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1. 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.

2. Milestones in microbiology 1546 to 1940

/ e 〃〃 e ・ The じ 4 〃ゞゞ 4 〃 d e が ec な Of オわ e 04 ー 0 な 0 じー〃 4 ビ 0 co 20 125 country, it not very unfrequently produces deformity of the skin, and sometlmes, under the best manage— ment, proves fatal. ・ These cn•cumstances must naturally create every instance some degree Of painful solicitude for its conse- uences. But as I have never known atal effects arise from the cow-pox, even 、 Vhen impressed in the 1 os [ un— favourable manner . . and as it clearly appears that this disease leaves the constltution in a state of perfect security from the infection of the Small-pox, may we not infer that a mode Of lnoculation may be intro- duced preferable to that at present adopted, especially among those families, WhiCh, for previous C1rcum- stances we may )udge to be redis- posed tO have the disease un avour- ab ly? Thus far have I proceeded in an inquiry, founded, as it must appear, on the basis of experiment; in which, however, has been occa— sionally admitted in order to present tO persons 、 vell situated for such discusslons, ObjectS for a more mrnute lnvestigation. ln the mean tlme I shall myself continue to prosecute this inquiry, encouraged by the hope of its becoming essentially beneficial to mankind. CO ″″〃 e 亡 Smallpox 込 a disease that has been known Slnce antiquity. lts symptoms are characterlStic, and because 0f this, it was easy tO observe lts transfer and to deter- mrne that it was infectious. We know today that it is caused by a large filter- able virus, although we use the word virus ln a different sense than did Jen- ner. The process of inoculation had been practiced for centuries in the Far East. lt was introduced into England from Turkey by Lady Mary Montagu, the wife of the British ambassador to Tur- key. Although the process first was greatly resisted, it eventually became es— tablished in EngIand. But because com- plications Often lnoculation ' as not without lts dangers. TOday we wouid consider cowpox Vlrus tO be 2 mutant Of smallpox vrrus which has 10S [ some of its virulence for man. lt still retains the ability to induce the production Of virus-neutralizing an- tibodies in man. lt is this fact which makes the process Of inoculation with cowpox possible. This process was called vaccrnation = COW), and this word has later been applied to all arti- fiCial immumzation procedures. Jenner's observatlons are qulte acute. He was able to bring together 2 number Of diverse facts, and in so d0ing, at a theory which seemed reasonable. He was fortunate that he could readily test his theory and clearly show that he was right. lt is interesting that Jenner attempted tO publish in the Transactions of the Royal Society the results of his first case. When the work was rejected, he then collected twenty-three cases and published his book. Because of the sim- plicity Of the vaccrnation procedure and its high degree of safety, it has eventu- ally become accepted as 2 common medi- cal procedure, although Jenner met much resrstance when his book , as first pub- lished.

3. Milestones in microbiology 1546 to 1940

Ko じ・ T ビ e 〃 0 ル g ア可 0 prepared by adding agar-agar t0 2 meat infuslon or peptone medium. However, on this medium the bacilli form 0 司 y irregular small crumbS' WhiCh are not nearly SO characteris— tlC as the growths on blOOd serum. Originally I cultivated the tubercle bacilli only from lung tubercles 0f guinea pigs which had been infected with tubercular material. Therefore the cultures from varrous sources had first tO pass through the intervening stage of the guinea pig before they were obtained in pure cultures. ln this way there was a possibility for error, in the same , ay as in the transfer Of a culture from one test tube tO another. This might occur through the accl- dental inoculation Of Other bactena into the animal, or through the ap- pearance in the guinea pig Of spon- taneous tuberculosis. ln order tO avoid such errors, special precautlons are necessary, which can be deduced from observations on the behavior Of this spontaneous tuberculosis. From hundreds of guinea pigs that have been purchased and have oc- casionally been dissected and exam- ined, I have never found a single case Of tuberculosis. Spontaneous tu- berculosis develops only occasionally and never before a tlme Of three or four months after the Other animals in the room have been infected with tuberculosis. ln animals which have become SiCk from sponstaneous れト berculosis, the bronchial glands be- come quite swollen and full 0f pus, and in most cases the lungs ShOW a large, cheesy mass With extensive de— composition in the center, SO that it occasionally resembles the similar . An- processes in the human lung. imals that have been lnoculated with tuberculosis show a completely dif- 蓄 [Koch did not at this time seem to be aware of the superiority Of agar as a solidify- ing agent•l 113 ferent picture. The place of inocula- tion Of the animals iS in the abdomen 、 close to the inguinal gland. This first becomes S 、 VOllen and gives an early and unmistakable indication that the lnoculation has been a success. Since a larger amount Of infectious material is present at the begmning, the infec- tion progresses much faster than thC spontaneous infection, and in tissue sectlons Of these animals, the spleen and liver ShOW more extensrve changes from the tuberculosis than the lun . Therefore it is not at all diffcult to differentiate the artificially induced tuberculosis from the spontaneous tu- berculosis in experimental animals. From a consideration Of these facts, it can be concluded that the develop- ment Of tuberculosis an experi— mental animal iS due tO the actlon Of rnoculated material, When a number of guinea pigs are purchased and in- oculated at the same tlme in the same way with the same material, and kept separated from Other animals in their own cage, and when they show the development Of the characterlstrc tu- berculosis symptoms Of inoculated an- imals in a short periOd Of time. ln this way, a substance can be tested for its virulence by inoculating four tO SiX guinea pigs with it, after making use Of 2 Ⅱ precautions, such as previously disinfecting the site of in- oculation, sterile instruments, etc. The results are uniformly the same. ln all animals which are lnocu- lated With fresh masses containing tubercle bacilli, the small inoculation site has almost always coalesced on the next day, then remains unaltered for about eight days, then forms a little nodule which may enlarge without breaking open, although it most often changes into a flat, dry abscess. After about two weeks, the inguinal glands and axillary glands on the side where the inoculation has occurred enlarge

4. Milestones in microbiology 1546 to 1940

126 The attenuatlon Of the causal agent Of fowl cholera IMMUNOLOGY The 0 g わ za / F 7 ルわ 4 れな叩 2 ”舫月 ppe 市も 24g 26 ター 272. ノ 8 砌・ん 0 〃な P ″ Pasteur, Louis. 1880. De l'atténuation du virus du choléra des poules. CO d 町 de / de 川 イ〃 c , 26 Oct0ber 1880 , VOI. 91 , pages 673 80. I WOULD LIKE TO REITERATE THE following results, which I have pre- viously had the honor 0f presenting t0 the academy: 1. FOWl cholera is a virulent disease of the first order. 2. 、 he virus conSlStS Of a nucrO— scopic parasite which multiplies read- il メ in culture away from the animal body. From this it1S possible t0 obtain the virus in a state Of purity and dem- onstrate irrefutably that it is the SO 厄 cause Of the disease and death. 3. 、 he virus may vary in itS viru- lence. At times the disease is followed by death, while at other times, after causing disease s.ymptoms Of variable lntenslty, recovery occurs. 4. These differences in the virulence Of the virus are not merely the result Of natural variatlons, as the menter can alter them at his will. 5. As is generally the case for virulent diseases, fOWl cholera does not recur, or rather the recurrence iS Of tensity with that Of the earlier infec- tion, and it iS always POSSible tO extend *[The causal agent of ん wl cholera is not a filterable virus, but a bacterium, no 、 known as ~ お el ″″ oc 4. ] the resistance SO far that inoculation With the most virulent VIrus does not produce any effect. 6. Without wishing to make a defi- nlte assertlon on the relationship be- tween the small pox and the COW pox Vlruses, lt seems from the above facts that in fOWl cholera, there exists a state Of the virus relative tO the most virulent virus, WhiCh actS in the same way as CO ' POX Virus relation tO small POX virus. COW POX virus brings about a benign illness cow POX, WhiCh immumzes against 2 very senous illness, small POX. ln the same , ay , the fOWl ChOlera Vlrus can occur ln a state Of virulence that is ciently attenuated, SO that it induces the disease but does not bring about death, and in such a way that after recovery, the animal can undergo an lnoculation With the most virulent virus. Nevertheless, the difference be- tween small pox and fowl cholera is considerable, in certarn respects, and 1 [ IS not amlSS tO remark that, with respect tO an understanding Of the principles, studies on fowl cholera will probably be mo 代 helpful. Whereas there iS still a dispute about the rela- tionships between small POX and COW

5. Milestones in microbiology 1546 to 1940

106 when converted intO nutrlent gelatin is easy or hard tO sterilize. Many, as for example alkaline urrne or Pasteur's fluid, are easy tO sterilize in the form Of nutrient gelatin. Others like meat extract or hay infusion are much more diffcult; one has to boil them daily for several days. This is because not 2 Ⅱ of the spores germinate at the same time. Occasional single colonies Will develo in the center Of the gelatin even ays after the last boiling, and their position shOWS that they were ⅲ there from the beginning and did not arrrve later. ー{0嶬℃ ver, if thiS should be the case, frequent examlna- tion Of the nutrient gelatin in the first week will allOW one tO notice these early enough and they can then be killed by another boiling. This fre- quent examination in the first week should never be omitted. Because it iS SO simple and certain tO prepare pure cultures using POtatO slices, I have preferred to prepare the nutrient gelatin in a similar form as a POtatO slice. lt can be poured intO flat watch glasses, small glass plates or the like. However, the most use- ful for the preparation of cultures, and especially for the microscopic ex- amination Of these, IS tO spread the nutrient gelatin as 2 long, wide drop on a mlcroscope slide, in WhiCh form it can be placed under the microscope When SO desired. ThiS iS done 、 vith a previously sterilized pipette, and 0f course the microscope slide iS previ- ously cleaned and sterilized by pro- longed heating at 150 。 C. The drops are about tWO millimeters thick. The gelatin hardens ln a few mrnutes and the slides are placed on a small glass shelf which will hold two or three slides next to each other. FinaIly a number of these shelves are placed in layers over each other and placed Under such ln a moist chamber. conditions the gelatin drops can be THE R 、 THEORY OF DISEASE kept two or three weeks before they dry out. The organism to be cultured is seeded by taking a flamed needle or platinum wire, picking up a very small quantity of the liquid or substance containing the organisms, and streak- ing this in three tO SIX cross lines on the gelatin surface. . The expres- SIOn ・ lnoculation" for thiS operation seems appropnate. The bell jar which serves as the moist chamber iS suffcient protectlon from contamrnatlon, even though it does not tightly. lt sometimes hap- pens that foreign organisms may fall on the gelatin during inoculation or manipulation 0f the slides. But these can only develop at the place on the gelatin where they have fallen and this iS usually not on the inoculation streak. lt is hardly possible that all of the cultures Of an organism will be- come contamlnated SO that thev can- not be transferred further, and this possibility is even more reduced if the bell jar is not opened often. Within a few days the pure cultures have de- veloped tO their maximum extent and can be inoculated further. There iS no purpose in allowing the cultures tO stand a long time, and this iS especiallv true when the bacteria being cultured are able to liquefy the gelatin, or when sporulation has set in. ln these cases a quick transfer iS necessarv. If it iS necessary tO keep single cultures for a long time without transfer, then it is necessar tO keep them in a contarner enclose with CO は on. At low temperatures the develop- ment Of the cultures proceeds quite S10 , 1 ! , and manv organisms require a certaln warmth in order tO proliferate well. The most luxuriant growth in gelatin cultures has been at 20 ー 25 。 C. , and I have not found anv organisms yet which are at 団 culturable, which could not grow 2 [ this temperature. However, if it iS necessary tO use tem-

6. Milestones in microbiology 1546 to 1940

124 care Of a horse affected with diseased heels. The active quality 0f the virus from the horses' heels iS greatly increased after it has acted on the nipples 0f the cow, as it rarely happens that the horse affects hiS dresser With and as rarely that a milk-maid escapes the infection when she milks infected CO る . lt is singular tO Observe that the Cow-pox virus, although it renders the constitution unsusceptible Of the 010uS , should, nevertheless, leave it unchanged With respect tO its own ac¯ tion. I have already produced an in- stance tO point out and shall no 、 v corroborate it With another. Elizabeth Wynne, wh0 had the Cow-pox in the year 1759 , was inocu- lated with variolous matter, without effect, in the year 1797 , and again caught the Cow-pox in the year 1798. lt is curious alSO tO observe' that the vrrus, which with respect tO its effects IS undetermined and uncertain prevl- ously tO itS passing from the horse through the medium 0f the should then not only become more active, but should invariably and com- pletely possess those specific properties which induce in the human constltu- tion symptoms similar tO those Of the variolous fever, and effect in it that peculiar change WhiCh for ever renders it unsusceptible Of the variolous con- tacnon. May it not, then be reasonablv con- )ectured, that the source 0f the Small- pox is morbid matter Of a peculiar k'ind, generated by a disease in the horse, and that accidental circum- stances may have and again arisen, still working changes upon it, until :t has acquired the conta- gious and malignant form under which we now commonly see it making its devastations amongsth us? from a consideration Of the change which IMMUNOLOGY the infectious matter undergoes from producing a disease on the COW' may ℃ not conceive that many contagious diseases, no 、 V prevalent among may owe their present appearance not tO a simple, but tO a compound origin? For example, is it diffcult tO imagine that the measles, the scarlet fever, and the ulcerous sore throat with a spotted skin, have all sprung from the same assuming some source, their fO ロ s according tO the nature Of their new combinations? 、 he same question will apply respecting the Of many Other contagious dis- eases, which bear a strong analogy tO each Other. At what period the Cow-pox was first noticed here iS not upon record. ()ur Oldest farmers were not unac— quainted with it in their earliest when it appeared among their farms without any deviation from the phae- nomena which it now exhibits. ltS con- nection with the Small-pox seems tO have been unknown tO them. Prob- ably the general introduction Of inoc- ulation first occasioned the discovery. lts rise in this country may not have been Of very remote date, as the prac— tice of milking cows might formerly have been in the hands Of women only; which I believe is the case now ln some Other dairy consequently that the COWS might not in former times have been exposed tO the contagious matter brought by the men servants from the heels Of horses. Should it be asked whether this in- vestigatlon IS a matter Of mere cur ト OSity, or whether it tends tO any bene- fic purpose? I should answen that notwithstanding the happy effects 0f lnoculation,t with 2 Ⅱ the improve- ments which the practice has received srnce its first introduction intO this ・ [This idea is now known to be wrong ・ 1 十 [ Meaning inoculation with smallpox it-

7. 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.]

8. Milestones in microbiology 1546 to 1940

90 sought tO determine whether these bOdies are the unique contagium Of anthrax. Recently Davaine has carried out a number Of inoculation experl— ments with fresh and dried blood con- taining the rods and has stated deci- sively that these rods are bacteria, and that only in the presence Of these bacteria can a fresh case Of anthrax be produced. The lack of proof 0f the direct transmrssron Of the anthrax diS- ease ln man and animals iS due tO the ability Of the bacteria tO remain alive for a long tlme in dry conditions and t0 be transmitted through the air by insects and the like. lt seems here that the mode Of transnusslon Of anthrax has been explained. Nevertheless, these ideas Of Davaine have found many opponents. Several workers have obtained experimental anthrax by inoculating b100d contain- ing bacteria, but have been unable tO shOW the presence Of bacteria in the b100d of the diseased animals. Others have been ab 厄 tO induce anthrax by inoculation with blood which could not be shown tO contain bacteria, but the diseased animals then had bacteria in their b100d. Others have noted that anthrax is not derived SOlely from a contagium WhiCh iS transmitted above ground, but that this disease IS related in some way with conditions of the soil. 、 hese experiences cannot be ex- plained by the hypothesis 0f Davaine, and because of this, many people feel that bacteria are Of no significance for anthrax. Since I have had the opportunit several times Of examimng amma S which had died of anthrax, I per- formed a series Of experiments WhiCh would clear up the uncertainties in the etiology 0f anthrax. Through these, I came tO the conclusion that the theory Of Davaine concerning the transmission Of anthrax iS only partly correct. THE GERM THEORY OF DISEASE I could show that the rods in the anthrax blood were not SO resistant as Davaine had believed. As I will show later, the blood, which contains only rods, keeps its ability tO induce anthrax on inoculation only a fe 、 V weeks in the dry state, and only a few days when moist. HO 、 V iS it possible then for an organism WhiCh iS SO easlly de- stroyed tO maintarn itself as a dormant contagrum for a year ln SOil and throughout the winter? If bacteria are really the cause Of anthrax, then we must hypothesize that they can go through a change in life history and assume a condition which ′ i Ⅱ be re— sistant tO alternate drying and I 月 01S - ture. What is more likely, and what has already been indicated by Prof. COhn is that the bacteria can form spores which possess the ability tO re- forl れ bacteria after a long or short resting periOd. AII of my experiments were de- signed tO discover this developmental stage Of the anthrax bacterium. After many unsuccessful expenments, I was finally able t0 reach this go 乢 and thus to find a basis for the etiology 0f anthrax. Since the life history of the anthrax bacterium Offers not only botanical interest but 引 SO much light on the heretofore uncertain etiology Of the soil-related infectious diseases, I 田れ publishing now the most important results of my experiments, although my work is still in progress. ll. LIFE HISTORY OF Ba じ〃 I ″ゞ 4 〃 th ac な lt has not been possible for me t0 Observe the multiplication Of the bac- teria directly in the animal. But it can be inferred that this occurs from the lnoculation experiments WhiCh fO Ⅱ 0 、 V. I have used the mouse as mv experi- mental animal, as it IS simple tO use. ln most experlments I inoculated them at the base of the tail, where

9. Milestones in microbiology 1546 to 1940

98 methods which he has used for the mrcroscopic Of blOOd and tlssues. The important points in this are the use Of aniline dyes for and the use of the Abbé condenser as a light source. He then describes his experiments 、 Vith infections in mice and rabbits, in which he has been ab 厄 t0 infect these animals by lnoculating them, and then them for the presence Of bacteria. ln the diseases studied—progressive de- structlon Of tissue (gangrene) in mice, septicaerma ln and spreading scess, pyaemia, and ery— sipelas in rabbits—he has been able t0 ShOW the continual presence Of unique types of bacteria whenever he has dis- eased animals. He mentions alSO hiS work with anthrax which showed the same thing. ] CONCLUSIONS AS regards the artificial traumat1C infective diseases observed by me, the conditions, WhiCh must be established before their parasitlc nature can be proved, were completely fulfilled in the case of the first five, but onlv par- tially in that of the sixth. For the in- fection was produced b such small quantities 0f fluid (bloo , serum, pus' etc. ) that the result cannot be attrib- uted tO a merely chemical poison. ln the materials used for inoculation bacteria were without exceptlon pres- ent, and in each disease a different and well-marked form of organism could be demonstrated. At the same time, the bOdies Of those animals which died of the artificial traumatlC infective diseases contained bacteria in such numbers that the sym- toms and the death Of the animals were ー 0 、 plained. Fu , , h00 , 市 0 suffcientl those which were present in the fluid used for inoculation, and a definite THE GERM THEORY OF DISEASE form Of orgamsms corresponded in every instance tO a distinct disease. . even in the small series Of ex- periments which I was able t0 carry out, one fact 、 vas SO prominent that I must regard it as constant, and, as it helps tO remove most Of the Ob- stacles tO the admissron Of the existence Of a CO 〃 tag ー″〃 Z VI 7 〃 for traumat1C infective diseases, 1 100k on it as the most important result Of my wor 長 . I refer tO the differences which exist between pathogenic bacteria and tO the constancy Of their characters. . A. distinct bacteric form corresponds, as we have seen, tO each disease, and thiS form always remains the same, hOW- ever Often the disease iS transmitted from one animal tO another. Further, When we succeed in reproducing the same disease de 000 by the inJection Of putrid substances, 0n1 ! the same bacteric form occurs whICh was be— fore found t0 be specific for that disease. Further, the differences between these bacterra are as great as could be expected between particles which border on the invisible. With regard tO these differences, I refer not only tO the size and form Of the bacteria, but alSO tO the conditions Of their growth, which can be best recognized by observing their situation and group- ing. I therefore study not onlv the individual alone, but the whole group of bacteria, and would, for example, consider a mrcrococcus which in one species Of animal occurred only in masses ( え巳 , in a zooglaea form) , as different from another which in the same variety Of animal, under the same conditions Of life, は S onlV met With as isolated individuals. AS, however, there corresponds tO each Of the diseases lnvestigated a form by its physiological action, by its con- ditions Of growth, size, and form,

10. Milestones in microbiology 1546 to 1940

114 until they are the size Of peas. From then on the animals become progres- sively weaker and die after four tO SIX weeks, or are killed in order tO exclude the later development 0f spon- taneous tuberculosis. ln the organs Of all of these animals, and most espe- cially in the spleen and liver, the rec- ognizable changes due tO tuberculosis occur. That these changes in the guinea pigs are only due tO the inocu- lation Of material containing the tuber- cle bacilli, can be see from expenments lll WhiCh inoculation was performed with scrofulous glands or fungus masses from JOintS, in WhiCh no tuber- cle bacilli could be found. ln these cases, not a single animal became SiCk, while in the animals inoculated with bacilli-containing material, the inocu- lated animals always showed an exten- SlVe infection With tuberculosis after four weeks. CuItures of tubercle bacilli were prepared from guinea pigs which had been lnoculated [ h tubercles from the lungs 0f apes, with material from the brain and lungs 0f humans that had died of miliary tuberculosis,* with cheesy masses from phthisistic lungs, and with nodules from lungs and frOI the peritoneum Of COWS af- fected with bovine tuberculosis. ln all these cases, the disease processes OC— curred in exactly the same way, and the cultures of bacilli obtained from these could not be differentiated in the slightest way. ln all, 15 pure cultures were made of tubercle bacilli, four from guinea pigs infected with ape tuberculosis, four with bovine tuber- culosis, and seven With human tuber— culosis. ln order tO answer the obJection that the nature of the bacilli was changed through the preliminary in- ・ [An acute, systemic form of the disease. ] 、 HE 見 R 、愛 THEORY OF DISEASÉ oculation intO guinea pigs, SO that they became more similar, experiments were set u tO cultivate tubercle ba- cilli direct from spontaneous cases man and animals. 、 his was successful a number Of trmes, and pure cultures have been Ob- tained from the lungs of two people with miliary tuberculosis, as well as one With cheesy pneumonia, tWice from the contents Of small cavrtles phthisistic lungs, once from cheese- like mesenteric glands, twice from freshly removed scrofulous glands, tWice from lungs Of COWS With bOVine tuberculosis, and three tlmes from the lungs of guinea pigs that had suf- fered spontaneous tuberculosis. AII 0f these cultures were quite similar and also resembled those that had been isolated through the preliminary guinea pig inoculation, SO that the identity of the bacilli occurring in the varrous tuberculous processes cannot be doubted. Up until now my studies have shown that a characteristic bacillus iS always associated with tuberculosis, and that these bacilli can be obtained from tuberculous organs and iSOlated ln pure culture. lt no , remained tO prove the most important question, namely, that the isolated bacilli were able to bring about the typical tuber- culOSiS disease process When inocu- lated again intO animals. The results 0f a number of inocula- tion experiments with bacillus cul- tures inoculated intO a large number Of animals, and inoculated in different ways, 2 Ⅱ have led to the same results. Simple iryections subcutaneouslv, or intO the peritoneal cavity, or intO the anterlor chamber Of the eye, or di- rectly into the blood stream, have all produced tuberculosis with only one exception. Further, the infection was not limited to only isolated nodules,