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1. Milestones in microbiology 1546 to 1940

Bordet ・ The じ行 0 ビ 2 ope Of ゞ e ″″ 2 f 07 〃 04 じ 0 ate イ 4 一″ m な 145 tious agent, following an ancient has been attributed tO a bactericidal substance dissolved in the fluids. This viewpoint has been especially de- fended with regard to the infections that are produced by microbes 0f the vibriO group, and it is the case Of cholera in particular which has been cited most Often in the battle between the doctrine of phagocytosis and the so-called theory of the bactericidal nature of the fluids. lndeed, in this special case, there eXIStS an evident correlation between the appearance Of an antiseptic PO 、 ver in the serum and the appearance Of immunity in the animal. This bactericidal power, which IS very 、 veak in the ummmunized ani— mal, becomes very marked in the vaccinated animal. And if it could be proven that the microbicidal material can be found during the life 0f the animal, uniformly distributed in the fluid, the cooperation of the phago- cytes , ould not seem tO be particu- larly necessary for the destruction Of the infectious agent. But this point has not been precisely demonstrated. The researches Of various workers, and 、 I. Metschnikoff in particular, seem tO show that the fluids of the body do not have the bactericidal power in the living animal WhiCh the serum posses- ses ITZ な ro. Nevertheless, it remalns tO be asked if, in the living organism, the active materials are not formed by fixed cells, and, for example, if it is not the leu- COCYtes which contain the bactericidal material, it is possible that the produc- tion Of this substance iS a special mam- festation Of their protective activity, and results from an adaptation under- gone by these elements under the in- fluence Of the vaccination. If such may be the origin Of this material, the appearance Of bactericidal PO ℃ r ln the serum Of a vaccinated animal may only be an indication Of an actual in- crease in phagocytosis. S じ第可 the んじ I カ 03 げ . Does the bactericidal power which has been found in the serum Of a vac— cinated animal act uniquely against the strain Of vibrio which は s used tO vaccinate the animal? ()r iS lt, the contrary, more general, acting on most Of the microbes belonging t() the vibrio group? M. Pfeiffer indicates that the bactericidal power of serum from a vaccmated animal is specifiC. have performed several experi- ments on this sub)ect. 、、一 e have lm- munized guinea or rabbits com- pletely With various strarns Of VibriOS. Ⅵ have collected the serum from the animals and have assayed the bacteri- cidal power Of each serum against each Of the vibrios. [These results are presented in Table 1. ] [ Similar results were obtained with Other strains and in guinea pigs ・ ] VVe do not believe it necessary to extend these experiments further, first because these results are in agreement with those Of Others, further because ℃ have devised a procedure for deter- whether an lmmune serum IS bactericidal against 2 given vibrio, which is much easier than doing plate counts in gelatin. ThiS procedure, WhiCh we shall stress later, consists Of revealing bactericidal action, in the case where it exists, by a granul•ar transformation Of the VibriOS (Pfeif- fer phenomenon), which the vibrios undergo when they are mixed with an lmmune serum alone, or an lmmune serum mixed previously With a cer- tain amount Of fresh non-llnmune serum. We can see in Table 1 that the bac- tericidal power Of a serum iS alwav ・ S the strongest against the vibriO identi- cal with that which was used tO vac- 物 [This serum-induced granular transfor- mation (Pfeiffer phenomenon) is probably lysis of the cells.]

2. Milestones in microbiology 1546 to 1940

Koch ・ The g 可 4 the skin is loose and covered with long h air. . I have made a large number Of inoculations in this way, using fresh anthrax material, and in every case I have had a ositive result, and I be- lieved there ore that the success of the inoculation could be used as an indication of the life or death of the bacilli inoculated. I will show through later experiments that this idea is true. Partly in order to always have avail- able fresh material, and partly to dis- cover if the bacilli would change into another fO ロ 11 after a certarn number Of generations, I inoculated a number Of mice ln senes, one from the other, each time using a mouse Which had just died as a source Of the spleenic material. The longest series Of mice treated in this 、 vay was twenty, which therefore represented that many gen- erations of the bacilli.* ln all animals the results were the same. The spleen was markedly swollen and contained a large number Of transparent rods WhiCh were very similar in appearance and were immotile and without spores. This same type of bacillus could be found 引 so in the blood, but not in so great 2 number as in the spleen. ln these experiments it was shown, there- fore, that a small number of bacilli could always develop into a significant mass 0f individuals of the same type . which appeared to reproduce by growing in length and then splitting after they had reached about twice the length of the individual bacilli. These results 21S0 indicate that it is highly unlikely that the bacilli would go through some change in form if 2 longer serres Of inoculations were made, and therefore it is unlikely that there is ultimately some alternation 0f generatlons. lt will take us too far afield to con- sider whether or not the actual cause ・ [We know now that the number of gen- erations Of the bacteria would be much greater than ℃ n . ] 91 of the death of the animals is due to the production Of carbon dioxide in the blood through the rapid growth Of the bacilli there, or, what seems mo 代 probable, that death is due to a metabolic product produced by the parasite through its utilization Of pro- tems as nutrrents, and that this meta- bOlic product is poisonous tO the ani- mal. [To study the life history of the bacilli away from the animal, ] a drop Of fresh beef serum or aqueous humor from the eye of a cow was placed on a mrcroscope slide. Then a small piece Of spleen which contained bacteria and which had been freshly removed from an infected animal was laced in this and a cover glass place on top. The microscope slide was then placed in a moist chamber to keep the liquid from evaporatlng, and this was then placed in an incubator. These preparations were incubated for 15 ー 20 hours at 35 ー 37 。 . At the end of this time, in the middle of the prep- aration between the tissue cells could be seen many unaltered bacilli, al- though in smaller numbers than in fresh preparations. } 孑 0 ℃ ver , away from the tissue in the fluid, one could see bacilli which were 3 ー 8 times longer and showed shallow bends and curva- tures (Fig. 2 ). 才 The closer to the edge 0f the cover glass, the longer the fila- ments, and these finally reached a size which was a hundred or 1 ore times the length of the original bacilli (Fig. 3 ). Many of these long filaments had lOSt their uniform structure and trans- parent appearance, and their contents had become finely granulated with the regular appearance of strongly 十 [Such 2 metabolic product, known today as a tOXin, IS usually associated in some way With most infectious diseases, including anthrax. ] t[This plate 2k0 contains the figures for the paper of Cohn, 1876 : "Studies on the b 同 0 , of the bacilli. " See page 49 for the text of this paper.]

3. Milestones in microbiology 1546 to 1940

D ' 〃 e 〃ビ・ / 〃切みなみ e 4 な go が立な地厩 e リみ 4 〃 159 Shiga bacillus. These latter properties ℃ re attenuated in succeeding cul- tures. ln summary, in certain convalescents from dysentery I have shown that the disappearance 0f the dysentery bacilli IS coincident with the appearance Of an invisible microbe endowed With antagonistic properties against the pathogen. This microbe, really a ml- crobe Of immunity, iS an obligate bac- teriophage. lts parasitism iS strictly specific, but if it iS restricted tO a cer- tain species, it is gradually able t0 acqurre actlV1ty agamst various gerrns. lt seems therefore that in the dysen- tery bacillus, as well as a homologous immunlty coming directly from the person infected, there eXlStS a hetero- logous immunity coming from an an- tagomstic microbe. lt is probable that thiS phenomenon IS not restricted tO dysentery, but that it is of general significance, because I have been able tO observe a similar S1tuat10n, even though weaker, in t ℃ cases Of para- typhoid fever. CO ″″〃 e 〃 t This discovery of bacteriophages, or bacterial viruses, was actually first made by Twort in 1905. But his work is not t00 clear and was not followed up, SO that I have included here the paper by d'Herelle, which was really responsible for the beginning Of extensive scientific work on these lnteresung organrsms. MOSt species Of bacteria that have been studied have been shown tO be hosts for bacterial viruses. AS d'Herelle sus- pected, the phenomenon widespread. But his early hopes that bacterial viruses would be useful in treating bacterial in- fections or ln conferring immunity have not been fulfilled. d'HereIIe had little understanding Of the word 'fimmunity as we use 1 [ , since the Of a bac— terial virus could not be expected to in- duce antibody formation against a bac- terrum. The bacterial Viruses have remained essentially laboratory ties. They have been used as model sys- tems for the study of viral reproduction and have made great impact on molecular genetics. They have also been used in diagnostic laboratories in the typing of certain widely occurring pathogens, such as & / 0 〃 el な p わ 0 and & 4 々たあ co ← C ″ゞ 4 ″ re ″ゞ . But all trials of their use as therapeutants have been unsuccessful.

4. Milestones in microbiology 1546 to 1940

・ T ゐ〃 0 可 4- な″ z 切 0 み 0 な 40 地ゆゐ〃 4 ビ T I Effect 可 & 4 れイ Age 可 0 0 れ S ″ゆら 4 れ〃イ e ル 0 Cultures on meat infusion peptone agar used as source of inocula. AII tubes contained 3.03 X 10 ー 4 M sulphanilamide. Age 可 ag 24 48 Ⅳ″川み 可 cells ーれ OC 耘イ 106 105 1 18 18 105 1 103 Growth 可′イ 4 十 0 0 0 十 0 0 0 十十十 十十十 0 0 十十十 0 0 2. 十十 十十十 十十十 十十十 0 十十十 十十十 0 0 3. 201 5. 0 0 十十十 十十十 0 十十十 十十十 十十十 Here and elsewhere 十 signs are roughly proporuonal to the mass of growth. Above experiment repeated in absence of sulphanilamide gave 十十十 in 2 Ⅱ cases in one day. infusion broth used for the stock cul- ture medium was freshly prepared in- hibition was again unsatisfactory; freshly-made medium was therefore avoided. 、、石 th these precautrons inhi- bition was com lete, with few excep- tIOns, for the uration Of the test ( 5 to 6 days). T な . Activity in antagonizing the inhibi- tory effect of sulphanilamide was fol- lowed by determining the minimum amount Of material necessary tO pro— mote full growth in the presence of a standard concentration ( 盟 / 3 30 の of the drug. ln each test the following series Of tubes were set up: ( 1 ) me- dium alone (full growth normally attained in one day or less) , ( 2 ) me- dium 十 sulphanilamide ()n duplicate) , ( 3 ) medium - ト top concentrations of test materials used ()O test for an ln- hibitory or growth-stimulating e ect) , ( 4 ) medium 十 sulphanilamide 十 test material in falling concentration from a 1 in 5 serial dilution. Tubes were incubated in air - 5 per cent C02 at 37 。 C. and readings taken daily for 5 days. lt was observed throughout that if growth occurred at , it always became maximal within 24 hours Of the appearance Of the first trace. ANTI-SULPHANILAMIDE ACTIVITY OF CELL EXTRACTS , ET CETERA The failure to obtain any significant activity with known essential sub- stances led tO a consideration Of the ossibility that the substance postu- ated might be one whose importance ln cell metabolism had not yet been recognized and a survey , as made Of cell extracts, et cetera. At this point Stamp ( 1939 ) demonstrated that ex- tracts Of streptococcal cells contained a substance which powerfully antag- onized the inhibitory action Of sul- phanilamide. As Stamp points out, this substance, which appears nor- mally to be present in the cell, may be a metabolite with which the action Of sulphanilamide is concerned. By ap- plying Stamp s method 0f extraction (incubation in / 25 NH3 at 37 。 C. ) tO baker's yeast we were ab 厄 tO ob- taln more active preparations than by Other methods. Such extracts were 引 SO more actlve than the Other cell extracts tried (TabIe Ⅱ ) , and seemed

5. Milestones in microbiology 1546 to 1940

160 lsolation Of a crystalline protern possessing the properties Of tobacco-mosarc VIrus 1935 ・Ⅳ印売〃 M. & 衂 I VIROLOGY Stanley, 、 V. M. 1935. lsolation of a crystalline protern possesstng the properties 0f tob acco- mosaic V1rus. Sc れ 0 ら VOI. 81 , NO. 21B , pages ょ CRYSTALLINE MATERIAL, WHICH HAS the properties Of tobacco-mosarc has been isolated from the juice 0f Turkish tobacco plants infected with this virus. The crystalline material contains 20 per cent. nitrogen and 1 per cent. ash, and a SOlution containing 1 milligram per cubic centimeter a positive test with Millon'S, xanthoproteic, glyoxylic acid and Folin's tyrosine reagents. The M01isch and Fehlings tests are even with concentrated solutions. 、 he ma- terial is preci itated by 0.4 saturated ammoruum su fate, by saturated mag- nesrum sulfate, or by safraninq ethyl alCOhOl, acetone, trichloracetic tannic acid, phosphotungstic acid and lead acetate. The c stalline protein iS practically insolub e in water and is soluble in dilute acid, alkali or salt solutions. solutions containing from 0.1 per cent. t0 2 per cent. Of the pro- tein are opalescent. They 2 代 fairly clear between pH 6 and 1 1 and be- tween pH 1 and 4 , and take on a dense whitish appearance between PH 4 and 6. The infectivity, chemical composi- tlOn and optical rotation Of the cr s- talline protein were unchanged a ter 10 successrve crystallizations. ln a fractional crystallization experiment the act1Vity 0f the first small po 山 on Of crystals tO come out Of solution was the same as the activity Of the mother liquor. When SOlutions are made more alkaline than about pH 11.8 the opal- 000 。 00 d P 「 000 and 市 ey b00 。 00 C 厄 ar. such SO utl()ns are devoid Of ac- tivity and it was shown by solubility tests that the protein had been dena- tured. The material is also denatured and itS acuvity lOSt when solutions are made more acid than about pH 1. lt is com letely coagulated and the ac- tl ⅵ ost on heating t0 94 。 C. Pre- liminary experiments, in which the amorphous form Of the protein was partially digested with pepsin, or par- tially coagulated by heat, indicate that the IOSS in activit iS about propor- tional tO the IOSS O native protein. The molecular weight Of the protein, as determined by two preliminary ex- perrments on osmotic pressure and diffusion, is of the order of a few millions. That the molecule is quite large is also indicated by the fact that the protein is held back by collodion filters through which proteins such as egg albumin readily pass. C0110dion

6. Milestones in microbiology 1546 to 1940

1 16 The etiology of tuberculosis [Koch's postulates. ] 1884 ・ Ro Ko THE GERM THEORY OF DISEASE Koch, Robert. 1884. Die Aetiologie der Tuberku- lose. M 耘舫〃 ge 〃 4 町イ Ka なわ e 〃 Gesund- ゐの肌ら V01. 2 , pages 1 ー 88. IT WAS FIRST NECFSSARY TO DETERMINE if characterrstic elements occurred in the diseased parts of the body, which dO not belong tO the constituents Of the body, and which have not arrsen frOI bOdy constituents. When such foreign structures have been demon- strated, it is further necessary tO as- certain if these are organized and if they shOW any Of the characteristics Of independent organisms, such as motility, growth, reproduction, and fructification. lt iS further necessary tO determine the relationships Of these structures tO their surroundings, the behavior 0f the neighboring tissue substances, the distribution Of the foreign substances in the body, their appearance in the stages Of the disease, and such similar Clrcum- stances, which 、 vould 2n0 , one tO conclude with greater or lesser prob- ability that there iS a causal relation- ship between these structures and dis- ease. The facts obtained in this study may possibly be suffcient proof of the causal relationship, that only the most sceptical can raise the ObJection that the discovered microorgamsm IS not the cause but only an accompani- ment Of the disease. However, many times this obJection has a certain va— lidity, and then it is necessary tO Ob- tain a perfect proof tO satisfy oncsclf that the parasite and the disease are not only correlated, but actually caus- ally related, and that the parasite is the actual direct cause Of the disease. This can only be done by completely separating the parasite from the dis- eased organism, and from 2 Ⅱ of the products 0f the disease which could be subscribed tO a disease-inducing in- fluence, and then introducing the iSOlated parasite intO healthy organ- lSms and induce the disease anew With all its characterlstic s.ymptoms and properties. An example will clarify the above statements. If the b100d of an animal dying Of anthrax is examined, one finds in it a large number Of reg- ular, rod-shaped, colorless, immotile structures. lt is not directly evident that these rods are plant-like, since in fact they were first taken by manv tO be non-living, crystalline structures. On ツ when it was possible to watch these structures grow, fO ロ spores, and then form new rods from the spores, could it be concluded with cer- tainty that they were living and be- longed to the class 0f lower plants. Further, if the rod-containing blood of an animal which had died of an- thrax was lnoculated in an extremely minute quantity intO another animal,

7. Milestones in microbiology 1546 to 1940

G 川・ T d ~ er 2 〃 al 立〃 g Of SC ゐ 0 〃リじ e オ e ″ e ゞ ec 〃 0 れゞ 215 ships with a typical fungus which reproduces either through basidio- develops a filamentous mycelium and spores or ascospores. CO ″′肥れ This paper illustrates some clear think- ing regarding the problem 0f bacterial taxonomy. Considering the limited knowledge of the times and the absence Of pure culture methods, it IS amanng that Cohn was able to analyze the prob- lem as accurately as he did. Throughout the nineteenth century 2 controversy raged regarding the varra— bility of bacteria. Some workers thought bacteria were highly variable (pleo- morphic ) and that all of the different fO ロ S that could be seen under the mi- croscope were different stages Of one species. USing modern genetlC concepts, this would mean that 2 Ⅱ bacterial cells contained exactly the same genes, and the different appearances which they sometrmes revealed were due tO envlron— mental influences. Other workers felt that different forms Of bacteria were ac- tually separate species with different gen- etic backgrounds. Cohn belonged to this latter group and presented his case here. The controversy could not be ended until the pure culture methods of Koch became available (see page 101 ). Only then could it be shown that different bacterial types bred true and could be considered separate species. COhn'S at— tempt here tO delineate several bacterial tribes and genera was premature but set the stage for later discoveries. The problems of bacterial taxonomy are not yet SOlved tOday. Our current bacterial classification, as presented in Bergey's Ma ″ of De 酣ビ Bac- ー e 0 / 0 , was devised on the assumption that genetlC recombination between bac- teria did not occur, making classification strictly artificial. We know now that genetlC recombination can occur. Future taxonomlC studies Will have [ 0 attempt [ 0 include this concept. Thus some day Cohn's objection that: "The genera of bacteria dO not have the same significance as do the genera of higher plants and ammals, since bacterra only reproduce by vegetauve reproduction, not sexu- 2 Ⅱ y , ” will no longer be valid. The differential staining of Schizomycetes ln trssue sectlons and in dried preparatlons 788 イ・ C な〃 4 〃 G 腕 Gram, C. 1884. Ueber die isolirte Färbung der Schizomyceten ⅲ Schnitt-u nd Trockenpråparaten ・ FO なな der Medicin, VOI. 2 , pages 185 ー 189. (I WISH TO THANK HERR DR. RIESS, Director of the GeneraI HospitaI of the city of BerJin for the facilities and equipment t0 perform the following studies. ) The differential staining method of

8. Milestones in microbiology 1546 to 1940

102 the inoculation Of an experimental ani- 1 al from a diseased animal tO transfer an infectious disease. Naturally in this procedure one has tO make several assumptions, Of WhiCh the first is that the culture vessel is really sterile. How lightly this sterili- zation has occasionally been treated can be seen from the controversy be- tw ℃ en pasteur and Bastian on spon- taneous generatron, and the well- known question Of the former tO the latter: "Flambez-vous VOS vases avant de vous en servir? ” which Bastian had tO answer in the negative. Second, one must assume that the sterile cotton is really mold-proof. As NägeIi has shown, this is not always the case. Third, it must be assumed that the nutrient liquid is bOth sterile and suit- able for the growth of the organism ln questlon. Fourth, it must be assumed that the substance used as rnoculum contalns no Other mcroorganisms than the one desired. Even a slight contamination Of the inoculum with another species which is faster growing than the or- gamsm desired will prevent anyone from ever obtaining 2 pure culture. Buchner has therefore developed his own methOd for the preparation Of an lnitial material for hiS studies on the anthrax bacillus. He inoculates the nutrient medium with such a high dilution Of anthrax infected material that, through calculations, it can be assumed that only one bacillus is placed in each culture vessel. Then from the characterlstlC macroscoprc appearance of the developing culture he concludes that he has obtained a pure culture. [This method also has diffculties which I shall go into later. ] "Do you flame your glassware before ナ [This is Lister's method; see page 58. ] THE 見 R 、愛・ rHEORY OF DISEASE Fifth, it has to be assumed that dur- ing the initial inoculation and alSO in the subsequent inoculations, that no foreign organism gets intO the culture liquid from the air. This is a danger which the experimenter will find dif- ficult to prevent with certainty, even when the protecting COtton plug is exposed t0 the air for only a very short time. Even if the first, second, and third transfers have success— ful, the probability that the culture will get contaminated will increase with the number Of transfers. ln order tO circumvent this eventuality as much as possible, it is customary tO prepare a number Of replicates, and only use those for further inoculations which appear by macroscopic or nucroscopic observatlon tO be pure. Unfortunately one cannot even rely on this proce— dure, because the macroscopic differ— entiation Of several cultures IS very uncertarn, and even the mtcroscopic examination iS fraught with diffculties, since one only knows that the very small drop of culture fluid under the mcroscope iS free Of contaminating organisms, and, as well, if the amount Of contamination IS small, there may be only occasional contaminants amongst the large number Of organ- lsms, and this makes them quite easv tO miss. Therefore the first initiation Of contamrnatlon cannot be distin- guished either macroscopically or mi- croscopically, and if one by chance uses for further inoculations 2 culture which is presumed to be pure but WhiCh has alreadv become contami- nated, and the contamlnating organisms are able tO overtake the experimental organism, then the pure culture IS completely lost. The microscope will reveal in the next generatlon, Without a doubt, that the culture IS contam- inated, but no ! lt iS t00 late, because it is impossible at this time tO rid oneself Of the uninvited guest.

9. Milestones in microbiology 1546 to 1940

40 SPONTANEOUS GENERATION AND FERMENTATION move With a gliding motion. During this movement, their bOdies ・ rigid or make slight undulations. They spin, balancing or quivering actively the two ends of their bodies. The undulatory nature Of their movements becomes very ObVious When they are longer than 0.015 mm in length. Fre- quently they are bent at one end, sometimes at bOth ends. ThiS latter is seldom seen when they are young ・ They reproduce by binary fission. lt is apparently because 0f their mode Of reproduction that they occur Often in chains. One Of the units attached tO Others may move quickly several times in order tO detach itself. These infusorn can be inoculated in the same way as beer yeast. They multiply if the medium is suitable for their nutrition. But it should be stated that they can be inoculated intO a medium containing only the crystalliz- able and mineral substances sugar, ammonrum, and phosphates, and they can reproduce simultaneously With the rapid appearance 0f the butyric acid fermentation. The weight 0f cells that are formed is significant, although 引 - ways small compared t0 the total quantity 0f butyric acid formed, but this is true for all ferments. The existence Of infusoria 、 VhiCh are able tO bring about fermentatlons IS already a notable fact. But in addition [These organisms are not animals, as Pasteur supposed, but small motile bac- teria. ] another unusual aspect should be men- tioned. ThiS iS that these infusorial animals are able t0 live and multiply indefinitely in the complete absence Of air or free oxvgen. infusona can not only live in the absence Of air, but air actually kills them. If a stream of carbon d ト OXide is passed through a medium which they are multiplying, their vi- ability and their reproduction are not affected in the least. On the contrar under the same conditions, if one su stltutes a stream Of air for the carbon dioxide, in one or ハ VO hours they 2 Ⅱ die, and the butyric acid fermentation WhiCh requires their viability iS stopped immediately. We have arrived therefore at the followincr double proposition: 1. The ferment which produces butyric acid iS an infusorium. 2. 、 his infusorium lives in the C01 - plete absence of free oxygen ・ I believe this is the first example known Of an animal ferment, and 2k0 the first example of an animal living in the absence Of free oxvgen. We will have to consider how the relationship 0f the mode 0f life and the properties Of thesc small animals, togethcr with the same aspccts Of the plant ferments which can 引 SO live without free oxvgen, arc rclatcd tO the processes Of fermentation. ln the meanwhile I would like to make no further commcnt on the ideas 、 vhich these new facts suggest until further rescarch has been done. CO 〃〃 e 〃 t This is the first report that any or- gamsm can live and reproduce in the complete absence 0f free oxygen. This discovery iS quite important for general biology, since it shows that oxygen gas IS not 2 requisite for life. This discovery opened up for Pasteur a new field 0f study, relating fermentative and biologi- cal processes tO the prescnce or absence of oxygen. lt led to his discovery that yeast can live either aerobically or an— aerobically, and that the yeast diffcrs in function under these [ 駅 ' 0 conditions (see page41) ・ There are a large number ()f organisms now known that will grow under an-

10. Milestones in microbiology 1546 to 1940

S じ衂〃・ / な 0 0 〃 0 角 rn 厩酣衂イ 24 可 4 じ〃 0 れ ( 3 ) ln grape juice the development Of gas iS a sign Of fermentation, and shortly thereafter appears a character- 1StiC filamentous fungus, WhiCh can be called a sugar fungus. 物 Throughout the duratlon Of the fermentation, these plants grow and increase number. ( の If ferments which already con- tain plants are placed in a sugar SOlu- tion, the fermentation begins very quickly, much quicker than when these plants must first develop. ( 5 ) Poisons which only affect in- fusoria and dO not affect lower plants ( E ェ . ー″じな 0 . ゞ p 耘 . ) prevent the manifestations Of putrefaction which are character1Stic Of infusoria, but dO not affect alcoholic fermentatron or putrefaction with molds. poisons which affect bo 市 animals and plants ・ [ No 記 the d erivation here of the Latin name Sacc 4 ′ 0 ~ ツ ce Which iS the genus name for common yeast. ] (arsenic) prevent putrefaction as ℃Ⅱ as alcoholic fermentation. 、 he connection between the alco- holic fermentation and the develop- ment of the sugar fungus should not be misunderstood. lt is highly probable that the development of the funous causes the fermentation. Because a nitrogen containing substance is alSO necessary for the fermentatron, it ap- pears that nitrogen iS necessary for the life of this plant, as it is probable that every fungus contains nitrogen. The alcoholic fermentatron must be considered tO be that decomposition which occurs when the sugar fungus utilizes sugar and nitrogen containing substances for itS growth, in the proc- ess Of which the elements of these substances which dO not go into the plant are preferentially converted into alcohol. MOSt Of the observations on the alcoholic fermentation fit quite mcely with this explanation. CO ″れ t From the simplicity and clarity of Schwann's observatlons, 1 [ seem obvious that his conclusions should quickly become universally accepted. However, this was not to be, as we shall see. ln his first experiment Schwann confirms Spallanzani S experiments, at the same tlme ans 、 vering any question that might arise about the availability of oxy- gen for the putrefaction process. He devises an ingenious SYStem tO prevent any unheated air from reaching hiS ves- sels, which surely required considerable patience and endurance. Many later 、 vorkers could not confirm Schwann's experiment, and this may have been due merely [ 0 their lack Of care running the experiment. Schwann then proceeds tO draw the conclusion that putrefaction is a by-product of the process of the growth Of organisms which use the or- ganiC materials as fOOd. He makes clever use of general and specific metabolic poisons in deciding What organisms were responsible for what phenomena. HiS experiments alcoholic fermen— tation are alSO outstanding. He was the first to observe yeast in the process of growing. He accurately describes this process, and draws the ObVious conclu— sion that this growth which he sees of a living organism. He shows that 21- COhOliC fermentation and the appearance Of yeast cells are always associated events, and draws the conclusion, later denied by Liebig, that the yeast cells make the alcohol. Schwann was many years ahead of his tlme, and it was not until Pasteur's work of 1860 that the scientific world gen- erally accepted these early conclusions. Two years after the preceding paper was published, Schwann published his classic work, "Microscopical researches on the similarity in structure and growth Of animals and plants," in which he de- scribed for the first time the cellular nature Of the higher animals.