7 /u, by 4 to 5 .u. ITp to tlie prt'sent, no zygosjuires have been observe*!. The optinuim temperature of development lies between 35 and 40" C When developed in .sju-charilied malt extnict or in a solution of dextrose and nutrient stilts, this fungus jirinluces an ap]>rocial)le amount of alcohol ; neither this matter, nor the 94 USE OF MUCORE^ IX THE SPIRIT INDUSTRY. capacity of the fungus for saccharifying starch, has, however, yet been thoroughly investigated. Until further particulars are forthcoming, we must regai'd Chlamydoiimrof ur//:ue as the piincipal agent in the diastatic process that goes on in the incipient Tapej. The gradually increasing quantity of sugar resulting from the activity of this fungus then furnishes the desired nutrient material for yeasts, which are either alread}- present in the Ragi or have found their way ^through the water or from the air — into the Tapej, where they reproduce freely and thus impart to this material the property forming the object of its preparation : namely, to quickly excite fermentation in the dilute molasses into which it is introduced when ripe. Further particulars respecting the yeasts in Tapej and the application of this material will be given in a later section. The natives of Java also prepare Tapej for another purpose. They express the sweet juice, allow it to thicken in the sun, and pack it in small twists made of Pisang leaves. Here it crystallises into crumbling masses, which are called Brem and furnish a favourite sweetmeat. The composition was ascertained to be in one case: dextrose, 69 per cent.; dextrin, 10.6 per cent. ; ash, 1.2 per cent. ; and water, 18.7 per cent. In addi- tion, Tapej is also eaten alone by the natives. § 242.— The so-called Amylomyces Process. The Amylomyces process (or Amylo process for short) is the name given to the process for the industrial utilisation of the diastatic activity of Macor Rouxii and several allied fungi. A company, the " Societe d'Amylo," was founded by A. Collette and A. Boidin (I.), who also, in 1897, took out in the name of this company a German patent for a " pi'ocess for producing alcohol from starchy materials^ by means of aseptic saccharifica- tion and fermentation with Mucedinece, characterised by the feature that, in order to prevent the combustion of starch during aseptic saccharification by means of Mucedinece, the sterilised raw material, treated with water, is subjected to the passage of a current of germ-free air, the material being meanwhile advan- tageously kept stirred by mechanical means ; after which the air supply is cut oflf and the material is pitched with yeast, which, by means of the consequent I'apid liberation of cai-bon dioxide, prevents the combustion of the alcohol already foi"med by the Miicedinece." We are indebted to A. Feexbach (II.) for a lucid description of the practical perfoi-mance of this process in the patentees' works, the maize distillery at Seclin near Lille. Of this de- scription only the main points can be reproduced here. The maize, mixed with twice its own weiglit of water, is steamed for THK SOfALLKl) A.MYLO.MVCES I'KOCKS- ,5 three hoiirs, umler u piessiire of 3.^ 104 atmospliereK, in a Henze hifih-pressiiio steamer, and is then dischargeil into a preliminary masliiii'j tun previously c-liarj^ed with i part, by weij^'ht, of ^reen malt for every 100 parts <»f maize, and sutlicient cold water to retluce the temperature uf the whole to 70' C Presumahlv the sole purpose of the ailded malt is to liides. In construction, this vessel resembles the ajiparatus iise escaping air (as also the caibon dioxide given off during fermentation) is removed through a pipe dipping into a w.iter sail, to prevent direct connection with the atmosphere. 15 v the end of five hours the temj»erature of the contents will have sunk to 38° C. They are then inoculated with a pure culture of the Anif/tdiinfreii, rich in spores, conUiined in a 3 litre ra>teur llask charged with 100 c.c. of beer- wort and 100 c.c. of boiled rice : this leaven is intiixluced thiough a pieviouslv closed special J>ipe in the upper part of the fermenting vessi-l, which i)ipe is immehaft of which passes through a germ-tight stufling box, is next .set in motion, and during the succeeding twentv-four hours a gentle current of air is pa.ssetl through the* mash, which at the siime 96 USE OF MUCOKEiE IN THE SPIRIT INDUSTRY. time is kept in motion to prevent the development of a mycelial herbaere on the surface of the mash, since this would lead to a loss of material by brisk respiration. In twenty-four hours after inoculation, the entire mash is permeated with the mycelium of the xbnyJoinyces. The forma- tion of sugar now proceeds with rapidity. Its fermentation, how- ever, according to the patent in question, is not (or at most not to any considerable extent) effected by A)nylomycetes capable of exercising this function, but rather by a suitable pure yeast, which at this stage is added to the mash cooled to 33° C, the amount taken being about 5 grams grown in about \ a litre of nutrient solution in a Pasteur flask. The reproduction of this comparatively small sowing proceeds rapidly during the next twenty-four hours, thanks to the uninterrupted admission of air. The latter is then excluded, whereupon both species of fungi act conjointly during the three following days : the Amyloimjces saccharifying the hitherto unconverted portions of starch and dextrin, whilst the fermentation of the sugar already present and that continually forming, is chiefly effected by the yeast. The principal task assigned to the yeast by the patentees is not this fermentation, but merely the formation of carbon dioxide, thereby ensuring the presence of an oxygen-free atmosphere within the fermenting vessel, and thus precluding the consumption of the alcohol by the Amylomyces. Collette and BoiDiN (II.) afterwards, in 1898, took out an additional patent, in which it is explained that the yeast may be enth-ely dispensed with, if the saccharified mash be freed from oxygen by stopping the supply of air immediately the iodine test reveals that the conversion of starch is completed ; then passing carbon dioxide through the mash for an hour, and afterwards leaving the Amylomyces to proceed with its task alone. It is neverthe- less a fact that yeast is still added in the Amylomyces process plant erected in Austria-Hungary since the date of this addi- tional patent. An English patent for a mechanico-technological modification of the process was also taken out by Collette and BoiDiN (III.) in 1S98. An important simplification of this process is practised in the Anker Distillery at Antwerp, as reported by 0. Saare (I.). In this case the sterilisation in the autoclave and the boiling with sulphuric acid are omitted. The mash, prepared from steamed maize with an addition of 2 per cent, of malt, is trans- ferred direct to the fermenting vessel, where it is boiled for a short time by steam under ordinary pressure, then cooled and aerated, the inoculation with the Aviylomyres culture being afterwards effected in the manner already recorded. When this has done its work and the mash no longer reacts with iodine, an addition of yeast is given, the kind used being the No. II. race of the Berlin Station (§ 245). THE SOCALLKl) A.MVLOMVfES PltOCRS- ,7 It is not the iiuthui's pioviiice now to puss judguit-nt on the value of the Aiwylouiyces process froni a technological ami economic stiimlpoint, the more so because the ivadei- interested in this matti-r will liinl more precise duta in thi* review pub- lished by M. Dkluuleck (111.). The chief advantiige of the Amylomyces process is the abolition of the expensive additions of malt reds. According to Hoidiu, a third species — nan»ely, that alrea«ly mentioned as y-Aiin/hnnycej', or Mit'Or y — is able to cjirry the fermentation o. i to 0.2 lialling further than is attainable by using the others aforesiiid. I^it- teily, however, this fungus seems to have V>een abandoned again, since it was reportehing the ascus fi-om the sporangium resides in the behaviour of the be^irer of the inherited properties of the organism, namely, the cell nucleus .luring sporulation. The sporangium (p. i , ) is polvnudenr from the outset, whereas the ascus cont^iins only a single nucleus at the commencement of sporulation. The nucleus-as will be more fully descnbe.l in .^ 250— repruluces bv suWivision into as many .laughter-nuclei as there will be ascospores. In the sporm- ,'uim, on the other hand, as many nuclei as there will subse- quently be endo.sporesare transferred from the aseptate mycelium to the sporangium before the s.'parating membmne is formetl It is only ,n the least highly .leveloped Asco„nreh',< that the ascus springs ,lirect from the mycelium ; in all 'the rest it is developed m or upon a special organ called the carpoascus, and all the AscoiuN-c-etes fructifying in this manner are grouped into the sub-class of Carjx>a.(i.tn\ which forms the connecting link with the Carjioa^ri, incipient fructification is already found, as well as the intermediate organ, wliicii we have alreadv encountered undi>r the name of ascogone. We thus obtain the following scheme of subdivision of the < >id('r ( Is nmoascea' : — Kio. 133.— CarUlKT;; Ihittou-Veut, Nu. .•. C'fllH taken from a culture ou wi>rt-;;i.-l:itiiie, ami i-xhiliitiiiK the formation df a to 3 asc'JsjKtrfs In eudi. .Ma^n 1000. (,A/tfr llantrii.) onler, or Sul>-clas8. Gyuiiioascea; (nsci naked) '(o) Without ascogone.-i [a^MyceUal cells themselves be- come Ojil'l Kamlly. Sacc Jin ro- ll! vcetfs. (b) Form nscogoues {,i) Special braiuhc.- of tlieniyct'liuni develop into asci .... 2. Exnasci. 3. Gymnoasci. I02 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. Of these three families of Gymnou^cew we are, in the present book, concerned with only one, namely the Saccharoinycetes, the Gi/mnomci on the other hand being of no importance so far as we are concerned, whilst the Exoa^ci may be dismissed in a few words. The genera of this last-named family have been divided by R. Sadebeck (IV.) into two groups : the one comprising parasites, the other saprophytes. The action of one representa- FiG. 123.— Endomyces decipiens Reess. 1. A mycelial branch, the lower part of which has developed three asci («), each of them producing four hat-shaped ascospores ; whereas the upper portion has separated into oidia (6). Magn. 320. 2. A fragment of mycelium entirely converted into oidia. Magn. 120. 3. A mycelial branch, which has formed oidia (V) at the top, but underneath has developed three chlamydospores {c). Magn. 240. 4. A mycelial branch with asci («) solely. Magn. 320. 5. Pair of spores from such an ascus. Magn. 350. (After Brefeld.) tive of the former group, namely Tayhrina Pruni (also formeidy known as E.roascu!< Pruni), will probably be familiar to the reader, viz. the malformation produced by this organism in green plums. According to Sadebeck (II.), the ascospores of this fungus, when grown in saccharine nutrient solutions, develop into a budding mycelium which excites weak alcoholic fermenta- tion. A second species of this group, Endomycei! decipienx, abounds in the lamellae of the fruit of Ayaricus {^Armillaria) SACCHAltOMYCKTES IX liOTANICAL SYSTEM. 103 tnelleiis, a jKuasiu- attackin'i; tirnbor and well known to foresters as " honey fungus." This secutnl specii-s of tlie /v.'uruft. Onu-ilay ulil culture in beer wort at 15' C Mnt:n. i^-vo. (Ai'U,- Sfhi'iiiitiivj.) regard to the Exi>a.<''i and lii/iiitiers, they otferelated as an intermediate link between the sub-kingdom of the fission fungi on the one hantl and that of all the other fungi on the other. The desire, which then .scnin arose, to find a connection between this outside grouj) and an order of the sub-kingdom Eumycetes, was then-fore not an artificial one, biit in harmony with exist- ing circumst;inc»'s and fully justified. The manner, however, of elYecting the gratification of this desire, and also the unscientific .standpoint adopted as the basis of procedure, cannot be termed praiseworthy. From the explanation given in the intrtnluctiiin to \'ol. i. the reatler is aware that the discoverv of veast as a living organism was coincident with a perional surremler was too much to be expected of them ; and in onler to preserve a little of the conception, they assumed that, even if the parentless origin io6 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. of the yeast cell from lifeless material could no longer be claimed with certainty, there was a probability of yeast being evolved from lower fungi on the one hand, and into higher fungi on the other. Two circumstances favoured the inception of this opinion and prolonged its existence. One of them consisted in the lack of positively reliable pure-culture processes for enabling the observer to start with a single individual (in this case a single cell) and trace its development with uninterrupted super- vision, to the exclusion of all other germs. It would be setting too small a value on the circumspection of the workers in question and the adherents of the assumption that yeast is descended from other fungi, wei'e one to suggest, in their justification, that they were not even dimly aware of the indispensable character of such a condition. They must there- fore have recognised that their method of working was un- reliable and led to deceptive results, and must have been inwai'dly convinced that the solution of this problem was the next task to be attempted. We have to reproach these ex- perimenters that they did not follow this inevitable knowledge to its conclusion, and did not devote their whole energy to first elaborating a truly reliable method of working. This we must do because the confusing results they conjured up by the aid of their oflicious culture methods, inflicted a twofold injury to science : first by aggravating the task of real research, and then by casting over the final results of the latter an anticipatory shadow of depreciation, which is solely due to these mycological necromancies. The second circumstance favourable to the endeavours of those who tried to show the descent of yeasts from other fungi was the discovery of pleomorphism by Tulasne. Since the year 1851, this worker demonstrated, by a number of examples, that certain of the higher fungi were capable, under difi'erent conditions of nutiition, of assuming different forms ; e.g. at one time appearing as a mycelial thread and putting forth conidia, at another as a sclerotium fiom which proceeded small pileated fungi. With such an instance we shall become acquainted, in the case of Sderotinia Fuckeliana, later on. In fact, in other words, it was thereby proved that many of the living forms which had hitherto been regarded as separate species of fungi, merely formed a phase in the cycle of develop- ment of one and the same species of fungus. This theory of pleomoi'phism — ^^which the inquiring reader will find fully described in the mycological handbooks already mentioned, more particularly in the treatise of A. Gilkinet (I.) recom- mended by A. de Bary — was established on an unassailable basis by its founder, by careful investigations of particular in- stances, and really marked a new epoch : not merely in mycology THE OKIOIN or THK SAC'CHAIIO.MVCKTKS. 107 alone. On tho othor hurnl, it ilj«l a guoj deal of luihchief at first ill the stmly of yeasts. The ohvious ()l)jectioii that the lack of reliaVile methods of jmre eultuie i-oukl also luiliUite against the certiiiiity of Tulasue's results, can at once ho disposed of hy the fact that this authority workt-d with comparatively large fungi; and, moreover, a study of his wcuks will very soon induce th»* conviction that such iloul)ts are incapable of shaking the reliability of his discoveries. Furthermore, his determinations in connection with the progress of development — which were mainly based on microscopical researches — were afterwards confirmed bv the cultures prepared by A. ile Hary and others. Tlie case was, however, dilferent as soon as a crowd of imitators began attempts to prove the e.xistenci' «jf similar pli-oniorphism in the less easily examined lower and lowest fungi. Whereas, in the case of the fungi examined by Tulasne— for instance a sclero- tium of (.'farin'ji.'i purpurea — it was ea.sy to select an individual and test its development, it became necessiiry, with the micro- scopically small fungi, to start with a multiplicity of individuals, e.ij. a sample of yeast, owing to the absence of any methoil of pure culture. The detectit)n of a small percentage of germs of other small fungi, <'.(/. a few conidia of a mould fungus, was not such an ea.sy matter, though the sjime nece>.siirilY made their pre.sence felt and even a.ssumed a predominating j)osi- tion when the environment underwent a change in theii- favour. Either in ignorance of, 01 stubboinly misunderstanding, this stiite of affairs, Bcchamp, in iSyijput forward the assumption that yeast cells could be developed from acetic bacteria. A year later, TuKm. (II.) announced having maile the .^ime observation with spores of P'tiirilliinii. C'tmver.selv. according to Dl'val (I.), y«ist cells were assumed wipable of undergoing conversion into lactic acid bacteria. More extensive fallacies were advanced in 1875 by Kuiu.v (1.), giving renewed vitalitv to the reports of ruuciiCT (I.) and lUii,, according to whoili yeast cells are able to change to Mu.;„\ I'mirillium, Aa/Hrt/i/hi.", \-c. (ireater success was attained by II. HokkMan (III.), who even obtiined a prize from the Paiis Academy in 1870 foi- his conversions. Foi- " st^iying power," however, the palm must be awarded to H. IIai.i.ikk (II.), who still continued to uphold, in 1896, the hypothesis which had been relegated bvA.de Harv to the "chronicpio scandaleuse " of .science more than fifteen yejirs ejirlier. One extenuating circumstance to be considered in jwissing judgment on most of the above-named, is the fiuctuations of meaning sustained by the term yeast in the cour-se of time. A backward glance on this {uiint is therefore nece.ssjiry. even on this ground alone. Apart from the vague terminology which permitted (5$ 13) every fermentative agent, even the fi.ssion fungi. io8 MORPHOLOGY AND LIFE-HLSTORY OF YEASTS. to be called yeast, sufficient indefiniteness attached even to the limitation of this term to the ferments belonging to the Eumycetes. For some years after Schwann's discovery, the sole botanical definition of yeast was : a unicellular organism, repro- ducing by means of a peculiar method (termed budding), and exhibiting the power of forming alcohol and carbon dioxide from sugar. In 1857, Th. Bail (I.) discovered the occurrence of budding mycelia (§ 219) in the family Mucoracece, and ten years later assumed the descent of beer yeast from Mucov racemosus. Doubt on this point seemed to him to be the more inadmissible, as he had noticed a weak alcoholic fermentation on submerging these budding Mucov mycelia in nutrient solutions containing sugar. Under the prevailing limitation of the term yeast, the name Mucor yeast was justifiable, though the same cannot be claimed for the generalised assumption, deduced from this ob- servation, that a connection exists between the life history of yeasts and that of the higher fungi. Soon afterwards a successful endeavour was made by M. Reess (I.) to become better acquainted with the yeasts, before disputing over their relationship to other fungi. Following up the previous discovery of the formation of ascospores (§ 247) in the cells of wine yeast and beer yeast, he recognised the import- ance of this phenomenon for the systematology of the yeasts, and did it justice by announcing this peculiarity as a principal char- acteristic of the now remodelled genus Saccliaromyres, which he relegated to the Eumycetes group. Subsequently, the establish- ment of this characteristic led to a separation, which was ob- jectionable from the standpoint of fermentation physiology : of the group of alcohol-producing budding fungi hitherto united under the common denomination " yeast," all those recognised as incapable of developing ascospores were rejected, and mostly, under the title " noii-Saccharoinyces of unknown systematic posi- tion," relegated to the group of Fungi imperfect i. The terms yeast and SaccJiaromyces ceased to be coincident. Many budding fungi capable of exciting alcoholic fermentation, e.g. several of the genus Torulci, which still have some claim to be considered as yeasts, are therefore excluded fi'om the Sarcharomyces family because of their inability to put forth ascospores. On the other hand there are certain true iiaccliaromycetes, which are unable to excite alcoholic fermentation and therefore have no title to the name yeast (in the above sense of the term), Saccharoniyres membrancefaciens being an example. After the publication of Schwann's discovery, that of Reess was the first advance in the systematology of the yeast fungi ; and it was left to E. Chr. Hansen to make the next move, viz. to separate the units (species, races, varieties) of the genus t'Sacchawinyces, introduce experimental investigation into syste- matic description, and, for the fii-st time, base this research on TllL URKJIX OK THK >ACCHA1U>MVlETE.S. 109 really pure culture;-. This he succeetled in accouipliishing by degrees, so that the Sarrharoinyretes now form a larjje uiid well- detiueil family. Of the objections laid ajjaiust the det4.'rmiuatioiis made by Reess, that put forward by Buekelu (V.) muht be briefly cou- .sidered. This worker observed, ami repoi-ted in 18S3, that the Kjxjres of smut fuuj^i ( 6V/ //«s (II.), that while Brefeld's observations in- creased the number of instances of yeast -like budding in the higher fungi, they by no means disproved the former worker's ilemonstnition of the indepen- dence of the genus Ha/^rliaro- inyrea and its allociition to the Agconn/i'^t'f! group. Eight years later, Buekelu (IX.) repeated his hypothesis that the yeasts must be regardetill unknown genus). He wa.s reminded by E. Chu. II.wsex (XVIII.) that the accuracy of this a.>ni/c€te{! and that of any other fungi could possibly have been proved hitherto. Similar expre.s>ions of opinion have been uttered by A. de Bary, Zopf, 11. Will, and others. As already .stilted on several occasions (§| 220 and 243), the ascus can be distingui-shinl from the sporangium by its more iletinite form and by tlie number, shape, and metho<^l of forma- tion of its spores. This precision of form must be prej>ent in any fungus before the latter cm be classified with the A.ores is not always Fig. I j6. — UsUIaco carbo, the cause of smut in oats I. The sporv W. Kruwn in a nutrient solution, has itn-duced a pt^-lyct-llular mvi-clium (f). which has put forth >ca^t- hke conidia (c) Matrn. 45^1. ;. Chains of l>iiii« from these conidia. Magn. 30CW (After Brtjcid.) no MORPHOLOGY AND LIFE-HISTORY OF YEASTS. the same in each ascus, and not infrequently an odd number is present. A similar lack of precision is also exhibited in other genera, such as Ascoidea, Protomyces, and TlieJeholus. Brefeld (IX.) would associate all these into a special group, to which he gave the name Hemiasci, intending thereby to express the im- perfect character of their asci, and that the development of the latter from the sporangia had been, as it were, arrested half- way. Therefore, according to his ideas, the Hemiasci formed a connecting link between the sporangiogenic Phycomijcetes and the true Ascomycetes. Brefeld then endeavoured to effect an analogous separation between the other Myco)7iycefef<, which, as we are aware, differ from the Aaroniyretes by lacking the capacity for producing endogenous spores, and fructify by means of conidia. In the highest of these, namely the Bai^idiomycetes, the conidiophore is developed into a basidium (§ 384). On the other hand, in a number of Mycomycetes that do not exhibit ascofructification (viz. the UMaginetn and the TiUetiece), the precision of the conidiophores is not so great as in the rest. Consequently the}' were separated — under the name Hemi- hasidii — from the latter, or Basidiomycetes ; and, according to Brefeld's ideas, they formed an intermediate link between the Phycomycetes group (with conidial fructification) and the Basidio- mycetes. They therefore constituted a branch of the Hemiasci ; and these latter were grouped by Brefeld along with the Hemi- ba^idii, to form an intermediate kingdom, under the new name of Mesomycefes. This conception, which is more fully detailed in a work by F. von Tavel, and according to which the SaccJiaro- mycetes should no longer be regarded as full-fledged Aaroniycetes, was opposed by "W. Zopf ; but the final settlement of this highly complex question has not yet been reached. For us the Sac- i-liaromycete^ will still continue to rank with the Ascomycetes, sub-class (order) GyDmoasceo', and not as Hemiasci. Any worker who attempts to trace a relationship between the Saccharomyeetes and other fungi must agree with Ileess"s classification of the foi'mer with the Ascomycefes. Neverthe- less, there is still room for diversity of opinion within the above limitation of the question. Thus, A. de Bary had already shown the great agreement, in structnre and other particulars, between the Sarrliaivmycetes and certain of the E.roascece, and remarked that the former might at once be ascribed to this latter group. Another matter worthy of attention is the question regarding any eventual connection between the Saccharomycetes and cer- tain Hyiiliomycefes which' have hitherto been included in the group of Fumji imperfecti. Some warrant may be accorded to such enterprise, though not without attention being drawn at the same time to the circumstance that a successful result must primai'ily be placed to the credit of these Hyphomycetes, since they would thereby obtain recognition as forming part of the THK oiiiory OK the salvhakomvcktes. 1 1 1 cycle of ileveloiniient uf im A-«omyre^, and cousecjuentlv t^ike a step upwards in the IxJtiiuical system. Tliis point, wliicli uiay also lay some claim to the attention of eaint'>t investi-rators will he hrietly toiahetl upon in a later chaptt-r dealing with Dematlum pullulang. At present, on the other hand, we luive to d.-al concistdy with a c-oui>1l* of recent assimiptions, or nither with the demonstration of thi-ir inapplicahilitv. In 1S95 J. Jlulek (I.) enlij:hteji»'d the world with the sen- siitional rejMirt that he had succee'j>'rijillu^ as to cause it to develop S(t'-rhaioiniires cells that produced alcohol. A njmmunication shortly afterwaitls issuets but on the part of a huge number of fermentation technologists, as can be seen, for instiince, in the case of a treatise by Eckenuoth and Heim.\.\.v (I.), who occupied themselves in a very .similar manner with a Peni'illium. In the interest of this branch it was nec-e.ssjirv ind indi.spen.vible that the.se hypotheses should be examine.! and refuted in all points by a number of workers schooled in l>otjiny. The credit of having shared in this ta.sk— a .somewhat thankless one from the scientific stjindpoint— is chieflv due to Kueckeu and S. hi.knmni; (I. and 11.), and al.s€r'fi/hui nrt/zii; or conversion of ^iurfuiru- fiiycea cells into this mould fungus. The same also applies to other .species of A.othesis that Siirf-luiromi/'-ti'S are derived from other fun"i. We therefore maint^iin that the distinguishing ch.inicteri.-tic of the term .Sot-rhai-onii/re^ is a purely lxjt4inico-morpholo"ic;il one. namely, the capacity of the cell for protlucing ascospores. The methiKl of formation of the latter will be fullv described in § 247- -^JJ the ^a'-'-Jiaroint/i't'tt,-' are budding fungi, i.r. thev ve"e- tate in the form of a budiling mycelium of the kind deicribetl in ^ 2 19, On the other hand, lu)wever. not every butldim; funt^us is a ^ao'hanniii/res. The term yeast has two i'miKntant chanic- teristics: one Uiorphological, the other physiological. In the 112 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. strictest sense the term is applied to such budding fungi as are capable of exciting alcoholic fermentation, and therefore the alcohpl-producing Mucors are not to be classed as yeast ; neither are the Saccharouu/eetes tliat lack fermentative power. Even in Pasteur's (III.) " Studies on Beer " no mutual limitation is placed on the terms Saccliaromnces and yeast, but both are regarded as interchangeable, so that in many places we are unable to ascertain which is meant. The chief interest attach- ing to the yeasts is in respect of their practical utility. In many treatises on the physiology of the ferments the question whether they should be considered as Sairharotnycetes, or as budding fungi of some other species, is left untouched. It is therefore impracticable to relegate the species there mentioned to one or the other group ; and in such cases there remains no alternative but to speak generally of " yeast." This has also been done, and should be so understood, in the following para- graphs. In laboratories where pure yeasts are cultivated for the pur- poses of the practical fermentation industry, it is very seldom that the rules of scientific nomenclatvire are adopted by sup- plementing the term Sarcharomyces with a specific name, even when the species of the organism is known with certainty, the usual practice being to name the yeast in accordance with the locality of origin. Thus the Johannisberg wine yeast No. i, illustrated in Fig. 124, was obtained from the sediment of a young Johannisberg wine. " Saaz yeast " is a bottom-fermen- tation beer yeast isolated by P. Lindner from the stock yeast of the Saaz brewery (Bohemia). This yeast will be frequently mentioned later, on account of its very low power of attenuation. The very high-attenuation " Frohberg yeast" originated at Frohberg's brewery, Grimma (Saxony). In many instances the yeast is simply given a number, under which it is registered in the laboratory collection (living herbarium) and is cultivated further. Thus, the distillery yeast known to distillery techno- logists and mycologists under the abbreviated title "Race II.," is the second of a series of yeasts given out by the Berlin Experimental Station, for practical testing in respect of their utility, the species in question finally proving superior to the others. It may be remarked casually that a proper discrimination between the three expressions : budding fungus, Saccliaromyces, and yeast, is often lacking in medical treatises dealing with a pathogenic budding fungus. Some of these are known to pro- duce illness, even attended with fatal results, when they find the conditions of development favourable in the body in which they have made their habitat. The literature on this matter has been collected by J. Raum (I.), and in a monograph by O. BussE (I.), as also in the different volumes of P. Baum- IJOTTOM YEASTS. 113 jjarten's Jahregheriflil. These species are oftt* iitiuies spukeii of as Sarcharomyre^, or yeust, altliouj;lj their connection with thin •jenus, or their f;ioulty of exritin^ feruientation, is more or less doubtful. This reiuiirk applies to the st>-e;illtHl Surrh. j'arri- ininosim TukUhiijf, SarrJi. iieofunnatig, and Saccfi. lit/ioi/tTiejf San- r'elire ; Sarr/i. splinriiius iind SivrJi. (/ml in run J/(M>ni, iVc. One species reco^fuiseil as exciting fennentjition, and consequently to be classed as a yeast, thou<.'h not as yet proved to be a true Sacrharomyres, is a budding fungus discovered by O. liusse, and observi'd by him to iiroduce, in tln« human body, a kind of general de]>ility i^SiVrharinnyi-usii') which may terminate fat;illy. That certiiin true yeasts are pathogenic when artificially introduced int(j the arterial circulation, was proved in 1S92 by Hut-ppe, in the course of expoiiments with Rauenthal wine yeast and young porpoises. This result has been repeatedly confirmed since, notiibly by L. Uabinowit.-ch, in a .series of experiments with about fifty stocks of dilferent species and origin, .seven of which proved pathogenic. § 245.— Bottom Yeasts. If a number of flasks be charged with a clear nutrient solu- tion, of a kind favourable to the growth of yeast and containing a ferment;ible sugar, and each of them be inoculated with a tnice of a pure culture of dilferent yeasts, such as are useil in brewing, distillery work, vinification, Ac, the cultures being then kept at room tempeniture for a couple of days, it will be found that cell reproduction and fermentiition — manifes^ted l)y the appearance of turbiditv and ;'as bul)bles— will occur in all. It will there- after soon be po.ssible to sepanite the flasks into two groups, according to the appearance pre.sented. In the one group the vaist crop developed from the .sowing will remain almost entirely within the licpiid throughout the entire periml of fermentation, and mostly at the bottom even from the start. Yejists of this kind are termed bottom yeasts, and excite bottom -fermentation, the yeast crop l)eiug sets are .ilTorded by the Munich lager-beer yeasts. On the other hand, the most highly VOL. II. H 114 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. developed top-fermentation yeasts are the species forming the bulk of the pressed yeast prepared by the old (Viennese) pro- cess. This pressed yeast consists exclusively of cells raised from the mash by the head, and of the daughter-cells of same, since it is impracticable to separate from the mash such cells as are left therein. These two extreme types of yeast are connected by a number of intei'mediate grades. The question of another, and fundamental, difference between top- and bottom-feraienta- tion yeasts will be discussed in the paragraphs relating to melibiase. As soon as the piimary fermentation is manifestly at an end, let us take a trace of the sedimental yeast from each of the flasks, thin each sample down with a drop of water on a glass ... r , i ^ m rap w ... ■5 .>■' 1 1 ■ •-*"" h ..„>. f^ Fig. 127. — Saccharomyces cerevisiaj I. Hansen. Cells from the sediment of a young culture in beer wort. Magn. 1000. {After Hansen.) slide, cover it with a cover glass, and examine it under a high power (250-500) in the microscope. In many of the specimens the cells will be found globular or oval in shape ; speaking generally, most of the beer yeasts and brandy yeasts will pre- sent this appearance. Kow because, since the time of Meyen, the name Sacch'iromi/ces cerevisite has been usually applied to beer yeast, it has gradually become the custom to say of yeasts exhibiting approximately globular or oval cells of large dimensions, that they are of the cerevisise type. Fig. 127 gives an example of this class, namely a top-fermentation yeast isolated by Hansen (XII.) from the stock yeast (of which it formed the main constituent) of a top-fermentation brewery in Edinburgh and called by him Saccli. eerevida' I. The sedimental yeast in other flasks will be found to differ from the foregoing, inasmuch as it contains cells which, instead of being globular or oval, are elliptical in shape. Yeasts of this kind were invariably found in fermenting must by Reess (I.), who called them Saccli. eJIiji-'^oiiJeii.^, which specific name gradually became enlarged to a morphological designation, so that we BOTTOM YE^VSTS. »«5 tliiMt'foro spL-ak of tliis or that yeafrt an hoinf? of tlie ellipsoideus type, uiivinii)',' tl)erel>y .soK-ly, in the tirst j.hice, tluit tliL- c<-lls of such species are generally elliptical in shape and i-uther smaller than those of the cerevisiie type. Fi^r. 128 gives an exiimplo in the »S(//v7/. eUiiiinpiilt'im I. obuiined hy Hansen (XII.) from the surface of ripe grape.s. This type is exhihited hy many species of wine yeast ; and it is therefore easy Xm umlerstiind why, in the absence of a nietlnxl of piue culture and the consequent iiupossil.ility of dot.-nuining the existence of divers species. KiG. 138. — Sacch. ellipsoiUeus I. Uanst-n. Cells fniiu the ftediiuental ycjist of a youiiB cultiu* in Uxt wort. Magn. iroo. (After Hansen.) Reess's aj)pellation of S(V'h. elliji-foideus very soon became a synonym for wine yeasts in geneml. This custom is, however, now no longer justiliable, since we are nowadays acquainteottles. The third group, consisting of the remainder of the fl:u;ks used in our experiment. dilTers from the other two, inasmuch as the cells of the young .sedimental yeast are of elongated form, something the shape of a sjiu.sjigo or a short tube with closed ii6 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. ends, and in some species slightly constricted in one or two places. Cells of this kind were noticed by Pasteur (XII.) during his researches on wine. They were also found by Reess (I.) in the secondary fermentation of certain wines examined by him ; and this worker named them Saccli. Pastorianus in honour of the French scientist. Cells of this type have been more fre- quently observed by subsequent investigators, and this specific name has been gradually modified into a morphological term. When it is said of a yeast that it exhibits pastorianus forms, or is of the Pastorianus type, the term merely implies that under normal conditions the sedimental yeast of the species in question chiefly forms cells that are sausage-shaped, and not globular, oval, or elliptical. An example of this kind is afforded by Saccli. Pagiorianus /., illustrated in Fig. 129. This species was discovered by Haxsen (II. and XII.), in 1880 and 1881, in the air at the Alt-Carlsberg brewery, Copenhagen, and was intro- duced into the literature, under the above name, by him in 1882, after he had succeeded in proving that it had also crept into the stock yeast of this brewery, imparting to the resulting beer an obnoxious bitter by-flavour and a smoky smell. It is therefore a virulent pathogenic yeast (in the technical sense). It would be a great mistake on the part of the reader to assume, from the foregoing sketch of the three main typical foi'ms of yeast cells, that each species of yeast invariably assumes the same form. This is not the case, a powerful influence on the form being exerted by the conditions of cultivation. This last fact was unknown, and indeed undiscoverable by the methods in use, at the time Reess set up his specific classifica- tion based solely on the form of the cells. Thus, up to the year 1882, it was thought that the bottom-fermentation yeast used in brewing always consisted of the one single species Sacch. cereviske ; and it was not until 1883 that E. Chr. Haxsex (XII.) showed that we have to I'eckon with a lax-ge number of species, and that consequently the names Sacch. cerevisice, Sacch. Pas- torianus, Sacch. elHjJsoideus, &c., could hencefoith be merely used as group names. Since that time, no small portion of this worker's investigations has been devoted to the question of the dependence of cell form on the conditions of cultivation, and to the elucidation of the fact that, morphologically, the character of a species of yeast does not reside in the form of the cell alone, but also in the manner of its dependence on the external con- ditions of which it is the result. If these conditions be known to a certain extent, then the foi'm of the cells constitutes a very valuable and fairly reliable indication. Since, like other mani- festations of vitality, the form of the cells is a I'esultant of two components, namely, inherited properties and the sum of all the external influences, it is evident that, even if the unifoi'mity of the latter conditions could be made absolutely certain, the L BOTTOM YEASTS. 117 foniR'i- leasuii alone woiiUl pii'tlude the expedjiiic-y of perfect refjulaiity in the cells of a culture. Moreover, owing to the iiii])erfectstjite of our knowledge, both in cheuiistry iinil physics, the production of ahsohitely identical conditions of existence in two cultures started at diil'fient times, is unattiiinahle. Again, even when working with a single cell, it will be found tluit the daughter-cells, grown in one and the sjinie nutrient medium, • lifVer among themselves; in one of them one of the inheriteil properties latent in the m(jther-cell makes its appejinmce, whil>t ill another of the cells other inherited properties prepondeinte. i ^ 1 Fic!. 129. — Sacch. Pastoriamis I. Hansen. Cells from the st'dlmental yeast o( a young culture in beer wort. Magii. ic^o. (Aftfr It is nccessjiry tacteria and all other living organisms, ami that too in no less a degi ee than with the yeasts. Xeverthele.s.s, within the limits alVoideil bv the aforesaid ditiiculty of control, one anproximatelv luii- form shape, s;iy, for instance, that of the .sediment;tl veust of the L'erevisia' type. That under these conditions, it is actually jnissible to trace dilVerontial characteristics, may be explained by the aid of the two drawings shown below, both of tliem representing sjvmples from the sediment;il ywist of a culture in beer wort, at the clo.se of tlie primary fermentation. Fig. 130 is the so-called Ciuds- ) ii8 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. berg Bottom- Yeast No. i, the first yeast prepared by the pure- culture method, and by means of which E. Chr. Hansen introduced his pure-culture method into practice, at the Altr Carlsberg brewery, in 1883. It was isolated as the principal constituent of the same stock yeast, which was found to be in- fected with the aforesaid Saccli. Pa4orianus T. A characteristic feature of this yeast is the preponderance of pointed oval cells, those of purely globular form being very much in the minority. Elongated cells also are very rare. On the other hand, the Carlsberg Bottom-Yeast No. 2, shown in Fig. 131, is character- ised by the more rounded appearance of its cells, and by the occuiTence of unusually large, or giant, cells, one of which can be seen on the left of the illustration. The appearance of these Fig. 130.— Carlsberg Bottom- Yeast No. i. Cells from the sedimental yeast at the close of primary fermentation. Magn. 1000. (After Hansen.) Fig. 131.— Carlsberg Bottom-Yeast No.''2. Cells from the sedimental yeast at the close of primary fermentation. Magn. 1000. (After Hansen.} giant cells is specially remarkable in some species, and then forms a good indication. Thus, Beyerinck (XX.) found large cells attaining as much as 20 /x in diameter, in old agar-agar cultures of a budding fungus known as Sarrh. Kefi/r, which he had isolated from the Kephir to be described in our final chapter, whereas the other cells measured only 5 to 6 /x. The sedimental yeast found deposited at the bottom of the fermenting vessels at the close of primary fermentation, in bottom-fermentation breweries, is drawn off — after the removal of the young beer — through an orifice in the bottom of the tun, and is collected in a vessel wherein it is washed with water, to be afterwards stored under ice water until required for pitching a subsequent brew. This yeast, it may be stated, is a highly diversified mixture. Apart from the pos.sible presence of several species of yeast and bacteria (.vrarma, c^'c), it also contains sundry other ingredients, the mo.st importiint of which are : First, salts of lime, chiefly the oxalate, immediately recognisable under the microscope by its octahedral, rhombo- hedral or flat tabular crystals. These have been precipitated during fermentation. Their origin is only to a small extent liOTTOM YKAS'rS. 119 attrilnjtjiV)lo to tlu* uietaljolism of tin* Vfust ; they ulsiiis, or eiuployiu',' it for .starting' a siii;.'le-cell culture. Secondly, the yeast mixture contains precipitiited hop resins, in the shape of very small ;,dobules, sometimes united as aggregations ; they give the resin reactions, and therefore assume a handsome retl coloration in presence of alcoholic tincture of alkanna root. Thirdly, there are the so-cjilled ghitin bodies, which are line ghdades of an albuminous nature, originating in the malt and precipitated fi-om the wort at the low temperatuiv (jf the fermenting cellar. They have formed the sul)jeet of some deep researches by 11. Will (111.) Fourthly, certiin dark brown fiagments, which are mostly lookeil on as hop resins by practical brewers, but in reality are said by 11. Will (IV.) to give all the reactions for albumin. When present in large (juantity they form a .source of trouble in brewing, by enveloping the yeast cells and rendering these latter inoperative. The upper layer of the sedimenUil yeast in the vat contjiining the beer in comlition for racking, is specially rich in such extraneous admixtures, and is con- sequently rather dark in colotir. This portion is generally removed in advance and thrown away, before the underlying "white" yea.st is drawn olT. Fifthly come mucilaginous mattens of diiTerent kinds (^ 354 and 255), which have been excretetl bv or extracted from the yeast cells. Sixthly are residual frag- ments of the mashed materials, hops, lupulin granules, and not infrecjuently aphides, and the like. It has alreaily been stated that all the Siiniples examined were taken from fresh cultures, /'.»'. cultures in which the primary fernientatit)n was ju.st terminated ami the dej>osited yeast crop was of recent date. On the other hand a diiTerent appearance is prestMited by the cells of a sediment that has lain for some time under the feiinented li«piid~ that is ti> s;iy, in old laboratory cvdtures, or the sludge found at the bottom of lager- beer storage vats, and therefore consisting of cells that have been expo.sed to the influence of the supernatant beer for some eonsiilerabh' time (often .sevenil nmnths). I'nder the.se circum- stiinces a large nundu'r of elongated cells of the I'tisttiriarnut type are produced. I'ven in yeasts that aic ordinarily of y the lilni vary uccunling to the species of yeast. The form of the cells from which the film is constiiu-ted ditVers in general from that of the sedimental cells, hy uttaitiiiig greater longitudinal de- velopinent(uj) to 150 fx and uvei), whilst the tnmsverse mea- surement is often less than in the cells of sedimentiil yeast. The second char- acteristic is a more or less abundant branching. An e.\- W amj K' is shown in Fig. 132. The time re«jnired for the ilevelojinient of the film to become manifest differs, other conditions being etjual, with the species of the veast, and is lonirer the lower the tem- pemture of the cul- ture. According to a series of determin- aitions made on species of ISarrharo- »«//'■»>■ bv II.\NSEN' (XVI.), " the time required for N about 9 to 18 davs ; at 20' to 28' v., about 7 to 1 1 days ; at I to 1:5 C I) / r ^ 00\^L> ir^ u about I ; to 30 davs ; iKiO. 133, — SarchAToniyce* ellipsoldeui I. Han««n. Cflls .-nut cb.iins o( cells trom the film of an old culture in beer wort. M.vrn. : -..-^ lA'^rr Ucin^m.^ 122 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. at 6° to 7° 0., about 2 to 3 months. N"o formation of film occurred above 34° 0. or below 5° C. These limits of temperature, which also apply to all the other species examined, are therefore more restricted than those wherein the yeast is able to bud and incite fermentation. Film formation is therefore precluded in the fermentin^^ cellars of bottom -fermentation breweries, the temperature here being, wherever possible, maintained between 0° and 2° C. It has already been shown by Hansen, and confirmed by H. Will (VIII.), R. Aderhold (I.), and other workers, that the time elapsing before the film makes its appearance, and the dimen- FiG. i33.-Sacch. Past. II. Hansen. Fig. 134.— Sacch. Past. III. Hansen. Cells of film grown on beer wort at 20° to 28° C. Magn. 1000. (After Hansen.) sions attained by the film, are very greatly dependent on the conditions of cultivation (composition of the nutrient solution, and also in a high degree on the method of sterilisation, supply of air, (fee). In some cases morphological peculiarities in the film cells afford a means for differentiating the species. With Sarch. Past. II. and Sacch. Past. III. this can be recognised in a beautiful manner, and at the same time a fresh in.stance is afforded of the dependence of cell form on temperature. The first-named, weak, harmless, top-fermentation species was isolated by Hansen from the air of the Carlsberg brewery. The other, which is of decidedly top-fermentation character, was obtained as a pure culture from a Copenhagen lager-beer suffering fi-om haze, and was recognised as the cause of the malady. It is difiicult to distinguish between them by the form of the cells present in the sedimental yeast, both being very similar to Sacch. Past. I. (see Fig. 129). The same also applies to the component members of their films grown at 20° to 28° 0. and illustrated in Figs. 133 and 134. The case is, however, FIL.M I oKMATlON. "3 (lifrt!t>- H ii;ir..ii)\ir« I'.ijit, 11. Uauscii. ?'/«•«.>■ /. and S(trrh. I'l- Colln in nim pmwn .>ii lH«cr »nrt at 1 ; to isT. \likfp\. lij>ti()i^ti'U.< J I. /laiisin. • The tacidty of developing a film on the surf.ice of suiu-ihle luitrient solutions i< shared l>y nearly all the budding fungi, 124 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. both Sacchawmyretes and 7ion-Saccharomycetes. Of these latter we shall deal (in the last section), under the generic name Myco- (lerma, with a special group, the species of which are widely distributed and grow spontaneously on the surface of wine or beer when the latter ai'e exposed to the air, a quick-growing, wrinkled skin (mould film) being formed. Air being highly essential to Mycoderma, these organisms normally grow ex- clusively in the form of a superficial film on the nutrient solution. Owing to this peculiarity they are the cause of disturbance in researches into film-formation in the true yeasts, when the latter are not grown as pure cultures but contaminated with the very abundant Jlycoderma. On this account objection may be raised against the reports of Reess (the first to observe the production of films by true Saccharo- myrete^), and also those of Pasteur (III.). In 1876 the latter characterised as aerobic or mould yeasts the film developing ou the surface of fully fermented wort. He halted between two opinions with respect to this phenomenon, one being that it was a special (i.e. aerobic) condition of development of the beer yeast residing at the bottom of the fermented liquid ; whilst the other looked on the film as composed of extraneous cells undesirably present with the sowing. It was not until Hansen applied pure cultures to these investigations that a decision could be formed on this point. The question of the convertibility of bottom-fermentation yeast into top-fermentation yeast, and vice vet'sa, is also touched by the foregoing explanations. Pasteur was of opinion that the " aerobic " cells constituting the film that had developed it at the close of primary fermentation in his cultures inocu- lated with (impure) bottom-fermentation yeast, were capable of exciting top-fermentation when transferred to a fresh nutrient solution. He even gave a recipe by means of which the brewer could prevent any such undesirable conversion of the stock yeast. This theoretically and practically important question was afterwards taken up by Hansen, who found that the descendants of the films of all the species of bottom yeasts examined by him in this connection, invariably produced nothing but bottom -yeast cells when inoculated into fresh nutrient solutions, even when exposed to a temperature (26° C.) very favourable to the progress of top-fermentation. The true Saccliawiaycetes can be separated — though not very sharply — into two groups : one of which does not form films until the primary fermentation is terminated and the sedi- mental yeast has all come down ; whilst in the other group growth proceeds on the surface fi'om the commencement, and indeed in many cases exclusively so. An instance of this latter kind is afforded by the Sacrharomyces Diemhrana'faciens, first discovered by E. Chr. Hansen (VIII.) in the mucinous FILM FOn.MATIOX. »2S ili.scluirge (^ 248) fiom elm, and Inter hi well - water by J. KuEULEH (1.). Till* sfpurute stages of tlio dovflopinent of film were closely iiivc>;tigatfil in four species of hottijui-feiiiientiition ye:ist by II. Will (VI II.). At the outset no tlillerence can We cleteeted between the cells of the seilimental yeast and those retained floating on the siufaee by Hakes of albumin and residual frag- ments (»f the •• head," and from which the development of the KlO. 1J7.— Cells of SfdiiiK'Htnl yca«t from a wort i-ulturc of- 9-0 >-§s 0, ^.i-®.3 oa ^ o PoQ,Oo9 o o Klennanent cells from the nini of a wort culture of — Bottoni-fermcntatlcn beeryeait No. 93 of the Muniih Ilrewinc Station. Masn. r yeast i.slets originates. Later on, however, it is ob.served that these floating cells produce daughter-cells, the chief feature of which — as may be seen from a comjiari.son of Figs. 137 and 13S — is that, instead of appearing singly or in pairs on the mother-cell, as they do in the seilimental yeast, a number are formed simultiineously thereon. They are al.so much smaller (»■.;/. only 7 /i as compared with 10 ft) than tho.se in the sedimental ye.ist. ai-e oval or s.iu.sjigo shaj>ed, and in t>n-n pro- duce similar daughter -cells, the whole remaining conntH."taireil in the first new generations, and in specially conspicuous ca.ses several recultivatious (repeated tninferences of the crop to fresh nutrient solution) are required in order to protluce a sedimentjil yeast equal in all respects to the original ance.stors of the film Cells used. Notice should al.so be til ken, e.ij. of an observation on this point by Ed. Kaysek (VI.). Further considenition will be given in subsequent panigraj)hs to this behaviour, from the standpoint of the theory of variation. At present we have to deal with the consequences connected with the pnicticiil cultivation of yejust, namely the restriction of film vegetiition and the exclusion of cells derived therefrom. To ellect this object it is nece.ssiiry to keep the stock yeast in the laboratory under such conditions as are unfavoui-able to the development of film, without being at the .sjime time inimical to the .sedimentjil yeast. The appeanince of the former may be countenictetl l)V frequent tnmsferences of the cultures to fresh nutrient solutions, and by keeping the culture at low tempenitures. According to Hansen, the best stiii the yi-ast riiie of a six-iiionlhs-dltl »i>rt culturi- I'f Mtiiiicli li<>tt<>iii'.vca. .•. aiul Rcrniiiiatol toa well-dcvclopctl chiiiii <•( liiulii III a drup uf wurt >>ii a inicri>»i-<>j>e slide in ^ixl)■•f(ll^r hours at 10' C. A &t.-ptuMi \\JiA fi>niied inside tl>re« of the nieint>ir> i^f Iho chain. Nearly all tlie cells exliiliit one or lwi> vacuole*, and tile two (lernmuent cclb(D) »how an even larger number. Magii. 750. (JJter Will.) 128 MOKPHOLOGY AND LIFE-HISTORY OF YEASTS. duction vessel, but must be first fi-eshened up by prepar- ing a re-inoculation, which in turn is used to inoculate a fresh nutrient solution as soon as development is in full swing. The operation is several times repeated, according to circum- stances, until one is able to assume that the film cells present in the first inoculation of sedimental yeast have been entirely suppressed. The beginner cannot be too strongly advised not to regai'd the task of yeast cultivation as completed by the pre- paration of the pure cultures, but rather to keep the latter under constant supervision, examination and care. Neglect of these precautions, and, in the case under consideration, the use of sedimental yeast containing film cells, may, under certain circumstances, lead to irregularities in the progress of fer- mentation on the large scale, diminution in the quality (flavour, &c.) of the product, and hence to unpleasant consequences for the yeast cultivator. Cases in point have been recorded by A. JOERGEXSEN (YII.). This, however, must not be held to imply that the film cells are the cause of all the unwelcome alterations that may appear in beer yeasts. On the contr-ary, other forces are here in opera- tion ; and from this side also, as already mentioned, we ari-ive at the wide field of variation in the yeast cell, a domain in which, as will be shown in a later chapter, Hansen, by his ex- tensive experimental researches, has been our pioneer. More- over, it should be recalled that Raymann and Kruis (I.) were able, by means of yeast derived from old film cells, to produce good beer that could not be distinguished from that obtained by the aid of normal yeast. This harmonises with the results obtained by Alb. Kloecker (privately communicated to the authoi') with Carlsberg bottom-yeast No. I. and No. II., Sacch. cerevisicB I. Hansen, Marienthal yeast, and Will's No. 2 stock yeast. Will's observations bring to mind the flying yeast (Flughefe) so dreaded by the brewer, i.e. yeast cells which are of smaller size than those of the sedimental yeast, and, instead of settling, continue to swim in the beer, and thus retard clarification. This presumptive relation has not yet been more closely investi- gated, but the researches of Hansen and others have placed beyond doubt that this phenomenon is in many cases attributable to the presence of wild yeasts. Another point that lequires closer examination is the part played, in the maturing of beer, by the film cells produced within the liquid. Finally, investi- gation from this point of view is also desirable on the problem of the cause of flocculence in the process of making pressed yeast by the new, so-called aeration or wort process, which differs chiefly from the Viennese method (§255) in the thick mash being replaced by a clear mash as nutrient medium ; this, after pitching, being well i-oused by aeration, whereby the reproduc- THK A.SCUSl'OKKS. 129 tioii of the cells is «ti>t crop in the chirifyin;; pans, and therefore its hepamtion from the licjuid, is obstructed l>y a so-called llocculenee, which is characterised hy the continued re-ascension of flocculent jij^fjref^ations of cells from tl»e dej»osit. The phenomenon has been descrilied by Stkmm.kin and JuEUUES in "' Alcohol" (i8y2, p. 2l8), and also by < >. I )i i;sr (1.). § 247.- The Ascospores. The first observation of the ascospores in yeast cells was made by Tn. Schwann (II.). He pointed out that the.se fungi reproiluce in two ways : one being by the process known as budding, and the other liy the formation, within the parent-cell, of daught»'r-cells, whicii are libcrateil when the niemltrane of the parent-cell opens. After this phenomenon had been described more closely by J. ije Skvnks (I.) in 1S6S, it was al.so observed a year later by ^I. Reess (111.) in i-ultures of beer yeast on Iwiled .sections of carrots, «\:c. He found the pi'ocess of development coincide with that of certjiin low Atfrumyceifs, and therefore das.seil these forms as ascospores, i-alling the mother-cells a.sci, and for this rea.son relegating the i>ui-r/iaroiiii/nttn (^\i\ 1 8 70) to the position of the lowest family among the Asroini/reteti. The earliest accur.ite investigations into the conditions under which this sporulation occiu's were carried on by E. Chr. Hansen (XII.), and, a[)art from the general biological results, led to the injportnnt fiu't that we have here a reliable meiins, hitherto lacking, of .separating the genus tSaccliaromyccs into its .sj)ecies. The conditions influencing the production of ascos|H>res in the i^airharomiji-etfs tu-e given below: (1) To obtain energetic sporulation, it is neces.s;iry that the sjimple should consist of young and well-nourished cells. (2) The supply of air must be abundant. (3) Tin- metlium nuist be moist. (4) The tempeni- ture of the envii-onment must be maintained within certain limits. (5) Within these limits the time required for the occurrence of sporulation is a function of the t4.«mpeniture. (6) Between the two extreme limits at which sp«irulation is still possible is an optimum temperature corresponding to a time minimum. The maximum tempeniture for sporulation is some- VOL. II t I30 ^yrOEPHOLOGY AND LIFE-HISTORY OF YEASTS. what lower, and the minimum temperature rather higher, than for the phenomenon of buckling. To examine the individual conditions more closely. That the time within which sporulation occurs should be a function of the temperature, requires no further analysis ; but careful attention should be bestowed on the point (in i) as to the condition of the cells, this being the prime factor determining the time limit. The time required for ascospores to be developed by any given species of Sarcharomi/ces, kept at any given temperature, differs according to the physiological condition of the cells themselves. Hence, if it be desired to produce sporulation (unconditionally) in any given species, all that is necessary is to take cells that are in vigorous condition — a state attainable by repeated pre- liminary transferences into fresh nutrient solution. The case is, however, different when it is a question of determining the time required for sporulation to make its appearance at one or another temperature. In such event, it must be borne in mind that this time limit is a function, not merely of the tempera- ture, but also of the physiological condition of the species under examination ; consequently this latter factor must be eliminated in order to enable the influence of the former to be determined. Experience has shown that sporulation occurs earliest and most certainly when the cells have reached the culminating point of their reproductive (budding) and fermentative activity ; and it is therefore in this condition that they should be employed for the experiment in question. On this account the cells to be examined for the time limit of sporulation should be subjected to the following preliminary treatment : the sample is sowed in sterilised beer wort and left to stand for several days at room temperature, Pasteur flasks being the best vessels for the purpose. A portion of the resulting sedimental yeast is transferred to fresh sterile beer wort and kept therein for twenty-four hours at 25° C, the fresh deposit being afterwards freed as carefully as possible from the supernatant liquid, and employed for stai'ting the spore cultures. One example will suffice to show the necessity of taking the condition of the cells into consideration. It is afforded by Hansen's experience, and relates to SaccJi. Pastorianus I. The cultiu-e was first conducted for a few days at room temperature, after which the sedimental yeast was retransferred, in the above manner, to two flasks, one of which was kept for twenty-four hours, the other for forty-eight hours, at 26° to 27° 0. before starting the spore cultures. The following figures show the time required for the commencement of sporulation in the two cases : — TJIK ASCUSI'OKKS. »3» Harr/i. Pcuifoi'ianini I. /lati/n^i. S]K>niliiticiri (Ki-iiri-fU al A(t«r previoiu Cultivation nt 36 to aj' C. for : 34 Hours. 48 Houn. 29° C. . . . 28° to 27.5° C. 2.;.5 to 23" C. 15 C. . . . At the end of 27 hours. It «i 24 ♦• II fi 20 ft 50 .. No Sporulution At the eml of 36 hours •• .. 30 •• »i It 54 ii The sample previously treatt-d fui- I'oity-eiglit hours gave no sporulation at a tempemture (29'^ C.) at which the other produced spori'S in aVMindance. Apparently this unfavourable etfect is attril)utal)le to the increasivl alcoliol content produced by the longer period of fermentiition. The relation in question has also been observed by other workers, e.7. by H. Mieller- TuuitGAU (III.), A. Adekhold (I.), lire. The (artificially effected) temporary or permanent loss of sporogenic capacity of yeasts will 1)0 dealt with in the chapter on variation. The best means of obtaining the moist medium and copious access of air essential to spoiulation, is afforded bv the gypsum block proposed by Enc;el(I.) and suit:il)ly improved by ll.insen. This block is a truncated cone, about 3 to 4 cm. in height, which is prepared, by means of an ungreased mould of sheets iron, from a mixture of S parts, by volume, of powdered, calcined gypsum and 3 parts of water. After the block has become perfectly dry through long exposure to the air, it is placed in a covereorulati(in hegins. C'oiu- jiiue whiit has heen stated in the la-^t jNinigi-u^th hut one of rp riie progress of develoimient in the ascosiKM-es will now lie followed in the ease of Sarr/i. rt-rcinititr I. Haun'-n. If, at the end of ahout twenty-four iiours after the streak has been laid on the blotrk and the latter placed in the thermostat at 25° C, a small portion of the y»*:ist strt-ak he sfrat<'hed ofY with a clean glass roil or a net'dlf. di>tributed in a siugle drop of water, and examined v.. fe ^<: ^£ 'S^^'^^^i Km. 141.— \\iiie Veast (n>ni WalixinJu-im. Assemblage "' buds from an oM nim ; some u( the iiiemlMTs have produced spurea. Magll. Soo (-1/Trr Adrrhnlil.) with a strong power (300 to 500) under a cover glass, decided in- dications »)f incipient sporulation will l)e iihservalijc in a larger or smaller numlier of the cell>. The plasma will he found to have sepanited itself into a number of Uills, as shown at a-orogenic cells to the totiil cells present differs -rreatly accordinj: to the specie.^, orijjin, kind of previous nutrition, A:e., but in most species fails U) attain even aj>pro.\imately to 100. In many of the cultivated yeasts, e.tj. five out of thii-ty-two species of lH)ttom-fermentJttion l)eer yea.sts e.xamineil by L.vsrni'; (IV.), no success has, so far. attended the attempts made to brin<; about sporulation ; con- sequently fen- the present these cannot be regarded as So'-chartj- iinji-i-ti't!. On the basis of his e.vperimental results in connection with sporulation. already alludeil to, Hansen has workeances on gaining acce.ss to the opei-ations of brewing, i^c. and are then spoken of as (technically) pathogenic. E.vaujples of this class are atVorded by SarrJi. I'(i. reinain sp»>reless. Ixith after seventy-two hours at 136 MORPHOLOGY AND LIFE-HISTOEY OF YEASTS i5°C and after forty hours at 25° C. Those of the second group Avhilst remaining sporeless after forty hours at 2C^ C exhibit .pores after seventy-two hours at 15" C. Finally, the members those last mentioned. On the other hand, the wild veasts exhibit spores under both sets of conditions (15° C and 2V C ) withm the time limits specified (seventy-two and forty hours respectively), or even much earlier. Consequently the desired ^T^T^^^^l^t^'"''''''^ ^'"" ""' ^^'^'^^^^ ^--"^^ According to Hansen's observations-which we shall have to deal with in the paragraphs treating of mixed sowings— the wild yeasts that have crept into the pitching yeast or the wort first make their appearance in large quantities towards the end of primary fermentation in the upper portions of the contents of the fermenting vessel Consequently at this period a sample should bedmwn from this part in a glass ; the suspended yeast cells must be left to subside and then immediately transferred to the prepared gypsum blocks, which are maintained at the tempera- tures of 15 C. and 25° C. respectively. At the end of forty and seventy-two hours respectively a sample is taken of each and should spores be detected in either or both series, then the presence of wild yeasts is demonstrated. When the brewerv is unprovided with a suitable laboratory and an expert, a sample ot the sedimental yeast must be sent to a laboratory, a drop of the yeast being dried on blotting paper in the manner described in a later paragraph dealing with this matter. Owing to the abundant sporulation of the wild yeasts, this method is very decisive, Holm and Poulsen having by this means succeeded in detecting the presence of as little as about 1 per cent, of added wild yeast (one or other of the above-named three pathogenic yeasts) in twenty different species of l)ottom-fermentation^beer yeasts For practical use this is sufficiently delicate. On the other hand, to be of any value for testing yeast in a pure-culture apparatus, a method must be absolutely reliable and capable of detecting and isolating even the slightest trace of infection. With this object the yeast from the apparatus is subiected to a preliminary treatment, by means of which the amount of any wild yeast present in the sample is increased. This matter will be further described in a subsequent paragraph treating of the influence of organic acids on yeasts. Differences also exist between the cultivated bottom-fer- mentation beer yeasts and the wild yeasts, in connection with the structure of the ascospores. Those of the latter are relatively smaller, and their contents are homogeneous and of high lustre whereas the ascospores of the culture yeasts mostly exhibit vacuoles and granulation, and the membrane is clearly dis- cernible. '' (JKK.MINATION ( )l' SAl'CHAllOMYl'ETES. »37 Till- examination of tlit* top - I'ci mentation beer yeaBts, to a considerable size. If they are still enveloped bv the |>art'nt cell, thi.■^ l.itter is stretched and assumes a .somewhat .angular contour, as shown at Kig. \.\.\ at a and ofu'„,u m honum- of its discoverer. This fungus is interesting in many respects, /,//,-/• alia as a very fine example of mvct-lial fahii an.l septation in the eells of s^ane. At present, how.-ver w_e have to do witl» another peculiaritv, mmjelv, the behaviom* of Its endnsjMjres during gei n.ination. Tlii-se sjK.res, iuhtead of putting forth loundc'd l.uds of a shiipc similar to their own a. m the type just deseribed. proiluce an elongated, sausage- shaped pK.mycehum of the kiiul illustmted in Fig. 146, 7"', ir T\\U prunm-elium it is that puts forth tlie buds, and i't therefore d i " "^ ^ Km. 146 — Saccharoniyce» LuJwi|;li Hbii6«u. (I. Twogpores. (temiinatiiiK on iht- ..no gl.lc (./ ), fiisii.K (,r >, gTowiiiK into • pronir cehun. (,f ). puttliiK forth a l.uJ (.* , at the crt.w... a.ul «-,.«r.tii.;' i.y the devclJpn"ni or a •eptiin, (,/ >. whirt-uiH.n one of them (./ ) H»»imu» a rounded form, which prv- Cell II il* i'*t"ii"f.f..t«.'\.'t..t>i,...i^^l^'""\ *^ !{• '' -• to « tulK- which pnMluce««launhtercell» hidependentlj. without • t "^r Uaiur,,.) ' constitutes an interme«liale stage between the bud cells and the siK)re. Contrary to the usual priHedtue. the veast cells of normal shape produced by this mycelium are not libenited as the result of constriction, but by the development of a septum •^t the place whei e sepaiation afteiwards occurs. This is shown lit ;/"". Very often, however, the piomycelium is not the residt of the germination of one single spore, luit t>f the coalescence of two germ tubes to form one new cell. This process, one of actual c.'ll fusion, is shown >i-^t>-^ exhibit greater powers of resistance to external influ- ences tlmn is possessed by the vegetative cells. Reference will be made in subsequent jiara- graphstoc-jises wherein the extract ion of moisture, drving, or chemical .stinndants, come under con- cnnieniT of whidi sidenition as influences of this nature. At pre- lHve.*'"wuir»"'nTw sent we are maiidy concerned with the elTects of i'!-"" ■■ • whiUtstiii tern pern tu re, and that too in presence of licjuids. I, ^^j ' "j^^ li.vxsEX (XII.) was the fir.st to point out that i;?'^'^"' ."" ("^o^*^ ., ^ - ' . , ... 1 ^ ,, - 1 • ">« "'J »P"re :iitni- tlie spores 01 Sufr/t. t'iltj'.-i>titi'ii.< It. perish m bram- «a« ruiiturtil five minutes when kept in sterili.sed water at ll'X' aniV" '*" ''''! 66° C, though they survive this periotl at 62' C. thi-oUur. On the other hand, the vegettitive cells tjiken \^tance is offered by the i>archaromi/rt\^ mentionetl in the }ireceding panigraph as Will's (IX.) No. Sir yeast, which was di.scovere^l in a beer of repulsive, bitter, irritant flavour, of which malady it was ivcogniscil as the cause. When kept in beer wort at 142 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. 70° C, an exposure of half-an-hour was required to kill the vegetative cells, whilst the spores withstood the influence of 75° 0. for the same period. Antithetical to this is Sacch. Jcergensenii, which was discovered by A. Lasche (I.) in a very turbid American, so-called temperance ale, and quickly perishes at temperatures above 30° 0. Similar experiments were made with French wine yeasts by E. Wasserzug (II.) and southern wine yeasts by Ed. Kayser (YII.), the latter of whom found the fatal temperature for spores to be about 5° C. higher than for vegetative cells. In order to ensure stability in fermented bevei'ages it is sufficient to so far weaken the yeast cells by warmth as to pre- clude subsequent repi'oduction, and thus practically eliminate them. To attain this end, experience has shown that the ne- cessary influence must be less stringent than is required for killing the cells ; and the case is facilitated when the liquid contains substances which, like alcohol and acids, exercise an injurious effect on yeasts and bacteria at a somewhat higher temperature than the normal. These latter conditions are ful- filled in the case of wine, beer, and wine-must, and an explanation is thus afforded of the fact that these liquids can be converted into a stable condition by a gentle heat, consider- ably below boUing point. The earliest application of such a process is traceable to the Japanese. According to 0. Kokschelt (I.), the Japanese rice beei', or sake (already mentioned in an earlier paragraph), is made to keep through the hot summer months by warming, a practice that has been pursued for more than a century. The first to adopt such a method in Europe was Scheele in 1782 (§ 11) ; and Appert, in 1810, recommended the warming of wine, in corked bottles, to 75° 0. It was, how- ever, soon found that this treatment injured the fine flavour, especially in the case of red wines. Then, in 1865, Yergnette- Lamotte (I.) proposed to employ a temperature not exceeding 50° 0., thereby avoiding the evil specified without missing the principal object in view ; and at the same period Pasteur (XX.), as a result of his researches on wines, found that they could be reliably protected against deterioration by warming them up to 55° to 60° C. With regard to the priority dispute between the two authorities, the reader is referred to the bibliography. Thanks to the reputation at that time enjoyed by Pasteur, the process patented by him was quickly introduced into practice, and called Pasteurisation in his honour. The inventor's primai'y intention was to use this process merely for imparting stability to wine, the killing or permanent weakening of the germs therein being effected by the conjoint influence of a temperature of 55° to 60° C. (in itself insufficient for the purpose) and of the toxic action (at this temperature) of the alcohol and organic acids present. Subsequently the name GKIIMINATION OF SACCHAHOMVCKThX 143 WHS exteiuled to tlit* attempts niiide, by gentle Wiinuiug, to weukeij or kill tin* g«*niis in other lic|uiditic4itionK), must be dismissed in a few weirds, the reader being referred, for further infijrinatioit, to the desciiptions given by Pasteiii (XIV.), li.vuo and Macu (I.), and F. Malvezi.v (I.). The forms of apjuinitus latterly constructetl for the jNisteurissition of wine have been describeottled Ijeer, will be found in Fas.sbender's "Techno- logie," and in the handbooks mentioned in !i Si. The dithculties encountered in the practical pa.steurisation of wine, beer and must are mainly of three kinds. Fir.^t is the selection of the lowest temperature at which it is jK)s.«.ible to attuin the end in view, viz. to kill the yejist and Ivicteria, or remler them incajxible of setting up any fuiiher action in the liquid in question. This teniperatuie, however, necessjirilv varies acconling to the species of the organisms present, and the chemical composition of the wine or l»eor. Hence no invariably applicable figure cjin be given, though 50° to 60" C may usually l>e adhered to. The fuilher this limit is exceeded in the direction of the boiling iK)int, the more decitled will be the boiletl — or in the ca.se of l>eer, the so-cnlletl l»reiMl-like — flavour of the liquid treated. This flavour larijelv owes its ori<'in t<> the mcMlitic.ition of certjiin constituent,s of the hot licpiid by oxygen. To afford a remeily — and this forms the second difliculty — not only must the acce.ss of air l)e prevented, i.e. by warming the liquid in corked lK)ttles or bunged ca.sk.s, but also the oxygen jtre.sent in solution hius to be eliminate*! — as was propo.sed in H. Gi-onwald's (Jennan Patent, No. 98,584 (1896). The third ditKculty, which we need not now consider, resides in the formation of a coagulum protluced by the heat, and con- stituting a deposit which, .since it would spoil the appejinince of the wine when poured out, has eventually to be removetl by liltnition, followed by a .second ]>a.steuri.sation. Some very tenacious bacteria are able to survive the jwsteur- isi\tion of the three liquids mentioned, but are saircely cni«ible of doing any subsiNpient damage. Thus, in a sjimple of Munich beer sufliciently pasteurised for the exjxjrt tnule, IXjUiEXS (I.) found 34 bj\cteria per c.c, all able to develop on nutrient 144 MORPHOLOGY AND LIFE-HISTORY OF YEASTS. gelatin. It may be remarked that the Mycoderma are very susceptible to this treatment ; and it has been proved that, under the conditions prevailing in the pasteurisation of beer, a tem- perature of 60° to 65° 0. is fatal to six different species, including Mycoderma rubriwi and M. Immuli ; as also to Sacch. Past. III. and several species of beer yeast (Lasche (V.)). In the case of several species of mucinous yeasts examined by Richard Meissner (II.), the limit was found to range between 51° and 61° 0. according to the species. Since it is easier to render the yeasts innocuous than the bacteria, a slighter warming will suffice when there is reason to believe that the latter are entirely absent from, or harmless in, the wine under treatment, and when, consequently, the task is confined to eliminating the yeasts alone, in order to prevent them afterwards setting up secondary fermentation and turbidity. Carl Schulze (I.) has shown that, under these circumstances and in presence of about 10 per cent, of alcohol, the yeasts are killed by exposing the bottled wine to a temperature of 45° C. for two hours. The samples tested in this manner did not exhibit any alteration of flavour, though a precipitation of coagulum occurred. The preparation of so-called unfermented and non-alcoholic grape and fruit wines, which in reality are nothing more than stabilised grape- or fruit-must — and which, thanks to the temperance movement, are more and more coming into favour — has also benefited by the observations just recorded. H. Mueller-Thurgau (XII.) in particular has occupied himself with this question, and has drawn up practical instructions for pasteurising these beverages, by exposure to a temperature of 60° 0, for half-an-hour to an hour. Here also it is necessary to filter off the resulting deposit, and pasteurise again. Fruit juices, e.g. the cherry juice so highly appreciated in North America, can also be rendered stable by similar treatment. The reader will now be in a position to understand and thoroughly appreciate the operation of warming the curd in cheese-making (as mentioned in § 182), and the influence of the temperature then maintained on the subsequent progress of ripening. i LllAlTER XLVll. ANATOMY OF THE YEAST CELL. ii 249.— The Cell Membrane. In tlio fiisf of younger cells, actively enjiageil in metiibolisnt, the cell membrane is very thin (only a few tenths of a ft) and has nearly the same refractive power as the enclosed plasma. It may be rendered deiirly visible and distinguishable by allow- ing some strongly plasnjolytic reagent, e (j. dilute acids, to act on the cell, whereby the contents are cau.sed to shrink. The meud)rane can l)e exposed in a simpler manner by merely pressing gently on the cover glass of the microscopical pre- panition, so that the cells burst and the empty membmnes become visible as jtale skin. The membrane of the jiernianent cells described in § 245 and § 246 is, however, di.scerniV>le with- out this preliminary treatment. According to the re.'-rearches of II. Will (VIII.). the membrane in these cells attains a thickness of 0.7 to o 8 /^, t.)r oven i /i occasionally. This thicken- ing of the cell membnme is a precautionary niea.sure against rtdveise influences, and occurs more paiticularly when the yeast cells are obliged to develop in ov u[M)n a concentrate/. .strong ale ("liockbier") wort or wort -gelatin. C. Heckek (I.) determined the thickness of the cell membrane of st(K'k yeast for tirdinary Munich l)eer, as 0.5 n, wiiereits that taken from a vat of strong ale measured up to o 7 /x, ami that from the still stronger Salvator beer 0.9 /i. The in- crease in thickne.ss is accompanied by diminished penetnibility, and consequently deciea.sed fermentative power. For this leason the sedimental yea.st from strong ales is nirely usetl again, or only for pitching worts of lower gravity (lager-beer). The influence of dilute acids (»•.(/. a i per cent, .solution of osmic acid) or alkalis, on yeast cells, especially the per- nument cells, is evidenced by stratification of the membrane, which was first observed l)y II. Will (Vlll.) and then confirmey Mii.kku (IV.), and afterwards by C, VOK WlKSELiN'tiU (1.) Tliu aljsence of any aiJitieciiihle action of ammoniac-al copjiei- oxido on tlie yejist cell had ahoady been obseivfd )»y LieMj;. According' to tlit' cont-ordant results fur- nished \>\ the researches undertaken by C. vox WissELlxuil and by Tamiet (111.), chitin, the second material taking part in the structure of tiie cell nienilirane uf the Kuiiii/rrftjs in genenil, also .seems to be lacking in the ca.•^e of yeastK The alleged observation, by Clutis(I.), c»f a violet colouration |»r(jut in an overlying uncousumey iodine and potassium iodide to the spore memVirane, and often to that of the vegeL;itive cells as well. The .s;ime behaviour was observed by ,). Ch. Holm in another species allied to the one just mentioned. Along with the.se exceptions must also 1)6 cla.s.sed the blue colounition ob.served by Likhei{M.\xx and BiTTo (I.) oji the addition of zinc iodochloride U) a macroscopical pre- paration of .so-called yeast cellulose (;< 225). In connection with this behaviour of the yeiv.st cell menibmne towanls colour- ing matters, (_'a.s;igrandi remarks tlmt Congo Red is not ab.sorl)ed, whereas, on the other hand, very good staining can be elTecttnl with Ehrlich's Methylene Blue or ll.instein's Aniline Violet. C Beckeu (I.), however, found only the latter of these two etTective. Solution of the mi'inbrane (M-curs on the cells being immer.sed in concentrated chromic or sulphuric acid, whilst other acids are inetfei'tual. The s;ime applies to the mixture of nitric acid and potissium chlorate, known as iSchulzes macer.itiou liipiid. through its mode of action having fir.st been ob.served and utilised bv Hrunnengi;eber in the l.iboi-.itorv of Profe.s,sor F. F. Schulze at Ho.stock. This is related l)y A. T.siiniKii (IV.) in an admirable historical review of microchemical methods. The foregoing, together with a series of other microchemical reactions, led Ca.sjigrandi to the belief that the yej»-'>t cell mem- brane contains a substnnce chemically .-illied to the pi*ctin recognised by Mangin as a constituent of other vegetable mem- branes. It is, however, dillicult to a.scertain whether and how 148 ANATOMY OF THE YEAST CELL. fai' this view is correct, owing to the absence of means for detecting this carbohydrate. Still more undecided is the ques- tion as to the divex'gent chemical composition of the various strata of the membi-ane. That some difference does exist may be deduced from the influence exerted by chromic acid (dilvited with its own volume of water) on the cell membrane, whereby the inner layer is more readily dissolved than the outer, especially in the case of permanent cells. This observation was first made by H. Will, and was afterwards confirmed by Casa- grandi and supplemented by different staining tests. It was probably owing to a similar observation that Nfegeli was led to surmise that the cell membrane in the films of jNIycoderma is cuticularised. The hypothesis founded ox\ the microchemical examination of the yeast cell membrane is undeniably supported by the macro- chemical researches of Salkowski (VI.) on the same point. He found that the residue obtained by extracting pressed yeast for half-an-hour (§ 2 5 4) with boiling 3 per cent, caustic potash solution, and amounting to about 3.1 per cent, by weight of the substance taken, was separable into two components — one soluble, the other insoluble — by prolonged boiling in a large volume of water, under a reflux condenser, or in the autoclave at a pressure of 2 to 2 1^ atmospheres. On separating and concentrating the solution, the soluble constituent was precipitated by absolute alcohol, as a white powder exhibiting the rotatory power a^ 173.3° ^^ i74-i°- The results of the ultimate analysis indicate the formula C^jH^yO^. On hydrolysis with dilute acids, practi- cally the whole is converted into ri'-glucose. Iodine in potassium iodide gives a powerful brown-red stain, on which account the substance has received the name erythrocellulose. It differs from the somewhat analogous glycogens in its lower rotatory power. The other constituent of the original residue remains in the form of a colourless jelly at the bottom of the liquid, and, as it is not altered by iodine solution, is called achroocellu- lose. On hydrolysis it furnishes a mixtuie, chiefly of glucose with compai-atively little mannose, and on this account is pro- bably not a uniform substance. From the above reports one thing seems clear : that the membrane of the yeast cell is a complex structure, both fi'om the anatomical and the chemical point of view. This fact will be^ regarded as so much the more certain when it is stated that no one has yet succeeded in obtaining the so-called yeast cellulose free from ash constituents and nitrogen. Schlossberger found i.o per cent, of ash, and the preparations made by Salkowski contained between 1.7 and 2.6 per cent., whilst Liebermann and BiTTO (I.) found 1.8 per cent, in theirs. It does not do, however, to launch out into theories respecting the significance of these inoi'ganic constituents, the more so because the com- THE CKLL MKMJJUANK. 149 j)u.sitii)u of this iisli is still unknown. Moreover, we have ulwi to reckon with tlie still uninvestipite«l puKsihility that the ash itself is introilueed into the meml>i-ane cess, and it is therefore not surprising that other experimenters of both sexes, »•.«/. Kit. Kit.xssxEit (I.) in 1S85, and Siuo.via KiSKXScniTZ ( 1.) ten ye.irs lati'r. tried it without success. No more fortunate in this respect was .Ion. K.\r.\i (11.) who. in 1S90, on the Ivisis of his staining te.sts (mentione'. 'futfii/iifuj<, luit are never found in Siin-h. Lwlici'jii and Si-hizugiu-charoinijrtfs Ol'tOKjHJI'lHt. A few remarks may here be made as to the division of the nucleus. The reports hitherto maroduction) of the hitherto cjuie.scent nucleus also bej^ins. This operation, which has l)een alreadv mentioned in |$ 46 and !5 219, may proceed in two ways: either the comparatively simple process of direct subdivision, known as fragmentation i(KLLKH (11.) in 1892, and this opinion was shared by 1)axge.\kd (II.) a year later, BrscALioxi (I.) taking' the s;ime view in i8()6, and H. Waijeu (I.) in 1S98. On the other hand, Ja.vssexs (I ) in 181,3 opined that the nucleus in the building yea,^t cell repro«luces Ity karyokinesi.s. Eipially conti-adictorv are the rejKjrts on nuclftir sulnlivision duriuir the formation of a.«iiin/r/.i Lwhriijii and Srhizn- mi'i'haronnji'fii orfosfmrits, are usually repitKluced bv karvo- kinesis, but that this prtK-ess may under<;o a more or less extensive sinipliiication and approximation to direct ilivision, more paiticularly when the cells are kept untler comiKuatively less favouiable conditions of exi.stence. Similar intermef observations on the protrie.ss t>f nuclear .miI>- division. The tirst thinir noticeable in a cell that is about to bud is the dis;ippeanince of the nuclear membrane, and the 152 ANATOMY OF THE YEAST CELL elongation and separation of the nuclear body into two parts. The two nucleoli, which are still connected by a thin thread, then move tow^ards that part of the cell at which the bud commences to protrude. One of them passes into the embryo daughter-cell, and develops into the nucleus thereof, enveloping itself with a membrane — as does also the other which has remained in the parent-cell. A view of the different stages of this subdivision is given in Fig. 150. During sporulation a similar subdivision occurs. Two daughter - nuclei are first formed, which, of course, remain in one and the same cell, and thereby give rise to the exceptional case, referred to above, of more than one nucleus being present in a yeast cell. Both these nucleoli eventually undergo a second svibdivision , the resulting four nuclei then consuming the existing cytoplasm for the purpose of developing into a corresponding number of Fig. 150.— Saccliaromyces Ludwigii Hansen. Cells at various slas'es of budding. Stained. a, Subdivision of nucleus accomplished ; the daughter-nucleus entered into the daughter cell still remains attached to the connecting thread ; b, the latter becomes detached ; c, the septum between mother and daughter-cells commences to form ; rf, the separation is complete. Magn. 1200. (After Janxsemi and Lchlane.) ascospores, each enveloped in a new membrane. In the event of eight spores being formed, the process of subdivision must first be repeated. In cells containing a different number of asco.spores {e.g. six or nine), it naturally follows that the process of subdivision has not been repeated the same number of times in all the nuclei, but has ceased in some eai'lier than in others. Finally it may be mentioned that the processes just referred to cannot be distinguished in the living cell without preparation — as-KiiASSER (II.) unsuccessfully endeavoured to accomplish — but that recoiu'se must be had to a suitable method of staining in order to render them visible. Since, as already stated, the nucleus in an unstained yeast preparation may readily be mistaken for a vacuole, and vice versa, by an unskilled eye, it is necessary to make a few remarks with i-egard to these vacuoles. As a rule — to which an exception is afforded by e.r/. Sarcharomijces a]>ictdahis — the appearance of one or more vacuoles in the yeast cell is coincident with ex- haustion and with a lack of nourishment. Up to the present no \ THK CKLL NUCLEUS. »53 ri'liiiMo iiifoi-iiiatioii is iivuiliible us t<> the luiture of tlie Iii|uid couipo.siu only in very youn>r '"H-^ that thov are not to be tound ; but they 154 ANATOMY OF THE YEAST CELL. appear in large numbers at the close of primary fermentation, and are usually very abundant in permanent cells (§ 245). Naturally, the method of nutrition has also some influence on their occvirrence. Thus, G. Hieronymus (I.) found only very few granules in the cells of pressed yeast that had been grown in a nutrient- salt solution containing grape sugar, whereas they weie abundant in such cells as had been kept in milk or a solution of beet sugar. According to a report by Joh. Raum (II.), no granules are formed when sugar is absent from the nutrient substratum. It is to the last-named worker that we are indebted for the first accurate investigations in connection with these inclusions. These researches are the more valuable in that they were ex- clusively performed on pure cultures, namely, of ten species of budding fungi, comprising six true ISacrharomycetes (8. cerevisice Fig. 152. — Permanent Cells of bottom-fermentation beer yeast, either partially or entirely filled with granules, wall considerably thiclcened. Magn. 2000. (Ajter M'iU.) Cell /. Hansen, S. ellipsoideus I. and 11. H., S. Pastorianus 1. H . pressed yeast from Warsaw, and a yeast from the air), a red budding fungus termed S. ghitimis, a so-called black yeast, and a budding fungus from Kephir granules. The occurrence of the inclusions in cjuestion was demonstrated in all these species. The number fluctuates considerably, and very often far exceeds a dozen. Their dimensions were also found to vary considerably. When a large number nre present, they are com- paratively minute (Fig. 152), generally only a few tenths of a micromillimetre in diameter ; but when the number is smaller (and it may sink to unity), the size attains considerable pro- portions. In the latter event they may readily be mistaken, by the inexperienced eye, for ascospores, owing to the high refrac- tive power they have in common with the latter. In doubtful cases the uncertainty may be removed by the aid of some of the microchemical reactions given below. Moreover, these granules are not always of the same shape ; for the most part they are globular, though specimens with an angular contour are by no means infrequent. In throwing doubt on the existence of this latter modification, and opining that it only appears on staiiiing, THE GK A.N LUX »55 Kill, isv — P«?riiittlii-lit Cell (if bottom • feriiK-iitation biHT \i'akt. ciiiiliiiiiiiiic nil iimiKUiilly liirKf ^rnmili*. Afler trt'iitiiieiit with Jaxssens !iuv llitMcjiiyimis ill the ^niiiuh-s of iiiistaiiu-i|, liviii'.' yi'ast t-i-lls 11. W'li.i. (\'lll.) was the lirst to imhlish fh-tjiils eoneernin^ the aiiatuiiiiral struetme of these fjniiiules. Whereas J. Kaiini had iiii'vioii.slv failed t^i ih-tfct aiiv stiuftui e. aiie of alliuiuinous substances and a fat like inteiior. which latt4.'r justities the name "oil drojis " liestowcd liy Will. The only point on which doubt exi.sts is with lejranl to the [ire.sence of filaments (Fii,'. 15^) which, accord- ing to the ob.servations of this worker, extend from the outer case to the interior of the ^'raiiule and there form a net- work enclosing' the fatty contents of the ijriiiude. O. Casackandi (I.) re','arils this network as lieinj;, not a jiecul- iarity of the unaltered ^Manulum, liut as a consequence of the iidluence of the rea^xents ('.'/. alcohol) emploved for dis- solvin;' out the fattv iioitions and reveal- alciliul. M gn. j,.ko. (A/irr ' • Will ) injj the alleged reticuhitions. For the rest, and in its chief jioints. Will's ob.servation was continue*! by C'a.sii^jnindi. The j)eculiar behaviour e.xhibited by the iii;/ri'.< ajiiru/atujt — are entirely agreed that the gi-anules are turned brown by the action of a 1 per cent, .solution of perosmic acid, which is the chief reagent for fat.s. The fatty content.s h.iving been extracted by a suitable solvent, the residual outer ca.se gives all the chanxcteri-stic reactions for albumin. When yemst cells are treated with concentnited sulplunic .acid the membnme swells up; the acid then gains .^ccess tc) the granules .and ile.-itroys their integument, whereupon the fatty contents of the individual granules coale.sce to form larger drops. These turn first green and then bluish-green to blue black, and. on the adtlition of a little to per cent, caustic potash, are partly or entirely dis- solved, /.*'. vani.sh fiom sight. According to Ca.sjigmmli's con- firmation of the results obt.:iin«>d by Kaum, a similar effect is obtained by treating yeast cells with artificial gastric juice: the outer case of the individual granules is digested, whereu|H>n the exposed contents mute to drops, which can then be removed bv t>ther-alcohol. 156 ANATOMY OF THE YEAST CELL. The action of fat-dissolving reagents on the contents of the still unaltered granules is, as may well be imagined, consider- ably impeded by the albuminous integument, and requires some time (often far more than twenty-four hours) to attain com- pletion. Lacking the necessary patience for this, one is easily led to the conclusion that the solvent has no influence ; as was the case, for instance, with Raum and the ether-alcohol mixture, with Hieronymus and concentrated caustic potash, and with Curtis (I.) and benzene, until Casagrandi, with a greater exercise of patience, showed the contrary. Moreover, it is not surprising that the requisite duration of exposure to these reagents should have been found to vary, not only with the granules of different cells, but also in the case of the different granules in one and the same cell. It is, how- ever, carrying matters to extremes, to do as was proposed by Sidoxia EiSENSCHiTZ (I.), for ex- ' ample, and attempt to establish a classification of the granules into dif- ferent groups on the basis of this divergent behavioui'. From the above- mentioned solubility of the outei' cases of the granules in artificial gastric juice, it may be concluded that they are con- structed of digestible albuminoids, and consequently that no nuclein is present in the granules. Nevertheless, according to the reports of Fr. Krasser (II.), this rule, which was established by E. Zacharias (I.), has certain exceptions. With regard to the composition of the fat-like contents of the granules, nothing precise can be said at present. Attention should, however, be paid to the reports recorded in the third paragraph from the end of ^ 253. The observations (especially those of H. Will) made on the consistence of the granules, show that the fatty contents of these inclusions are not perfectly liquid, but rather semi-fluid. The application of pressure to the cover glass, under which a yeast cell has been placed, causes the granule to burst, as is shown in two examples in Fig. 154. With regard to the distribution of the granules within the yeast cell, it was found by J. Raum that a certain order prevails, inasmuch as these structures are arranged in series which represent arcs or portions of arcs. Closer investigations, con- ducted by Hieronymus, supplemented this discovery by showing that the granules are arranged in spirally wound chains, embedded Fig. 154. — Dead Permanent Cells from bottom-fermentation beer yeast ; each con- taining a larfie granule which has burst in con- sequence of the application of pressure to the cover glass. Magn. 2000. {After iVill.) THE OHANT'LHS. «57 o*»\ / ,^^v^o^rr^ f i«j. ^6„ in ;i loutinuuu.s [Jiotophismic liluuifiit, as ilepict4*sivo sub- limate, tlu' fatty contents of the granules are then exti-acte( tlu- jnTiiiule» are deciileilly inultiiiUKolHr. l»o <eer wort ; oiiscrvi-J in the live state. Kxhlhit* the |ila$nial framework, with ap- purtenant kii'ts. nferreil t<.> In the •• " 1 >'■ ■ ■ '1 '. 1. li-us is not (Ajtf, 158 ANATOMY OF THE YEAST CELL. fuchsine, decolorised, and finally stained by immersion in a 30 per cent, aqueous solution of picric acid. This colours the integu- ment red and causes the granules to stand out clearly fi'om the surrounding plasma, which is stained yellow. Even during the life of the cell, the granules will absorb certain dyestuffs, for instance when, as recommended by Eisenschitz, cells of yeast are cultivated in beer wort containing an addition of a i per cent, solution of Benzopurpurin. Some of the granules will stain red by this method in one to two days. In addition to these granules, which can be detected at once in the unchanged living cell, there are, according to Hieronymus, other similar inclusions, which only become visible after the cells have been fixed with, say, chromic acid or hydrochloric acid. When this treatment has been followed by staining, the " central filament" is found to be surrounded by an abundantly looped chain of granules embedded in the portions of the plasma adjoining the wall. Nearly every one of the workers hitherto named has re- corded a certain power of locomotion on the part of the struc- tui'es in question, though the reports on the extent of this power differ. It was remarked by Raum and then confirmed by Hieronymus, that in many instances (though not invariably) a gradual migration of granviles from the parent-cell into the young bud goes on during the formation of a daughter-cell. This is represented in Fig. 155. The movement of the granules, however, proceeds very slowly, and bears no comparison with the brisk saltatory motion (Brownian movement?) noticeable in the bodies often observed in the vacuoles of many yeasts, and which are indeed so plentiful in the cells of Mycoderma as to constitute a characteristic of this mould fundus. Divergent opinions prevail on the nature of these vacuolous inclusions (so- called saltatory bodies), some regarding them as being invading bacteria, others as endogenous cells, or as dead plasmal excre- tions. J. Raum was the first to observe the entry of these bodies from the plasma into the vacuole; and it was then shown by E. KuESTER (I.) that this migration could be induced by allowing the yeast cells to dry on some substratum. According to the reports of Eisenschitz, a reversal of this migi-ation — namely, from the vacuole back to the suiTOunding cell plasma, may occasionally take place. The chemical construction of these saltatory bodies can no longer be regarded as identical with that of the plasma granules, since E. Kuester has shown that the two differ in certain I'espects as regards their behaviovir towards dyestuffs. This notwithstanding, neither he nor Symmers (IL) later on, succeeded in arriving at a definite solution of the problem. In many instances it is observed that the granules offer a greater resistance to decomposition than any other constituents I THE CllANri.KS. 159 of the yeiust t-ell, >o tlmt, even when the cell uiembmne Im8 become wm-pfd hy moisture aut investigations concerning the percentage projxjrtion of nuclein to the tobil nitrogenous constituents of ye^ist were pi-rformed by A. Sii rzKit (1.). According to this worker, the dried resiilue obtained from beer yeast bv several days' cold exti'action with 95 per cent, alcohol followed by drying over sulphuric acid, contained 8.65 per cent, of totjil nitrogen, of which 2.26 per cent. ((»r more than a (juarter) was in the form of nuclein. Still richer in this respect was the thallus of a mould fungus of undetermined species, which settled fron» the air into a nutrient solution containing t;irtaric acid ; since, out of 3.7S per cent, of total nitrogen in the dry residue, 1.54 per cent, (or nearly 41 per cent, of the whole) was present as nuclein. It is to the labours of A. Kossel and his pupils that we are mainly indel>te«l for information on the chemical constitution of nuclein in general, and of yeast nuclein in particular. Their discoveries furnished the basis for Kossel's (II.) clas.sification of the nucleins into two groups. The fiist includes substances, which, so far as is known, do not occur in cell luuhi. a!id which were named paranucleins by Kossel, or pseudonucleins by Ham- MAUSTE.v (ill.). When decomposed by dilute acids they > ield only phosphoric acid and all)umin. On the othei- hand, under the .same treatment, the true nucleins, which alone form the subject of the following lines, furnish, in addition, basic sub- .stances to which the name nuclein bases has been given. An import4int elucidation of the constitution of nuclein wa.s first presented by li. Ai.i.Man.n (I.) by the discovery that the.se proteids are decomposed by dilute alkalis intt^ albumin anise. xanthin (C^II^X^O.,), ty K0.SSEL and Xei.mann (U.) that thymin is also i)re- sent in the molecular complex of the nucleic acid of yesist, has not been confirmed, though, according to A. Ascoli (III.), the parent sub.stjince of thymin, namely uracyl (C^H^N.O.,) is found therein. It may also be remaiked bv the way that the fourth of the new nuclein bases, cytosin, has so far only been found as a constituent of nucleic acitls of animal •origin. In the crvstiilline stiite this substance has the formula C.,,H.,oN,,0,. 5H.,6. Apart from the la.st-named, which has not yet been suffi- ciently investigated, the nuclein bases mentioned may be dassifieil into two groups in accordance with their constitu- tion Adenin, .xanthin, hypoxanthin, and guanin are deriveil from the atomic complex to which Emil Fiscuer (I.) gave the name Piirin : — 1 64 CHEMISTRY OF THE YEAST CELL. N = C . H I I H. C C — N.H il II \c H Purin. H. N— C : 0 H.N— C : 0 II' 11 C:C C-N.H H.C C-N.H I It \p TT II II ^C H Xanthin. Hypoxanthin. C5H4N4O0. CgH4N40. 2-6-dioxy-purin. 6-monoxT-purin. H. N-C : O N = C . NHo II 11 NHo.C C— N.H H.C C— NH 11 II \p H II il \o H Guanin. C5H5N3O. Adenin. CsHgNs. 2-amino-6-oxy- 6-amino-purin. puiiii. They accordingly belong to the large group of purin bases. On the other hand, Thymin and Ascoli's yeast nuclein base are of more simple construction : — H.N — C:0 H.N— C:0 II II : C C . H 0 : C C . CH3 I II I II H.N— C.H H.N-C.H Ascoli's base. C4H4N.2O2. Thymin. CsHgNsOo. Uracyl. 5-methyl-uracyl. Of these, thymin seems the most widely diffused, since only three nucleins not containing this base are known, one of them being yeast nucleic acid. The second is the above-mentioned guanylic acid, which, according to I. Bang (L), furnishes guanin alone (and of this about 35 per cent, by weight). The third is inosinic acid, which was discovered by Liebig in meat juice, and which, according to the experiments made by F. Hasier in 1895, probably contains hypoxanthin solely. The percentage content of xanthin, guanin, hypoxanthin, and adenin (of course in the combined condition specified) in yeast, was first asceitained by S. Schindleh (I.) by the aid of his quantitative method of separation, and was found to be 0.024, 0.029, °-093) ^^^^ 0.043 grii™ respectively per 100 grams of pressed yeast, the character of which was not more accurately defined. These values should not be considered as more than merely approximate, the reliability of analytical methods having been proved doubtful by 0. Wulf (I.). CHK.MISTKV or THE YEAST CELL NUCLEUS. 165 To the reossible to arrive at more than a hypothesis jKJs.se.ssinjj a certjiin proliahility, it Wing found that cells or aj^filomej-ations of cells, cont^iining either a large number of nuclei or nuclei of con.sideiiiV»le size, yield a higher j»roiH)rtion of nuclein than such as conUiin only a few nuclei or tho.se of small dimensions This was the conclusion formed l>y Mieschei-, who succeeded in separating the cell nuclei from the pus cells on which liis exhaustive experiments were performed. liy similar companitive methods, A. Kossel (L) came to the conclusit)n that nuclein, instead of being a resei-ve material, plays an active part wherever new cells are in course of formation and nuclear reprmluction is consequently in pi*o- gress, i.e. in all phenomena of germination. Thus the formation of nuclei and nuclein proceeds simultmeou.sly. This opinion was also shared by H. A. Laxdweuk (I.). The sole method of obtiiining reliable information with regard to the .situation of the nuclein within the cell is by microchemieal examination. With this ol>ject use can be made of the high re.sistiince olfered by the nucleins towards pep.sin. For example, E. Zachakias (L) detected the presence of nuclein in the nuclei of pressed yeast cells by the aid of .so-called artificLil ga.stric juice (a .solution of pepsin in 0.2 per cent, hydrochloric acid). The stiiining methotls, howevei*, are caj)able of more extensive ajipliwition. The various indiviilual chemical constituents nf the cell ex- hibit divergent absorptive allinities for dilTerent dye.stulTs. It is known that fiom a mixture of red, blue, or green dyestuffs one cell constituent will ab.sorb chiefly or entirely the first-named, ij\ erythrophil, whereas ant)tlier cun.stituent will prefer the Idue (or green), and is therefore termed cyanophil. This preference, however, in each case is determineil, not l)y the .shade of colour, but by the chemical constitution (or reaction) of the dyestuff. on the one hand, and by that of the cell cc»nstituent,s with which it is brought into contact, on the other. A cell con.stituent is therefore .sjiid to be acidophil when, from a mixture of acid and ba.sic dye.stutTs, it absorbs the former and becomes stjiined there- with ; in the conveise case it is termed basophil. Chemical atlinity is almost the sole factor influencing the occurrence or non- occurrence of .st:iining, so that, in presence of a mixture of an acid red dyestulT and a Imsic blue dyestulf, a given ba.sophil cell consti- tuent will behave as a cyanophil, but, in presence of a mixture of a basic red and an aciil blue dyestulT, as an erythrophil. An instance of this kind is alTorded by the nucleic .-kmiI of yea.st, which is strongly basopliil, and. according to ob.servatitms made by H. Malkaiti (L), E. Zachakias (II.), and L. Liliexkeud (I.), is stained (blue or green) by a mixture of Acid Fuchsine 1 66 CHEMISTEY OF THE YEAST CELL. and Methyl Blue (or Methyl Green), but red by a mixture of Safranine and Pale Green. Such behaviour having been macrochemically observed with the known dyestuffs, the same can also be employed microchemically for examining the cell for the presence of one or another chemical substance occurring in local accumulations therein. In any event, care is necessary in the preliminary treatment of the preparation, since in many cases this has a decisive influence on the results. For more detailed information on this point the reader is referred to the monograph by A. Zimmermann (II.). In so far as these micro- chemical reactions enable a reliable decision to be formed, it has been shown in this manner that the nucleus of the yeast cell is rich in nuclein, and that the latter is probably not present in the cytoplasm. Now, as already shown in §250, the nucleus consists of the reticulated framework, the sap (or juice), the membrane, and the nucleoli. The first-named, in tiu'n, is com- posed of the reticulated matter known as linin, which is difficult to stain, and of the globules situated at the intersections of the network, these globules consisting of a substance which is readily penetrated by dyestuffs, and which has therefore received the name chromatin (§ 35). The nuclein is located in this chromatin, whereas the filaments of linin and the nucleoli con- tain, in addition to albumin, a substance termed plastin, which differs appreciably from, although closely allied to, the nucleins. The question as to the amount and nature of the other nitrogenous substances associated with the nucleoproteids in the nucleus of the yeast cell, cannot at present be satisfactorily decided, the reports on this matter being few in number and not reliable. The same applies to the nitrogenous constituents of the cytoplasm (§ 2 1 9), the scanty communications referring to which will be dealt with in § 255. The composition of the yeast water prepared in the manner described in § 82 will be briefly considered here from the standpoint of the reports mentioned in the foregoing para- graphs. Even mere boiling in water is sufficient to break up the yeast nuclein, with formation of nuclein bases and free phosphoric acid, which therefore occur as constituents of yeast water. The appearance of phosphoric acid in decoctions of yeast had already been observed by A. Biechamp (VII.), who found, in 1865, that 100 grams of dry yeast lost, on extraction, 2.8 to 3 grams of phosphoric acid, part of which was in the free state. This was confirmed by A. Kossel (III.). He also found that this liberation proceeds rapidly at first, but later on very slowly. With regard to the extraction of nitrogenous sub- stances (xanthin, guanin, sarcin, carnin, levicin, and tyrosin) from yeast cells by the water in which they have been boiled, certain experiments were made in 1874 by P. Schuetzenberger (I.), who, by repeatedly extracting yeast with boiling water (•iii:mi.sti;\ (»k thi; vkast ckll nuclkin 167 iiiilil t'xIiiiustiMl, toiuid thiit, of the 2.78 jji-ains of nitrofjeii piL'soiit in 100 ^niiii.s of fifsh yen-st (coiitiiinin^ 30 per cvut. of dry matter), 075 {;niiii couM lie extnu-teil in tlii.s uiauiier. In distilliiiir fermented distillei y mash, a larj^er or smaUer amount of the nuch'in of the c(»ntainet is similarly decomjMjseil, so that the still residue eontains relatively little nuclein, liUt a hirge amount of nuelein bases and free phosjthorie acid. This fact must he kept in mind, not : the purity of a given meat extract and detectinj^ the presence of a sul'Stitute preparetl from yeast. The (juantity of beer yeast annually produced in the great brewing countiies has been approximately estinuiteil by Feron at about 305,000 tons. Of this amount about 132,000 tons have l.itlu'rto been utili.setl, partly for pitch- ing in breweries and molasses distilleries, and partly us an adidterant of pressed yeast. This leaves .still nn less than 173,000 teen made to convert this surplus beer yeast, which is rich in proteids and phosphoric acid, into preparations either capable of utilisation as a yeast fix)d in the distillery (especially in mola.sses dis- tilleries) instead of the usual addition of grain, or el.se for huuum consinnption as a sul)stitutt« ft)r meat extnu-ts, or finally as a concentnited cattle food. With this t)bject the yea.^t is either converted into extnut, or is worked uji in its entirety alone or with certain adjuncts. A number t)f proces.ses liave been devi.st'd for working up yeast into nutritive preparations; antl of these, mention may be made of the following foi- making yeast extracts, viz. those of E. Baieii (II.), Gilliiaisex. K. Waul and M. IIeniis (1.). K. Kkessel (I.), Pecteus (1.). .1. Cood- FELLOW (1.). T. HlM.-.JoNES and K. KuESSEI, (1.). K. .loHXSOX (I.), C. OSii.MVAN (i.), I). Watson (I.). A. Penavkk (1.). and Lebiun (11). In the methotl of (!. Ek iiei.hai m (I.), yeast is subjected to the action of species of Asft^'ri/illtt." before lixivia- tion ; and according to that of O. (J. Oveuheck (1.) to the inlluence of peptase from malt cuhus. The folh)wing methotls deal with the ye;ist as a whole, without extniction : that of Wegener, which is conceriunl with the jirtxhiction of a cofTee 1 68 CHEMISTRY OF THE YEAST CELL. substitute from yeast ; that of K. Kleinschmidt (I.), and that of Siebel. According to the hxtter, a pulp which he calls yeast sugar and which has the appearance and consistence of con- densed milk, is prepared by grinding pressed and de-bittered beer yeast with sugar and starch. The preparation of concen- trated fodder is the object of the methods of J. Steickel (I.) and C. Brucker, as well as of the English Patent No. 20,060 of 1893. The purpose of several of the methods first-named above is the recovery of a yeast extract to serve as a substitute for meat extract ; and as a matter of fact some of the prepara- tions of this kind already sold are undistinguishable by the laity from real meat extract, so far as the smell, flavour, and external appearance are concerned. Such are, e.(j. Bios, Eurostose and Carnos, according to A. Eichexgruen (I.); and, according to Lebbin (II.), Ovos as well. An intelligent manufacturer should obviate all chance of such confusion by giving his yeast pre- paration a special and indubitable title, e.g. Vegetable Peptone. This, however, is not always the case, and it would appear that auv(IV.) in 1S6}, enl:ir;(ed our insi;;ht into the hot^inical siile of the question by the iinportjint dis- covery that the suljstjince referred tuted throu<;hout the plasma, hut is contini'd to one portion thereof, which he proposed to call the epiplasma. At the same time he expressed the opinion that the suhstiince in question was a c.irl)()hydrate. Five years later W. KfEUXE (la.) reported the occurrence of the '' matirre glycoijcne,"' or j,'lyco;rcii, in At'tlt(tlinin sfjifimni. This discovery, however, did not atfoid any certain proof of the appearance of fjlycogen in fun«;i, the last- named organism beloni,'in^' to the Miji-fficnn ("^ 23), and there- foie merely a connecting' link between the fun^i and the lower animals. Up to that time no deiinite proof had been adduced of the e.xi.stence of <,dycoj;en in vt-^'etible cells (those of funjji in jtarticular), and therefore the term "animal starch, " applied to this substance, was still in general use. It was not until 1882 that L. Eureka (11.), working in A. de IJary's laboratory, showed the principal constituent of fungoid epiplasm to be a carbohydrate, agreeing with animal glycogen in all the properties examined by microchemicjil means. He also demon.strated its wide distribution, not only in the (first examined) da.ss of As<-i)iiiiireli\<, but also in the JifL^iiliomi/- fffes anil the Mui-oi-(t<-<(r {\l\. and IV.). His di.scoveries were confirmed by .severjil other workers, r.i/ \>\ Kkaekukk (I.) and afterwards l»y E. Lai'KEXT (VI.). who found this carbohydrate also in Oidinm /aai-:ition be carefully wjirmed to 60' to 70° C after the addition of the iodine, the \M\\e yellow due to albumin lyo CHEMISTRY OF THE YEAST CELL. will remaiu, whilst the red-brown will vanish — to reappear in its original intensity on cooling. If the cells of the treated preparation be i-uptured by applying pressure to the cover glass, a rapid examination under the microscope will show that the browned constituents quickly dissolve in the surrounding fluid on escaping from the cells, the residual portion exhibiting the customary yellow coloration furnished by albuminoids on treatment with iodine. If present in any considerable amount, the glycogen becomes visible to the microscopist by virtue of its optical properties, even in unstained preparations. In such event it is seen in the cells as a semi-fluid, whitish opalescent mass of strong refractive power. Sometimes it takes up a position in such portions of the cell contents as are nearest the walls; but in others, as recoi-ded by Errera (V.), it collects in one place and assumes the shape of a semilunar aggregation. The separation and recovery of fungoid glycogen by macro- chemical means was first successfidly accomplished by Errera (II.), though the quantities obtained were insuflicient for the lequirements of an accuiute macrochemical examination. This difficulty was first overcome by M. Cremer (III.) in 1894, who was then able to show that the glycogen of yeast is identical with that in the liver of animals, and that too, not merely in respect of the propeities aforesaid, but also with reference to its I'esistance to the action of Fehling's solution and its behaviour on hydrolysis by dilute hydrochloric acid (as also by diastase, saliva, and the panci'eatic enzyme), dextrose being produced. E. Salkowski's report (lY.) that yeast glycogen and liver glycogen differ, inasmuch as the former is partly conveited into cellulose on being heated to i 7.0" C. alonsr with a little water, was not confirmed l)y Cre.meii (III). At all events, the optical rotatory power of yeast glycogen was found to be lower («,,= + 198.9°) than that of animal elvcogen, for which the values in the literature differ and are in pai't considerably higher (up to 235"). For comprehensive researches into the chemistry of this carbohydi-ate we are indebted to a pupil of Errera's, namely G. Clautriau (I.), to whose work the interested reader is referred, especially as regaids the choice of the best method of isolating glycogen from the different species of fungi. Glycogen is amorphous, and therefore cannot be prepared in the pure state by the crystallisation process, but cont;iins an admixture — larger or .smaller in accordance with the oriijin and care bestowed on the manufacture — of other organic and inorganic cell con- stituents. This })ossibility should be kept in mind when mention is made of small discrepancies, similar to those found in animal glycogen by earlier workers, and also by Clautriau in comparing yeast glycogen with that obtained from other fungi. Thus the angle of rotation found by this worker, in the case of yeast glycogen, was 184.5°, whereas in that from Amanita muscaria tiLYCOCKN AND FAT. 171 it wii.N 196.2 . The sjniie uj»i>Ht*s tt) the telMpemtuie at which the iixliiie stailiilijr tli-S!ijtpeius ; in the mse «if ;rl_vt*<>^eli fmijj yeast u teiiijieiatme nf 72 1** 73 C is ie !ihv:i\s the ptx>v>iliility of having; to reekiai witli the exi>tein.e of >evei-:il isoiiieiic glycogeus, ill re^rd to which .*>ee an observation by K. Bhaix (I.). The qiiatititittive ileteiuiiuation of the plyco^ien content by the Kiielz n»etho«l— which, as nHHlifiele results — is a Very troubles^aue task. L'Liut]i;iu workeound, and tinally estimating the aniouut by compaiing the colour with that obtained In ajijilying the sjiuie treatment to a i;lyeo«ren solution of known streu'rth. In this manner he ol»taiue dry resitlue : 20 per cent, of glycogen in a sample of liohtua etiulia ; 14 per cent, in a sample of Amanita »/,'■ ■ •. and 51 per cent, in a sjimple of yeast. This la.^t fi;. - 1 ees with that obtained seveml yeai-s earlier in a different manner by E. L.xikent (VIII.). whti found that the riche.st of seveml yeast samples containeil alx)Ut 32.6 per cent, of glycogen. On the instigation of I^ Erkeka (VII.), LAurent al.so en- deavoured to investigjite the conditit>ns favouring the enrich- ment of vea.^t cells in glycogen, and found that, fi^r this purjxise. the metliod of culture on wort gelatin is particulaily useful. The following substances were recognised as glycogen formers : lactic acid, succinic acid, malic acid, aspanigin. glutamine, e^^ albumin, peptone, numnite, glucose, hevuh»se. saccharokse. and maltose. Acconling to M. Chemek (1.), the.se mu.st be supple- mented by (/ pilacto.se and o.se, as well as lactovSt» and glycerin, to be unsuitable; though a contrary opinion with regartl to the two last was expressed by E. Ljiurent. E. Kaysek and E. lioiLLAXOEu (l.)then inve.stigateil the influence of other conditions, e.ij. the presence of air, and the percentage content, of tartaric acid, malic acid, or citiic acid, on the inception an»l progress of glycogen enrichment. In twenty-eight st<.K"ks of wine yeJtst examined with this object. Hi< hahd Meissxek (111.) found that glycogen makes it.s appeaiance, even in the young buds, when the latter have attaine*! aKnit one-fifth the diameter of the jvu-ent-cell. A notable obser\iition. and one worthy of further investigation, was made by M. Ckemer (VI.). n.imely. that the ;;lvco:;en reaction tK-cumnl in alH>ut twelve hours in a glycogen-free presseil yeast juice that had been treatetl with 10 per cent or more of fermentable sup\r (dextnxse) and kept at 172 CHEMISTRY OF THE YEAST CELL. the ordinary temperature. Possibly therefore we must assume the presence of a synthetic enzyme in the cell contents. In view of the amorphous nature of this carbohydrate, some surprise will be manifested at Laurent's report that the yeast cells are capable of absorbing and accumulating the glycogen present in a nutrient solution. And in fact this assumption was contradicted both by M. Cremer (I.) and by A. Koch and H. Hos^us (I.). The last-named observers found in the case of a pressed yeast, a bottom-fermentation beer yeast, and the so- called Frohberg yeast, that the glycogen added to the nutrient solution (wort or meat extract solution, with or without an addition of grape sugar), whether obtained from calves' liver, rabbits' liver, or pressed yeast, not only remained unutilised, so far as could be ascertained, but also retarded the cell reproduc- tion and fermentative power, so that the yeast crop and the percentage of alcohol in the treated samples were smaller than in those left without addition of glycogen. They failed to detect alcohol in the sugar-free cultures ; and concluded that yeast is incapable of secreting a hydrolytic enzyme by means of which the glycogen in the nutrient solution could be converted into fermentable sugai'. General application cannot be accorded to this deduction, since M. Cremer (TV.) noted hydrolysis of glycogen on keeping yeast in chloroform water, and found that dextrose was thereby formed. The contrary assumption, by E. Salkowski (II.), of the formation of Isevo-rotatory sugar, is not altogether free from objection, as the latter himself (VII.) admitted. Since the conversion of the accumulated arlycoeen withm the cell is probably connected with such an enzyme, a strong presumption exists in favour of the occurrence of a similar enzyme, produced by the plasma. This probability has been brought to almost a certainty by the discovery of Buchner and Rapp (II.) that glycogen can be fermented by expressed yeast juice. The presence of a similar enzyme may also be assumed in other fungi that store up glycogen, and consequently will have to be reckoned with in the separation of this carbohydrate, especially when a quantitative determination of the same is in question. It may be remarked in passing, that, as was first shown by A. KoCH and H. Hos^us (I.), the faculty of hydrolysing glycogen is also possessed by various species of bacteina. This at the same time explains Salkowski's (II.) discovery, previously reported by ScHUETZENBERGER and Destrem (L), as also the results obtained by N. VON Chudiakow (I.), and urged by him against the assump- tion that autofermentatioii occurs in yeast. Glycogen has been rightly termed a reserve material. In times of superfluity of nutrient materials the glycogen is stored up in the cell for the purpose of consumption in case of need, either for maintaining the vitality of the individual in an environment destitute of the necessary foodstuff, or for the (;ia'cu(;kn and fat. 173 construction of new imlividiials ui»«l ft>e«liiig them until such time as thev iire able to eater for tliemselves. This hitler t-vent takes phice for insUmee when a sclerotium fi^erminates. In the case of Cuj/nnun nirt'iin and Clat i'-tj>s }>ur/tun a, L. Kuukua (VI.) accurately traced IIr* gradual migration of the glyo^gen from the colls of those hard mycelia into the pileated fungi developed therefrom. Ho also observed the same in the germiiiation of various fungus spores, f.ij. those are able to use up this store, since they .sometimes perish befoie the same is exhau.sted. According to the observations of II. Will (X.), dead permanent cells rich in glycogen can always be found in the sedimental yeast of old cultures in wort or (liquefieil) wort gelatin. Finally it may bo rem.irked that the commencement of the working up of accumulated glycogen does ni»t always coincide witli the occurrence of a lack of the nocessiiry foodstuUs outside the cell, but seems to be bound up with other circumstjinces, the age of the cells in particular ; so that the glycogen content of a .sjimple of ywist may decline, although sugar is still pre.'^ent in the nutrient solution. In this connection observations have been made by N. Jouluaikk (1.) in the cour.so of some researches which will be referred to in a later section; and these were conlirmed by ( Jontscharuk, who>e results have been reported by 1{. Mkissnkk (ill.). The importance of fats for the life of the yeast cell is equal to that of {jlvcoyen ; they al.so are valuable reserve material. Their presem-e in wine sediment was observed by l?raconnot. Till' percentjige of fat varies with the monu'nt;iry conditions nf nutrition of the cells. Payex (11.) fixed it at 2.\ per cent, of the dry residue in the case of beer yeast; but, by the aiil of a better metlunl, ensuring complete extniction, N.KUKLi and LoKW {\\.) succeeded in separating about 5 per cent, of fat from a bottom-fermentition beer yeast. Acconling to the researi'hes of P. Dauf.xv (I.), alone, and in collabiiration with E. (Jkkakd (I.V the cliief constituents of yeast fat are steaiii- 174 CHEMISTRY OF THE YEAST CELL. acid and palmitic acid (in almost equal amounts), along with a small quantity of butyric acid, part of the acid being in the free state and part combined with glycerin. The chief seat of the fat is in the granules, whose albuminous membrane prevents lixiviating fluids gaining access to the enclosed fat. Consequenth', as remarked by Ntegeli and Loew, a simple treatment with ether is ineffectual. Still less likely to effect extraction is the small amount of alcohol present in wine ; and, in fact, as shown by Mueller-Thurgau (XVIIL), wine does not contain more than about o. i gram of fat per litre. In a case ob- served by H. Will (YIII.) in 1898, drops of fat were found in both dead and living cells and spores of a ten years old culture of Sacch. Ludioujii in beer wort, their colour being reddish- yellow, so that the culture gradually became of a brick-red shade. A substance closely allied to the fats, namely lecithin, which is the cholin ester of palmito-stearo-glycerophosphoric acid, and is therefore constituted in accordance with the formula : — (C13H31COO) (Ci7H33COO):C3H5-0- PO.OH- C - CHo.CH.iNlCHslsOH Palmitic Stearic Glycerin. Phosphoric Cholin, acid. acid. acid. was first discovered in yeast by Hoppe-Seyler (IY.) in 1866. It was afterwards prepared by him (Y.), to the extent of 0.25 gram from 81 grams of air-dry pressed yeast, in the course of I'ebutting a contrary opinion launched by 0. Loew (YIII.) and IST^GELi and Loew (II.). The first discovery of cholesterin in fungi was made in 1867, by O. KoHLRAuscH (I.), in a species of Morel; and -soon after- wards it was found by Hoppe-Seyler (IY. ) in yeast. Later on, this same worker (Y.) recovered 0.44 gram of this monovalent alcohol (C.jjH^o.OH) from 81 grams of air-dry pressed yeast. The name cholesterin, which was originally bestowed on a single substance of animal origin, afterwards became enlarged to a collective term, in consequence of the discovery of several isomeric and homologous cholesterins, which, tliough agreeing with the first cholesterin (from gall, ttc.) in their chief pro- perties, yet behaved differently in several reactions. Thus, Hesse isolated from peas the cholesterin to which he gave the name phytosterin ; Reinke and Rodewald another (para- cholesterin) from AethaUum sepficiun ; C. Tanret (IY.) a third (ergosterin), from ergot of rye. According to the re- searches of E. Geuard (III.-Y.), these latter are identical with all the cholesterins hitherto isolated from yeast, as also from Mucor mucedo, Penicillium glaucum, Staj^hylococcus albut>, and indeed from Crypfotjams generally. THK ('AIII'.OHVDKATE rJUOl'l'. 175 ■^ 254. Mucinous Substances of the Carbohydrate Group. The Gelatinous Network. Umlfi- tin- iKtim- \ca>l j.'uui, >f\L'iiil \vuik«*r> have isoliiU'd mucinous carbohydrates from yt-ast. Thou;,;!! Mjuie of tliese products have not been closely t'xuiiiined, they Imve been so fur characterised that we may assume the alx»ve name to be nothing; more than a collective term. They all behave in the sa^ue manner towards Fehlinij's solution, a circumst-mce of some value in connection with their iirepanition and isolation, since all are preci]»itjiteer compound, from neiilial oi- faintly alkaline solutions. On leaving yeast to uiidergo autofermentJition (see a later para<:raph) in water, J. liKCIIAMl' (VIll.) in 1874, discovered in the liquid (whieh was protecte.;/. in Vieing precipitable by alkaline i««pper solution, though its rotiitory powei- was much h-.^s. namely + 78°. The formula 3(C^H,„0,,) + 2H.,0 was deduced from the results of ultimate analysis. This gum was gradually convertt-d into glucose by the action of acids; w.is stained brown-red l>y iotline ; and therefore— as Eureka (II.) surmised with gootl rea.son — alsj»ale.scent litpiid, the rotatory jmwer of which, referred to about i per cent, strength, was found to approximate to 0^= +285.7'. A fifum (laevulan), dilTering from the foregoing carl>ohy}»tion that different kinds of gum are pre.sent in different s;imj»les of yea.st, the results of Fiirrz Hesskxi.and's (I. ) experiments permit the further conclusion that several kinds of glutinous carlH)h\di-at*'s 176 CHEMISTRY OF THE YEAST CELL. may be simviltaneously present in one and the same yeast, whether of the top- or bottom-fermentation variety. This worker boiled yeast over a naked flame three times in succession for six hours each, in Avater containing a small addition of lime, then precipitated with ammonium oxalate the lime from the filtered extract, filtered and concentrated the solution, faintly acidified it with hydi'ochloric acid, and treated it with an equal volume of 96 per cent, alcohol. The resulting precipitate of brown gum was decolorised by washing with alcohol, and was found to exhibit a percentage composition corresponding to the formula C^H^QOg. The gum from top - fermentation yeast had the rotatory power cId = + 283.7°, that of the pro- duct from bottom - fermentation yeast being -f 287.6'. The precipitate thrown down from the aqueous solution by Fehling's solution had the composition 2(C,.H-,,p^).CuO.HoO. This gum furnishes on hydrolysis, not one sugar only but two, namely, a little glucose and a large quantity of J-mannose ; it therefore chiefly (but not exclusively) consists of mannan, the percentage of which, referred to the dry residue of the yeast, amounts to 6-7 per cent. E. Salkowski (V.) classed as in the main identical with this mannan the yeast gum which he obtained in 1894 by boiling pressed yeast for half-an-hour with a tenfold volume of 3 per cent, caustic potash. This treatment brought into solution the whole of the cell contents, including the gum. When cold this extract was syphoned off from the residue (of so-called yeast cellulose), treated with 15 per cent, (vol.) Fehling's re- agent, well mixed and heated. The blue copper compound of the gum separated out in lumps, which were immediately taken out, rinsed with a little water, triturated in a basin with a few drops of hydrochloric acid, and thus converted into a cloudv liquid, from which the gum could be thrown down by a three- to four- fold volume of 96 per cent, alcohol. Repeated solution and re-precipitation finally gave a white, ash-free mass, which was dried with alcohol and ether, and then amounted to about 7 per cent, by weight of the dry residue of the yeast. The results of the ultimate analysis corresponded to the formula CjoHooOj^. In contradistinction to the yeast gum obtained by Keegeli and Loew, this product dissolved readily in water to a filterable, but very glutinous liquid. The sugar furnished by the hydrolysis of this gum seems to have been regarded by Salkowski as r/-mannose. Na^geli and Loew thought themselves justified in assuming their yeast gum to be a conversion product of the yeast cell membrane, because they found that fresh quantities of this gum could be obtiined from beer yeast by repeated boilings in water, the total amount (including the so-called cellulose) being about 37 per cent, of the dry yeast. It was then found by E. Sal- THK CAKJiUUVJ.KATK OKOUl' / / KowsKi (\'.) that the if>iilu.' left iiftfr lixiviiitiiig veast with dilute caustic potiisli \ ieMt-d no further j^'uuj on " prolonged boiling; thus proving that, with a uioro jKiWerful solvent tlwn mere water, the whole of the yeast guiu can he e.\tr;ieted at one openition. This, however, tloes not solve the prol.lem with regaitl to the location of this unicinous substance; nor at present is any reliable information avaihible as to the seiit of the pentosjins, which were detecteil, to tiie extent of 2 to 3 per cent, (referred U) the dry residue) in i>oth top- and Ujttom- fennentition yeast, by Hessexland (I.), with the furfural methotl. Sciii KTZEXHEUOKii (1.) found in yeast extmct a gum, which proved convertible, by hydrolysis, into a redticing sugar. The conclusion that this carbohytlmte is an arabin is opposed by the further report that the same was converted, liy b.jiling nitric acid, into a mixture of oxalic and mucic acids. From this behaviour it is probjible that he had to do with a galactane. At all events the amount of carbon fouml. namely 4J.7 per cent., does not correspond with the formula C^;11,„0,' H. Mkissneu (II.) has described a luimber of budtling fungi, whidi, when sown in wine mu.st, convert the .sjime into an oleaginous ropy liquid, by the aid of a mucinous sub.stJince, the composition and method of formation of which are .still un- known. Whereas until very recently the ropiness of beer and wine h.is been attributed almo.st exclusively to bacterial activity (.^ 164), this worker succeeded in .showing' that this malady can also be induced by budding fungi. He subjectemir,f,-jt, In a later paragraph mention will be made of the circumsUinces under which they become a .source of danger. The gummy substance.s leferred to in the foregoing lines make their appearance in quantity more particularly when the fermentjitive activity of the cells h":us drawn to a clos'e. In this event, whether as a result of the swelling of the invariably mucinou.s outer layer oi the membrane, or of an excretion from the interior, the mucinous envelopes develojied by the individual cells coale.sce to a sort of honeycomb in which the cells appear embedded. 'J'hese formations were first tO.served bv E. Cu. IIaxskx (X.), who gave them the name "gelatinous network." They were found in the so-called yeast ring and in the films (ji 246) on old cultures of ^^arrftarotni/reteji and a few other building fungi, grown in nutrient solution.s. They jtre also not infrequent in the inoculation streaks that have been drawn on yoL. u. jj 178 CHEMISTRY OF THE YEAST CELL. gypsum blocks for the purposes of spore analysis (§ 247) ; and a similar gelatinous network is often observable in yeast samples (about the size of a pea) that have been taken by the brewer and dried between blotting-paper in order to be conveniently sent by post to a laboratory for examination. Their detection is facilitated by the aid of staining, either with Methyl Violet, which stains the cells only (Fig. 167), or by the so-called capsule staining method (§ 33) described by Hansen (XVI.). The latter method stains only the mucinous network, and thus renders the latter visible in cases where it could not be detected in the un- stained preparation. It sometimes happens that, during the preliminary treatment of the sample of dried cells to be stained, Fig. 157.— Network in Carlsberg Bottom-fermentation Yeast No. i. Hansen. The majority of the cells have been washed out of the network in the course of staining with ^Methyl Violet, only thirty-one cells being left. These, having been deeply stained, have a dense black appearance in the Fig. Magn. 1000. {After Hansen's original draivings.) the cells themselves get washed out of the network, thus leaving the latter isolated and empty. Fragments of evacuated net- work are not infrequently observed (Fig. 158). I^eveitheless, it should not be assumed, without further inquiry, that every formation with this appearance is a residue of this kind ; but one miist bear in mind the observation made by H. Will (VII.) to the effect that the glutin bodies present in sedimental yeast frequently exhibit, when dried, an appearance resembling frag- ments of the network in question. In such cases, decisive information is afforded by an addition of acetic acid to the preparation, this acid dissolving only the remains of the glutin bodies. Will has also shown that several different kinds of reticulation, and substances composing same, are found in the films and yeast ring of wort cultures of beer yeast. This matter will be reverted to in the next paragraph. TJIK CA1M;()11VJ)KATK (JJiOrP. ,;9 Tho a.Mitioii of a lai-u,. .juaiititv of water to the ywu,t -ll-ssolvt-s the network. NeVLMtheless/as HaiiHeii has Kh.mn, it tonus anew, unles.s the treatiinMit be repeated suflicieiitlv often •md prolonj^'cl. Acvonlin^r Uj II. Wm.l (VII.), lioweve'r, it is pernuiiu-ntly remove.! wh.-ii tl»e wa.shii.g is repeated t;e of this albuniiik and the aforesaid mucinous substances present. Yeast from the upper hiyer takes up two to three parts by volume of ether, but core yeast only about one part, the mucinous substances forming bubbles charged with ether. In tlicsi- laboratory experiments the ether plays the sjime part as caibon dioxide iloes in practical fermen- tation in the vat ; and as the mucinous substances do not remain in the .sey Hubich, and then proved, by isolation, by C. LiXT.VEU, sen., and Kkischaueu in 1S76. This froth glutin (Krauesengliitin) is .sjiid to originate in the wort or the malt. liy means of comparative fermentixtion experiment's with «lif- ferent beer yeasts, it was then est;ibli.shed by Alb. Reicii.^kd (III.) that, in adtlition to this malt albuminoid, the presence of certain mucinous excretiims (especially those of an albuminoid character) from the yeast is essential to the formation of a normal " head " composed of tine bubbles. The condition of these formative materials in the head at different stiiges of fermenta- tion has not vet been more doselv investigated. In the case of a liquid ab.solutely tlevoid of vi.scosity and free from mucinous constituents, the bubbles of carl>on dioxide libei-iteoneut of unadultei-ated pressed yeast. The main features in the preparation of this article by the old or Viennese metiiod are as follow : Alnjut three to four hours after the wort has Ijeen pitched with yeast — a so-called "lUtilicial " yeast (§ 148) of suitiible ijuality — the mash begins to work. The scum of husks and grains that has accumulated on the surface in the mean- time, is now penetnited liy an ascending white head, the development and growth of which, during the next twelve hours, presents a picture of eViullient motion of ci>nsideiiil)le briskness. Under ordinary circumstunces the head at the end of this time will have attained a thickness of twelve to fifteen inches, whilst the gravity of the wort will have decreased to about half its initial value. This head foriiis the vehicle containing the major part of the yeast crop reprot., and therefore on the yeast crt>p. More detJiiltnl information on this matter will be found in the volume on pressed-yeast manufac- ture, issued by O. Dlkst (I.). The so-calleil rit)t«.>us or blaildery fermentation of beer yeast may be left out of consideration here. The sti-uctur.il maty' ruite and jitii-i inoducts of f?uorar works with- out any recognisable cause, iiml wliich is reganii-d \)\ sumo tochnicists as a purely cheniical process of decomposition, some information will he found in a communication by O. Laxa (I.). The fact, of which mention has already l)een made, that certiiin albuminoid constituents are extractexl from yeast cells by ethyl ether, has V)een utilised in pnictiee by H. Hlchneh and M. (luruKK (I.), who manufacture nutrient preparations therefrom by a patented process. j 256.— "Break" and Clarification in Beer and Wine. In the formation of head on fermenting liquids tlie aforesaid mucinous excretions from the yeast cell are ass(X"iated to a »rreater or smaller extent, aceordin-,' to circumst;inces, with other mucinous bodies already initially present in the nutrient sub- sti"atum. Their dependence on such assistiince is diminished by the inception of tlie phenomenon known as *' break " in brewing and wineinaking. In proportion as the liV)eration of carbon dioxide gradually decreases towanls the end of fermentation, there ensues a diminution of the force by means of which the yeast cells float- ing in the fermenting liquid have so far been kept in motion. Consecjuently the atti-action of gi-avitiition can now make its influence on the cells, which have a higher specific gnivity than the young V)eer or new wine. The resulting sulisidence of the cells is, however, opposed by two forces, namely, the vis- co.sity of the li(juid and the friction l)etween the licpiid and the individual cells. These forces suffer diminution when .sevenil cells adhere to form a small ball, since, whilst the weight of this agglomemtion is e(pial to the sum of the weights of the individual component cells, the surface is c(»nsidei-ibly smaller than the total superficial area of the component parts. Hence, while the downward pressure of the weight remains the sjime jvs before, the surface of contact with the litpiid, and therefore the amount of resisttvnce, is les.s. Now the afores:iid gummy and all)uininous mucinous matters favour, and indeed are in- dispensjible to, this agglomemtion of the cells. When they have accomjilished this task and united the cells into colonies that are visible tttom- fermentiition yeast that, on their first introduction into the brewery, they exhibit little or no " break " until they have been pitched on two or even three successive batches of wort. Now, it is easy to underst;ind that, in presence of this difficulty, the brewer, l)ein^ already suspicious of the value of the innovation, should decide to revert to the "good old .stock yeast " to which he is accustomed, oblivious for the moment of the many di.siippoint- ments the latter has already caused him. The causes of imper- fect " break " are manifold, and, in part, still undetermined ; for iustjince, an insufficiency of lime (§ 258) in the wort, too low a formentinj.; temperature, or imperfect rousing during the cultiva- tion in wort. On the other hand, some stocks of yeast exhibit a defective " break " under all condition-^, and furnish a thin sedimentjil yeast instead of a firm deposit in the tun. Despite the greater care involved in working with yeasts of this type, they are still employed, especially when they exhiVnt other valuable tpialities and furnish, for example, a beer characterise*! by great stability and therefore suitiible for export. Such a yeast is met with iu the Carlsberg bottom yeast No. i, V>y means of which E. Ch. Hansen first iutro<.luced his pure-cidtui-e method into the fermentation industry. On the other hand, for the production of (piick-runiiing ales, it will be found economically advantiigeous to make u.-^e of a yeast that chirifies rapidly. Yeasts exhibiting coarse " break" and gooil clarification are still more important in the preparation of champagne than for Ijrewing. since in the former case imperfect clarification cannot be remedied by the use of strips or by filtration. Presupposing a knowledge of the rudiments of champagne-making, it may be recalled that the wine for this purpose is treated with sugar, and is subjected whilst in bottle to a secondary fermentation in order to protluce the requisite amount of carbon dioxide. In the old process the fermentiition of this sugiU* was left to the few cells usually present in the wine; the result.s, however, fre- quently failed to come up to the exj>ectations fonaeu\\>) witli inust and Howing it with yeast. When tleveluiiiuent was comjilete, he washed ofV the superficial shells ami intitMliu-t'd thf '* fermentiition tibro " (richly interspersed inteinallv with fettered cells of yeast) into the wine cask, where it carried out the desired seciindary fermentiition without afTecting the brightness of the liijuid. END OK Vol.. II.. TAUT I. PrinU'«l l>y nALL.\STTSK, Hanso.v 6^ Co. Edinburgh ^ London i A >^ i:j, i^c'v \ (jx FK'.M TIIF SCIENTIFIC AND TECHNICAL WORKS i'L'ULISUKU UV CHARLES GRIFFIN & COMPANY, LIMITED. (Bemg Sectious 4-10 from Messrs. Griflin's •• General Catalogue.") ) MKSSRS. CHARLKS GRIFFIN .k COMPANY'S PLriiLICATl()N\> may be oht&iiie.l thmu!,'). aav Books.'ll.M- ui the L niteON (Prof.), Manuals, . . 34 Electrical Tramways, . . . 34 JENKINS (H. C), Metallurgical Machinery, .65 JOHNSON (J. C. F.), Getting Gold, . 59 KASSNKR (T), (+old Seeking, . . 60 KERR (G. L.), Practical Coal-Mining, 58 Elementary Coal- Mining, . . 58 KNECHT & RAM^SON, Dyeing, . . 82 LAFAR, Technical Mycology, . . 73 FAOB LAWN (J. G.), Mine Accounts, . . 68 LIVERSIDGE (J. G.), Engine-Rooni Pra.ctic6 ... . . • . 29 LYON (Prof.), Building Construction, 54 MACKENZIE (T.), Mechanics . 42 M'MILLAN (W.G.),Electro-MetaJlurgy,88 & BORCHERS, Electric Smelting, 68 MILL (Dr. R. H.). New Lands, . 54 MILLAR (W. J.), Latitude & Longitude, 43 MITCHELL (C. A.), Flesh Foods, . 74 MORGAN (J. J.), Metallurgical Analysis 87 MUNRO & JAMIESON'S Electric*! Pocket- Book, 4» MUNRO (J. M. H.), Agricultural Analysis, 75 MUNRO (R. D.), Steam- BoilerB, . . 33 • Kitchen Boiler Explosions, . . 3S NAYLOR(W.), Trades' Waste, . . 77 OPPENHEIMER (C), Ferments, . 74 PARK, (J.), Cyanide Process, . 59 PEARCE(W. J.), Painting, . . . 80 PETTIGREW (W. F.), Locomotive Engineering, 30 PHILLIPS & BAUERM AN, Metallurgy, 60 PHIPSON (Dr. T. L.), Earth's Atmos- phere, 53 POYNTING (Prof.). Mean Density, . 50 & THOMSON, Physics, ... 50 PRAEGER ( R. L. \ Open Air Botany, . 86 RANKINE'S Works, . . .35,36 RAWSON, GARDNER, & LAYCOCK, Dictionary of Dvestufife, . . .82 REDGRAVE (G. R.), Cements, . . 76 REDWOOD (Dr Boverton), Petroleum, 61 & THOMSON, Handbook, . . 61 Petroleum Lamp, . . . .61 REED (Sir E.J. ), Stability of Ships, . 38 REID (Geo., M.D.), Sanitation, . 78 RICHMOND (H. D.), Dairy Chemistry, 73- ROBERTS -AUSTEN (Prof.), Metal- lurgy 63 Alloys 62: ROBINSON (Prof.), Hydraulics, . . 37 ROSE (T. K.), Gold, Aletallurgy of, . 63 ROTH WELL, (C.F.S.),TextilePrinting, 83 SCHWACKHOFER and BROWNE, Fuel and Water, 47^ SEATON (A. E.), Marine Engineering, 45 & ROUNTHWAITE, Marine Engineers' Pocket-Book, . . . 45 SEELEY (Prof.), Physical Geology, . 58 SEXTON (Prof.), Metallurgy, . . 66 Quantitative A- Qualitative Analysis, 70 SHELTON (W. v.). Mechanic's Guide. 36 SMITH, (J.), Shipmaster's Medical Help, 43 SMITH (Prof. R. H.), Calculus, . . 46 Measurement Conver.'f Forces — Bending; Strain — The Graphic Kepresenlation of Bending Moment ">. Pari II.— Ge.nkral Principles of Bridge-Construction :— The C'">niparitive Anatomy of Bridges — Combined or Composite Brid(jei — Theoretical Weight of Bridges — On Deflection, or the Curve of a Beaded Ciirder -Continuous Cjirdci^ Pari III.— The Strencih op Materials :— Theoretical Strength of Columns — Design and Construction of Struts — Strength and Construction of Ties — \V. irking Strength of Iron and Steel, and the Working Stress in Brid^«8 — WbhTcr's Kxperiments. Pari IV. — The Design of Bridges in Detail:— The Load on Brides —Calculation of Stresses due to the Movable Load — Parallel liuiiers — Direct * alculation of the Weight uf Metal — Pambolic Girders, Pulvgonal Truaaes, and Curved Girders — Suspension Bridges and Archea : Flexible Conatrurtion — Rigid Construction — Bowstring Girders usei.1 as Arches or aa Su^oension Bridges— Rigid Arched Ribs or Suspension Ribs — Continuooa Gliders and Cantilever Bridges — The Niagara Hric^i^e — The Forth Bndge — Wind- i'lessure and Wind-Bracing: Modem KxperimeiUs. "The new edition of Mr. Fidler's work will a^n occupy the sauiv OON- BPi'Tois i-tJsiTiu.N aiiiong professional toxtlKXjks and treatices as ha« been accorded to it« l>rc«lecessors. The instruction imparted is scHXD, siuplr, AM HI L. The volume will bo found valuable and useful alike to tlu«e who may wish to study only the theoretical jirinciples cniinciateil. and . • . to others whose object and business is . . . practical-" — The Engwrrr. Mr. Fli>LEH's ^rcCE^>^ iirii«(>8 from the cyimbination i>f ixPKRiR5r» and Bl^-UClFT or tkkatmknt diniok is. tberefure. aa u*«ful to girdrr nii^krra «e to itndentji of Hmige Conntnution " 7"^* Arrhittci. " Ab lM>i8rKNaABLK HAM>EkK>K for the practic&l Engineer ' Satw-* LONDON: CHARLES GRIFFIN a lu.. LiMiItu. lamlu iintti. oTRAHO. 28 OHARLES O BIFFIN ds CO.'S PUBLICATIONS. Works by BRYAN DONKIN, M.Inst.C.E., M.Inst.Mech.E., &e. Third Edition, Revised and Enlarged. With additional Illustrations. Large 8vo, Handsome Cloth. 25s. GAS, OIL, AND AIR ENGINES: A Ppaetieal Text - Book on Internal Combustion Motors without Boiler. By BRYAN DONKIN, M.Inst.C.E., M.Inst.Mech.E. GsNBMAL CoNTiNTS.— Gas EngfineS :— General Description— History and Derelop- tuerit — British, French, and German Gas Engines — Gas Production for Motive Power — Theory of the Gas Engine — Chemical Composition of Gas in Gas Engines — Utilisation of He it — Explosion and Combustion. Oil Motors : — History and Development — Vaiioui Types. -Priestman's and other Oil Engines. Hot- Air Engines :— History and Develop- ment- -Various Types : Stirling's, Ericsson's, &c., &c. " The BBST BOOK NOW PUBLISHED oD Gas, Oil, and Air Engines. . . . Will be of ruRY GREAT INTEREST to the numerous practical engineers who hare to make themselves faiailiar with the motor of the day. . . . Mr. Donkin has the advantage of long PRACTICAL EXPERIENCE, combined with HIGH SCIENTIFIC AND EXPERIMENTAL KNO^'LBDOB, and aa accurate perception of the requirements of Engineers." — The Enginttr. " We HBARTILV K3C0MMEMD Mr. Doulain's work. ... A monument nf capofiil liib^ur. . . . Luminous and comprehensive." — J eumal of Gai LigJiiint, . " A thoroughly reliable and exhaustive Treatise." — Enginetriti!;. In Quarto, Handsome Cloth, With Numerous Plates. 25s. THE HEAT EFFICIENCY OF STEAM BOILERS (LAND, MARINE, AND LOCOMOTIVE). With many Test§ and Experiments on different Types of Boilers, as to the Heating Value of Fuels, &e., with Analyses of Gases and Amount of Evaporation, and Suggestions for the Testing of Boilers. By BRYAN DONKIN, M.Inst.C.E. General Contents. — Classification of different Types of Boilers — 425 Experiments on English and Foreign Boilers with their Heat Efficiencies iiown in Fifty Tables — P'ire Grates of Various Types — Mechanical Stokers — Combustion of Fuel in Boilers — Transmission of Heat through Boiler Plates, and their Temperature — Feed Water Heaters, Superheaters, Feed Pumps, &c. — Smoke and its Prevention — Instraments used in Testing Boilers — Marine and Locomotive Boilers — Fuel Testing Stations — Discussion of the Trials and Conclusions — On the Choice of a Boiler, and Testing of Land, Marine, and Locomotive Boilers — Appendices — Bibliography — Index, With Plates illustrating Progress made during recent years, and the best Modern Practice. "A WORK OF RKFBEENCit AT PBKBKNT UNiQCB. 'Will glyo an aDBwef to ainiO' t any j[ueBtlon oonnected with the performance of boilers that rt is possible to ask."— .Fnotr'a/T. "Probably the most exhaustive resume that hae ever been collected. A PB>CTiaAi. Book by a thoroughly practical man." — Iron and Coal Trades Review. lONDON: CHARLES GRIFFIN & CO.. LIMITED. EXETER STREET, STRAND. ttSUlSKtnUSO ASit MKUHAMOti. 29 T«imi> Ru^noa, Ktrntiiif8 Qi'amtitt ■■f Tsf .hiht' .s ^rr^al,.I•.| In a rerj convenlrnl form. . . A Hoar ca»DL TOLUMi . . . p - . nuwhere •!■«."— Z1U AnyMiMr. Third /tnprestion. L*rge Grown Svo. With numerous IllustrationB. 6«. ENGINE-ROOM PRACTICE : A Handbook for Engineers and Officers in the Royal Navy and Mercantile Marine, Including the Management of the Main and Auxiliary Engines on Board Ship. By JOllN.G. LIVEllSIDGE, Kiiirlno«r, R.N., A.M.I.C.E., Iiistnictor In Applird Mecliaiilc* at the Boyal NaraJ CullrK^i lireenwlch. C'on(«n(<.— 0«ner«l DetcripUon of Marin* Machinery —T^(• C"ti1it1onii of S»>rT!c*i and OuMftn uf Elaflne«ni of tlio Kov li Navy — Entry ai^ J t'onlr . ■ of v." L*ainpAnl-s. — lUl^inl.• Ste*ni — I 'utirt >•' .. -.« and Hollari — ShutUriv ofl Stomn. — Harbour I)ul:i'« an 1 \\ ii:.-:,c« - . ^nj ftopairv of Kntrluia — lYB»*r»aii . iinil 1 ci'»ir» if "Tanls ' l^>il<>rti.- 1 ilt Uttliixi - i'loantni,' and I'aini.iiL: Machinery — K«>oipri'0«'Hi|{ J'umi" *• ■ ..\ Autni' 'I.- Fe'd - Water U^K'nlatora — ETapiir:iU>ra — St.-am B ^.l Maaiuu TV — HyJrauil.- MachiiKMv - A;r-Cijni(Tei'fitic I'uinj •. — 'ir' .^. — Machinery of Di-t'royrra — 1 hr .ManitKeni'-nt of \\ atrr 1 ut« !l<>li«>r*.— U<>i(uUsioi.t for Bntry of Anaiilant Kn>;iiieer«, 11. .\. — Qupttion* kI^**" '■> Kiaininatlun* for rnmiotlOD of KnKlne«ia R.N — liAK-iiliiti'ni re>.p-ctlD)( Uoaril uf Trn ' ui for KuKlnpcrs. Ae " 1 he oontenU iasnot fail Tt» rk api^R'Iatrk " "Thll VBBT URirVL BiMiK. . . . I I.Ll'ITKATlUil.x :.-^. IMl^BTANCK ID a WOrk of (hia kind, an 1 it i« Katufactory to Dud thai sricciAL attextiom baa ht«a civ'n in tbii re^p•ot."— A'lVirMeri' Ojtttlt. In Crotni .?!•(., fxtrn, 1, r/. .Wvi^o^D IU\tstrnt\on». [Skorii^. GAS AND OIL ENGINES: An Introductory Texl-Book on the Theory, Design, Coustruclion, and Testing of Internal Combustion Engines without Boiler. FOR THE USE OF STUDENTS. By Pkof. W. II. WATKINSOX. Wurr. Sen., M.In8T.Mk<-h.R, Ulaagow and Wctt of Scotland Technical College. lONDON : CHARLES GRIFFIN A CO., LIMITED. EXETER STREET, STRAND. 30 CHARLES ORIFFIN aji A^a^f. "The inrork contains all that oak be lkaknt from a book upon sucii a sub.V^ct. It wll at once rank as the standakd vtork upon this impobtant subject."— .fiaij«ra|f Ui-ga&tn*. In Large 8ro. Handnome Cloth. With Platen and IVniMratxons. 16». AT HOME AND ABROAD. By WILLIAM HENRY COLE, M.Inst.O.E., Lite Deputy-Manager, North-Western Railway, India. (7on-oiau!io will |irorc a rtrj raluatil* aid, atid t-od U (ti« prodactioD ••: Eiiflun uftcii.iTiric DHio'i and BcoiioiiiCAL woasmo. . Wttl tf iatrr'T •oQfhf kt'r by 8lud«uU mod l>c«ltnien "— i/arMa« KnQMttr. " I'sayt'L and thurocohlt fkacticai. Will undoubtedly b« found of ockai vaixv Uy All eoi'< emcd with lii« dsaifn of \ alTs-goarlnx "- Ute/umiein Wend. ■ ■ Imoit iTiBT TTj-i of VAi VI c "^ r '' : Is rlfarly •«! forth, mi ' '^d o •■ob ■ «'a> at u> b« Kmat>iLT csuak.- ^icallt A>-n.iii> bj •Uh'-' np>pr, Drauc! L«nian or Btudant . . >L .. : , : :h LBcriLaMl vallabli i ,....»«• M-ok'.ii. r^T kt: iiBUi and OLits lnf>'miaUoQ oo ine aubjecl 111 nioderaM pncr bruiya r wltblD '• m.'j 0' *U " — IndutU-Mi an i lr(m " Mr nrkn • work li akkibablt iulted to ti.e no»