CORNEL L UNIV ERSITY THE Sflotucr Hctcnnary Slibrarg FOUNDED BY ROSWELL P. FLOWER for the use of the N. Y. STATE VETERINARY COLLEGE 1897 Cornell University Library QR 6S.H88m The methods of bacteriological investiga 3 1924 000 340 046 I Cornell University ^ Library The original of tiiis book is in tine Cornell University Library. There are no known copyright restrictions in the United States on the use of the text. http://www.archive.org/details/cu31924000340046 THE METHODS OF BACTERIOLOGICAL mYESTIGATIOI^. BY Dk. fekdinand htjeppe, SOOBNT nf HYGIEfiE ANT> BACTEKIOLOGT IN THE GHEMIOAL LAB0BAT0B7 OF B. TBE8EN1UB AX WIESBADEN. TRANSLATED BY • HERMANN M. BIGGS, M.D., msTsnoioB in the oabneoie xabobatoby, and assistant to the obaib op patho- I.OGIOAL ANATOMY IN BSLLEVUE HOSPITAL UEDICAL COLLEGE. ILLVSTBATBD BY THIBTY-ONE WOOD-OUTB. NEW YORK: D. APPLETON AND COMPANY, 1, 8, AND 5 BOND STREET. 1890. A/<5- /^^^/ COPYBIQHT, 1886, By D. APPLETON AND COMPAKT. DEDICATED IN THANKFUL RESPECT TO THE GEHEIMEK EEGIEKUlfGSEATH, DE. EOBEET KOCH. AUTHOR^S PREFACE. Urged by the wish of my highly esteemed teacher, the Geheimrath Koch, I have attempted in the fol- lowing work to meet the lack of a comprehensive representation of the methods of bacteria-investiga- tion. It was my endeavor, as an historical and ex- perimental critic, to sift the whole of the literature, which was extraordinarily scattered and in part very difficult of access, and to select the good from the hardly conceivable confusion of useful and useless communications, in order to give to the iadependent investigator a useful hand-book, and to the beginner a trustworthy introduction into this territory. The Aitthoe. Wiesbaden, February, 1885. TEAE-SLATOE'S PEEFAOE. This translation of Hueppe's excellent work on the "Methods of Bacteriological Investigation " was suggested by the want in English of a satisfactory text-book on this subject for the use of students working under my direction ia the Carnegie Labo- ratory. The preparation of the original was under- taken by Dr. Hueppe, at the request of Prof. Eobert Koch, and the work has been thoroughly and care- fully done. It shows a complete familiarity with the subject in hand, is comprehensive in character, and treats carefully all of the approved methods of investigation. Some difficulties have been met in the transla- tion which those acquainted with this kind of work wiU readily understand. It is literal so far as is consistent with clearness, and no attempt has been made to attain elegance in style or diction; but it is weU known that many German terms have no exact English equivalent, and can only be ren- dered accurately by a roundabout expression. This is weU illustrated by " Massenkultur " (as translat- ed by quantity-culture on page 101 et seq.), which means a culture, whether pure or not, where a great 6 TBANSLATOB'8 PBEFAOE. quantity or bulk of bacteria are growing ; but, since this expression can not be inserted each time in the text, quantity-culture wUl be used wherever it occurs. The labor of preparing the translation was per- formed in the short intervals of leisure found in the midst of numerous other duties, and was un- avoidably interrupted just before its completion, so that some errors may be found in the book, but I think that none of serious importance have escaped correction. The author has requested me to emphasize the fact "that the short, concise form chosen in the work is based on an extensive historical and ex- perimental review of the whole subject, and that if any methods, stiU much used, h^ve been omitted or only briefly considered, it is because they have not now the significance or importance that has been ascribed to them by other writers." I here desire to acknowledge my indebtedness to Dr. L. W. Hubbard and Dr. S. IST. Nelson, both of New York, for valuable assistance kindly ren- dered me in the translation. The work has been very favorably received in Germany, and if this translation meets only a small part of the same consideration in America, 1 shall feel well repaid for the labor expended. Hekmanw M. Biggs. Oaenegib Labobatoet, New Yoek, December 1, 1885. 0l!5"TE]S"TS. PA8K Inteodttotion 9 True Saprophytic Forms 11 I. Spontanboitb Gbneeation and the Peinoiples of Steeil- IZATION 15 II. FOEMS OP BaOTEEIA and MiOEOSOOPIOAL TECHlflQUB . . 28 A. The True Endospore Bacteria 29 £. Arthrospore Bacteria 29 1. Arthro-Cocci 29 2. Arthro-Bacteria 29 8. Leptothrix 29 4. Cladothrix 30 DetermiDation of the Presence of Bacteria Unstained . . 34 Staining Bacteria 40 General Principles of Staining 42 Preparation of Staining Fluids 48 Other Eeagents and Apparatus 51 Oover-glass Preparations 55 Examination for Tuhercle Bacilli in Sputum . . 61 Examination of Blood for Bacteria . . . .67 Methods of Staining the Flagella 73 Methods of Staining Spores 74 Preparation of Sections 78 III. Culttjeb-Mbthods ; Puee Oulttjees 92 1. Transparent Fluid Oulture-Media 92 2. Fractional Cultures 100 3. Opaque Solid Culture-Media 101 4. The Gelatin-Culture of Klebs and Brefeld . . .105 5. Method of Dilution 113 Method of Isolation by Heat 119 8 CONTENTS. 6. Cultures in Capillary-Tubes, after Salomonsen . . 121 7. The Infection-Methods 125 8. The Cultures upon Transparent Solid Nutrient Media, according to Koch 128 A. Transparent Solid Media made by the Addition of Gelatinizing Substances — " Nutrient Gelatin " . 132 a. Slide-Cultures . . . . . . .134 h. Plate-Cultures 138 c. Test-Tube Cultures 142 Improvised Means 146 B. Transparent Solid Media, without the Addition of Gelatinizing Substances — Blood-Serum . . . 148 IV. Inooulations fob the Dbteemination op the Cattbal Eelation of Baotbeia-Geowth to Decomposition and Disease 160 A. Septic Bacteria 160 Ana§robiosis in Fluids 164 B, Parasitic Bacteria 172 Inhalation Experiments 174 Feeding Experiments 175 Cutaneous Inoculations 177 Subcutaneous Applications 178 Subcutaneous Injections 179 Direct Injection into the Circulation .... 180 V. Geneeal Biological Pboblems 183 Enzyme 185 Ptomaines 187 Behavior to Temperatures 188 Disinfection vrith Fluids 194 Disinfection with Gases 195 Drying 197 Action of Low Temperature and High Pressure . . . 197 Electricity 198 Phosphorescence 198 Light 199 VI. Special Hygienic Investigation 200 A. Earth 200 B. Water 202 O. Mr 205 VII. Bactbeiologt as an Object of Insteitction . . . 210 BACTERIOLOGICAL INVESTIGATION. INTRODUCTION. The perfection of peculiar methods for the study of bacteria was conditioned, on the one hand, by the minuteness and rapid multiplication of these lower organisms, by reason of which the formerly approved methods scarcely at all sufficed to determine their morphology ; and, on the other, by the biological processes in which these micro-organisms take part, and which the methods for their study must also consider. In regard to their biology, the bacteria may be separated into two great groups : the septic (sapro- phytic) bacteria, which feed on dead organic bodies, and the parasitic, which are found in living organ- isms. Among the septic bacteria, the true bacteria of putrefaction should be distinguished from those which cause a more typical decomposition of life- less organic matter. In the latter it is possible to even separate chemically the products formed. These last septic forms are designated as ferment bacteria. Aside from these, the pigment bacteria also deserve special notice. 10 BACTERIOLOGICAL INYESTIQATION, Many of these septic micro-organisms need, un- der all conditions, tlie oxygen of the air for life and activity — the aerobic forms ; some species can for the time being be deprived of this, and are able even then to bring about their specific decomposition, but they can also live and multiply with a free access of air. These may be designated as the optional anaerobic (facultativ-anaerobiotische) forms. Again, of other forms it has been asserted that the absence of the oxygen of the air is necessary for their life and ac- tivity, and that they are immediately destroyed by oxygen — the obligatory anaerobic (obligat-anaerobio- tische) forms. We know of still other forms in which, directly contrary to this, a transference of the oxygen to the medium in which they grow takes place with their life activity. These cause an oxidation fermen- tation. Among the bacteria living as parasites, there are forms which do not complete their development upon the animal organism, but only occasionally appear as parasites, or pass through a portion of their exist- ence in living organisms — the facultative parasites of van Tieghem; other forms find, as a rule, only in living organisms all the conditions necessary for their existence, but can occasionally, or in certain stages of development, also live as septic bacteria — the facul- tative septic forms of de Bary. Still other forms, finally, seem to be fitted only for the parasitic mode of life, and appear quite incapable of passing any period of their existence as septic' bacteria. These are designated as the strongly obligatory parasites of de Bary. Although it is very diflicult to determine in individual cases whether a parasitic micro-organism belongs to this or that group, yet in such cases as the recognition of the minute diiferences also depends INTBODUOTIOHr. H more or less upon the subjective opinion of the ob- server, it thus permits this grouping to be regarded as much freer from constraint as to their real condition than the division of the pathogenic infectious micro- organisms into endogen and ectogen, which has ref- erence only to the extremes. These last designations permit only insufficiently, either a valuation of the investigations concerning the accessory causes of the infectious diseases, or a strong emphasis beiag placed upon one or another factor, and do not allow the at- tainment of an unprejudiced judgment upon matters all-important in the aetiology. There is also the great- est difference as to the need of oxygen among the parasitic bacteria. It is to be noted, likewise, that the parasites may be endophytic — i. e., can live in the interior of organs or cells ; perhaps also epiphy- tic — i. e., can live upon the surface. These different phenomena of adaptability can be theoretically deduced from the simple septic mode of life ; but it is to be remembered that direct inter- media,te Hnks can seldom be determined, and that individual forms can produce different actions. True Saprophytic Forms. / I \ I. Ferment hact&ria. II. Pigment bacteria. III. Parasitic bacteria. Aerobic. Facultative parasites. Ponns produc- Facultative ana§robio. Facultative septic forms, ing oxidation | | fermentation. Obligatory anaerobic. Strongly obligatory parasites. The most general problem which is presented in bacteria investigation — viz., the determination of the group to which a form of bacteria belongs — can now be quickly and accurately solved. I. It is to be determined whether; in decomposi- 12 BAGTERIOLOOIGAL INVESTIGATION. tion or disease, bacteria are present or not. This question associates itself essentially with tlie question of spontaneous generation and abiogenesis, teaches us the value and general principles of sterilization, and makes us acquainted with the indispensable re- quirements for reliable work in bacteriology. II. If bacteria are present, it is to be determined what forms they possess. This general morphologi- cal question demands special technical skill, as the ordinary histological teoJinique does not suffice. III. Each form found to be present is to be culti- vated by itseU, free from all chemical and morpho- logical admixtures— " pure cultures." The problem to be solved by the aid of pure cultures is a double one— i. e., with the help of these the general mor- phological investigation is completed and extended, and, IV. By transfers of really pure cultures to decom- posable materials or susceptible animals, it is to be determined whether the bacteria found are the cause of the decomposition or disease. By this investigation it is certainly shown to which of the described groups a form of bacteria belongs ; then, extending likewise from the pure cultures, there are yet, Y. A further series of more exact biological prob- lems to be solved later, which, in union with the first questions, afford the broad basis for theoretical con- sideration and practical treatment. The solution of all these questions is to be aimed at ; but experience has shown that this is not possible in every case, and that cases may come up in which one or the other of these cardinal questions remains unanswered. For instance, as regards the parasitic bacteria which, in the highest degree, represent the INTRODUGTIOK 13 parasitic adaptability, viz., the strongly obligatory parasitic forms, perhaps only the presence of the micro-organism may be determined, wMle pure cult- ures can not be obtained. In other forms of this group, a further step has already been taken, by which, to a limited extent, transfers to animals have been made. With individual pathogenic bacteria of the other groups, on the other hand, transfers are not successful, because as yet no species of animals has been shown to be susceptible to the disease which they produce, although pure cultures of these bac- teria have been obtained. (Compare Methods of In- fection.) In these cases the greater attention should be given to those problems that can be solved ; and it is always to be borne in miad that, with a more complete mas- tery of the methods in the seemingly most unprom- ising cases, a classical solution of all questions has sometimes been obtained. The facts ascertained by means of a perfected technique must form, in every branch of natural sci- ence, the solid foundation upon which the theories are built up. Wo investigator, although in possession of many facts, can dispense with the guiding ideas that these afford ; but the aversion, of many investi- gators to every speculation is immediately aroused, for the reason that by many the deductions of natu- ral philosophy are stated as scientific facts. Even the subject of bacteriology has in this respect passed through an experience too sad to aUow us to forget that the observations in natural philosophy can be nothing else than provisional explanations of phe- nomena not yet really understood, the value of which, however, in suggesting lines of investigation, is very great. Eesolutely must we guard ourselves against 14 BAOTERIOLOQIOAL INVE8TI0ATI0N. placing under restraint sucli deductions, often con- fused, but yet in harmony with facts ; against bring- ing into discredit the solid foundation of facts by cheap badinage of the endeavors of "learned ones" in search of "little facts"; or against citing ever anew agreeable theories or refuted statements as proved facts, as has often happened in the domain of bacteriology. Resolutely also must we see to it that speculations do not form the foundation of the practical treatment of hygiene. Against such sad errors of speculation there is no better remedy than the familiarity to be obtained with the methods (constantly becoming more accu- rate) which are so closely united to the real advances in the knowledge and possibilities regarding the caus- al relation of micro-organisms to decomposition and disease. SPONTANEOUS GENERATION AND THE PRINCIPLES OF STERILIZATION.* Spallanzani t opened the series of scientific ex- periments concerning spontaneous generation and. the principles of sterilization. He placed infusions of organic substances in flasks, which were corked and sealed, and then boiled for an hour in a water-bath. These experiments, which form the groundwork of the Appert method of preservation of organic substances, can be better made in flasks with long-drawn-out necks, which are closed during the process of heating. Now and then one of these experiments (where the dura- tion of the heating is only one hour) fails. But the chief objection to the conclusions drawn from these experiments lies in the fact that no oxygen can gain admission to the flask. In 1810 Gay-Lussac expressed the opinion that, for the production of fermentation, oxygen must have admission. The next methodical advance in this direction con- sisted in the admission of air into the vessel after heating, this air having been previously so treated * "ZusammenfassendeDarstellnngen derFrage fiber Ablogenesis; Generatio spontanea flnden sioh bei Gscheidlen." '• Physiologische Kethodik," Heft 2, 1876, S. 274 ; Heft 4, 1879, S. 499 ; und bei Tyn- dall, "Essays on the Floating Matter of the Air," second edition, 1883. t " Physikalisohe und mathematische Abhandlungen," 1769. 16 BACTERIOLOGICAL INVESTIGATION: that it could contain no germs. Franz Schtilze * de- signed a flask wMch had a double perforated cork, in which were placed two glass tubes bent at a right angle, and which terminated close underneath the cork. The decomposing infusions or substances sus- pended in water were heated on a sand-bath until aU portions had reached the temperature of boiling wa- ter. Then, while the steam was still escaping, a Lie- big's globe apparatus was fastened to each of the glass tubes. One of the globes was filled with concentrated sulphuric acid, and the other with a solution of caus- tic potash. Now, by the application of suction-force on the side to which the apparatus containing caustic potash is attached, air is drawn into the flask which before its entrance must pass through the sulphuric acid. When this is done, no decomposition takes place, notwithstanding the presence of the air ; but it soon occurs if, after the heating, ordinary air is allowed admittance. Schwann t showed that "it is not the oxygen, or at least not alone the oxygeii, of the atmospheric air " which causes fermentation and putrefaction, by pass- ing the air through mercury which was heated to the boiling-point. No change occurred in the fluids after this was done. To meet the objection, that by this procedure the air might be altered chemically, Schroder and von Dusch:j: passed the air through cotton, which was * " Voriaufige Mittheilung der Resultate einer experimentellen Beobachtung -aber Greneratio aeqnivoca." Poggendorf s " Annalen der Physik," 1836, Bd. 39, S. 487. t " Vorlaufige Mittbeilung, betreffend Versiiche fiber die "Wein- gahrung und Faulniss." Poggendorf s "Annalen," 1837, Bd. 41, S. 184. X " TJeber Filtration der Luft in Beziehnng auf Faulniss und Gab- rung." " Annalen der Ohemie und Pbarmacie," 1854, Bd. 89, S. 387, SPONTANEOUS GENERATION. 17 either placed in tubes connected with the right-angled glass tubes, or they stopped the neck of the flask with cotton during the process of boiling. Pasteur * boiled the infusions in flasks, the necks of which were long-drawn-out and curved in different ways, with only this precaution, that the open end always looked downward. In this way the air could enter, after the heating, unaltered and unfiltered, and no putrefaction took place. In milk, Pasteur succeeded in preventing decom- position certainly only by elevating the temperature to 110° or 112° C. in a pressure of one and one half atmospheres. Schroder also showed the inefficacy of boiling to prevent decomposition in respect to single substances, which he found could be sterilized with certainty only after a long-continued boiling, or by elevating the temperature in a digester with a press- ure of about two atmospheres. The digester, or steam-kettle, for expanded steam, is used for sterilization with a high temperature. The temperature required for sterilization ranges be- tween 110° and 112° C, corresponding to a pressure of from one to two atmospheres. Since an equaliza- tion of temperature is produced by the strong cur- rents in the fluid brought about by the differences in temperature, the best execution, reasoning a pri- ori, ought to be expected from this apparatus, since, in accordance with the dynamic theory of heat with the elevation of the temperature, the tim,e required for the equalization of the temperature, and hence, the time for sterilization, is diminished. Many * " Mdmoire sur les eorpnsoles organises qui existant dansl'atinos- phere." "Annalesde chimie et de physiques," III. Sep., T. 64, 1863, S. 66. And shorter article, " Oompt. rend.," Bd. 48, 1859, S. 837 ; and ibid., Bd. 50, S. 849. 18 BACTERIOLOaiOAL INVESTIGATION: forms of apparatus* correspond to this theoretical postulate, but aU f do not satisfy it in all respects. The further consideration is added to this uncer- tainty that many substances are chemically altered by the elevation of the temperature above 100° C, so that the apparent advantages of the apparatus, as compared with others, are in many cases quite illusory. Any one who possesses a reliable, tested steam-kettle (Dampfkessel) may safely use this, especially if the substances to be sterilized wHL bear an elevation of the temperature above 100° C. If it is desired to use a temperature above 100° C, without employing a steam-kettle, a salt-, oil-, or parafflne-bath can be employed. If boiling water is to be used, then the larger flasks, smaller flasks, and test-tubes are boiled di- rectly in the water-bath, but the water in it should have a somewhat higher temperature than the con- tents of the glasses. The equalization of tempera- ture in the water -bath is not only attained cor- respondingly slower than at a lower temperature, but it is so uncertain that it should be practically tested. If a sufficiently long boiling affords a real steril- ization, then the temperature of the boiling water can be better used in the form of streaming steam. The steam sterilization-cylinder of Koch, Gaffky, and Loffler answers this purpose (Fig. 1). This consists of a cylinder made of strong tin plate, about haU a metre high and from twenty to twenty-five centime- * Fitz, " Ueber Spaltpilzgahi-ungen." " Berichte der deutschen chemisohen G-esellsohaft," Bd. XVII, 1884, 8. 1188. t Koch, Gaflky, Ldffler, " Versuche uber die Verwerthbarkeit heisser Wasserdampfe zu Disinfectionszwecken." " Mittheilungen aus dem kaiserlichen Gesundheitsamte," I, 1881, S. 322. SPONTANEOUS GENERATION. 19 tres in diameter, which is surrounded by an asbestos covering to prevent the loss of the heat, and is pro- vided with a copper bottom. In the interior at R, in the lower third, is placed a grate ; the space under this is filled three fourths full of water, which is brought to the boiling-point by a number of gars -flames (three or five burners) placed under the vessel. It is closed above by a cover {H) made of block-tin covered with as- bestos, and is not hermetically sealed, so that the steam can escape around the edges. In a hole in the cover a thermometer (t) is placed. The apparatus may also have somewhat larger dimensions. But, if these dimensions are very much increased, it is necessary to use salt solution in order to keep the temperature of the outer currents of steam at 100° C. If the escape of the steam is not quite free, and the radiation of the heat is prevented, the temperature of the interior of the cylinder in this way may be kept equal throughout ; and since the cover is not hermetically sealed, the temperature of the steam does not exceed the boiling-point, but gives the temperature of boiling water, correspond- ing to the conditions of pressure — that is, with the barometer at nearly its normal height, about 100° C. The advantages of this apparatus, in comparison with the steam-kettle, are its cheapness and the im- possibility of exceeding the temperature of 100° C. when water is used, so that all substances can be sterilized with this, which will bear a temperature of 100° C. The equalization of the temperature is very soon reached, and does not undergo such oscillations 20 BAGTEBIOLOOIOAL INYESTIGATIOm as vdth the steam-kettle, because the technical use of the apparatus can scarcely be siiflpler, and on this account this element is reduced to the minimum. The currents of steam are far superior to the water- bath, through the certainty of their action and the relatively short time required. These practical advantages render this apparatus the most desirable one for all cases in which high temjieratures are used for sterilization, in spite of the fact that, theoretically, to compensate for the some- what lower temperature, it must be used longer than would be necessary for expanded steam at a tempera- ture above 100° C. The beginning of the heating not being included, the time required for the sterili- zation varies from one half to two hours, depending on the size of the object. A vessel which fits in the cylinder is incljided with the apparatus. In this are placed the smaU flasks or tubes to be sterilized, and to the handle of the vessel or to the neck of the larger flasks a string is fastened for conveniently raising and lowering them. This string is made fast to the hook h, to be found on the rim of the cylinder. But many substances are altered by exposure to a temperature of 100° C., and especially coagulation of the albumen is always brought about. In order to avoid this, the discontinuous sterilization of Tyndall is used. The principle of " Sterilization by Discon- tinuous Heating " (Z. c, pp. 210 and 337) was founded on the observation that living bacteria are killed by exposure to a relatively low temperature, below the point required for the coagulation of albumen, while the spores are not destroyed by these low tempera- tures,* but are easUy killed after germination. If * Oohn, " Untersuchungen fiber Baoterien," IV ; " Die Bacterien SPONTANEOUS GENERATION. 21 Fio. 2. the fluid to be sterilized is exposed for one or two hours to a temperature of from 52° C. to 65° C, only the living bacteria are in this way destroyed, and per- haps not even all of these the first time. The resistant spores possibly present in the solution thus treated germinate some on the first and second, others on the third and following days. If now the fluid is exposed to the same temperature as before on the second and third days, the living bacteria, or those that have de- veloped from the spores, are killed each time, so that, if this operation is continued long enough, it is pos- sible to sterilize with certainty all fluids below the temperature producing chem- ical alterations. In general, exposure to a temperature of about 68° C. for one or two hours, on from flve to eight successive days, is recommend- ed. This may be done by the use of the water-bath, but more conveniently with the appara- tus shown in Fig. 2. This con- sists of a double-walled cyl- inder made of copper. The chamber between the two walls is about half filled with water, and the cylinder itseK well closed with a double-walled cover, which is also filled with water. The cover has upon its side a hol- low tube {d), whose lumen communicates with the chamber in the cover. This is warmed by the flame {(F), while the cylinder itself is warmed from below by a flame placed under it. There are three tubes in the cover, one of which (c) is used for fiUiag the cover nnd die TTrzeugnng." " Beitrage zur Biologie der Pflanzen," Bd. 11, Heft 2, S. 249, 1876. 22 BACTERIOLOGIGAL INVESTIOATIOK and for receiving a thermometer which indicates the temperature of the water in it ; a second (5') receives a thermometer which passes into the air-chamber (6) of the cylinder ; and the middle tube {a}) receives a thermometer which passes into a small central cylin- der (a), the cavity of which communicates vrith the water-chamber on the exterior. The outer water- chamber is filled by means of a tube placed on the side. The principle of discontinuous sterilization may also be used in many cases as discontinuous boiling, since there are many substances which are altered by long boiling that are not jnaterially changed by brief but often repeated boiling. If such substances — as, for example, gelatin — are boiled for a short time on four or five successive days, it is possible to sterilize them with certainty. For refuting other objections which are made as to the existence of spontaneous generation, it may be desirable to use substances which have not even once been subjected to the lower temperatures— i. e., tem- peratures below the coagulation-point of albumen. This object is attained in two ways : first, by free- ing the solution by filtration from possible admixt- ures ; or, secondly, by endeavoring to obtain the same uncontaminated from the beginning. In respect to the first, Helmholz* observed that the fermentation produced by yeast did not pass through a membrane ; on the contrary, this did occur with putrefkction. Consequently such membranes are not available for this purpose. Positive results were first obtained by Tiegel,t who succeeded in separating * " ITeber das Wesen der Faulniss nnd Gahrung." MuUer's "Arohiv far Anatomie und Physiologie," 1843, S. 453. t " Correspondenzblatt fur schweizer Aerzte," 1871, S. 275 ; nnd SPONTANEOUS GENERATION. 23 mechanically— by filtration of septic fluids through clay cells, using positiye or negative pressure on one side— the septic material from the quite inactive fluid. Miquel and Benoist * endeavored to remove the germs by a gypsum filter. They took glass balloons with drawn-out necks, in the narrow portion of which an asbestos-stopper was placed, and beyond this a layer of gypsum. Before use the apparatus is slowly heated to 170° C, then the fluid to be filtered is slow- ly poured upon the gypsum-stopper. The stopper consists of 1-6 asbestos, 52-1 gypsum, 46 water. The diluted juice of flesh and plants and urine filtered rapidly, serum and albuminous fluids somewhat more slowly ; all were germ free and free from life. A cap- illary tube, cemented underneath the constriction, by being united to an aspirator, permits the reduc- tion of the pressure in the balloon. Grautier f used a very long-necked flask of faience or unglazed porcelain, which tapered below into a cone. Through this porous cone, the real filter, the fluid to be filtered passed from without into the inte- rior of the porcelain flask. For rarefying the air in the neck of the flask, a glass tube, bent at a right an- gle, is fastened by vermilion-paint cement, so that the shank reaches down to the bottom of the cone whUe the other outer end tapers into a small cone and exactly flts into a corresponding conical expan- sion of a second tube. This second glass tube is like- wise bent at a right angle, and the end, which is " Ueber die fiebererregende Eigensohaft des Mikrosporen septioum." Dissert. Bern, 1871. Oitirt nach Klebs. * " Bulletin de la sooi6t6 chimique de Paris," 1881, Bd. 35, S. 652. t " Sterilization k froid des liqnides fermentescibles." " Bulletin de la soci6t6 chimique," 1884, Bd. 42, S. 146. 24 BACTERIOLOGICAL IN7ESTIQATI0F. united with the portion of the first tube, possesses a conical expansion, while the other end reaches to the bottom of a glass flask with a narrow neck. To the side of this glass flask a tube with a conical expan- sion is cemented. The two conical expansions are closed with cotton, and then this glass balloon with its projecting portion and the glass tubes are steril- ized. In the same way the porcelain flask with its glass tube is heated, and, after removal of the cotton stopper from the cone of the first glass tube, this is inserted into the conical expansion of the second. In the conical expansion of the projection from the glass flask, after the removal of the stopper, a glass tube, extending out into a corresponding cone, is intro- duced, which is filled with heated asbestos. The joints uniting the conical expansions with the corre- sponding conical constrictions are covered with shel- lac. By aspiration at the free end of the asbestos- tube, the air in the entire apparatus is rarefied, and, when the cone of the porcelain flask is immersed in a fluid, by the existing negative pressure, fluid which is quite free from germs is aspirated into the porce- lain flask. The vemiilion-paint cement, prepared with oil of turpentine, consists of Boracic acid (crystallized) 8 parts. Silicic acid 2 " Vermilion 13 " Most of the filtrates obtained free from germs in this way have experienced no material alterations ; but some of them do not remain unchanged after the filtration, as the filter does not allow aU materials to pass through equally well, and albuminous solu- tions are often quite seriously altered quantitatively and qualitatively. In order, also, to eliminate this possible error, the SPONTAFEOUS GENERATION. 25 substances should be obtained, quite uncontaminated, by the use of the greatest cleanliness. Before each manipulation the hands are cleansed with a one per cent solution of corrosive sublimate, and are then rinsed in sterilized water, or the sublimate is removed by alcohol, the alcohol by ether, and the latter al- lowed to evaporate. AU vessels are well sterilized ; the manipulations and aU operations are performed very rapidly and with the antiseptic precautions of modem surgery. In this manner it is possible to ob- tain blood, mUk, urine, etc., without anything ever being mingled with the same, or without decompo- sition taking place. Some details follow later in the methods of infection and in the inoculation experi- ments. No organism is ever formed, not even a mi- crococcus, either from the unorganized material, or from ''molecules of nitrogen," or from microscopic forms of life, or from the anamorphosis of proto- plasm, which fact certainly has not prevented many authors from mistaking, for real cocci, bodies of dif- ferent origin showing molecular motion, from which also later, bacUli, etc., should develop. Whoever has obtained contrary results, upon the ground of a few experiments, must first show that he has mastered the technique, as was the case in the many positive results of van den Broek, Pasteur, Roberts, Lister, Cheyne, and Meissner. The technical dexterity nec- essary for this is only to be obtained by many indi- vidual experiments. By these experiments, arranged for spontaneous generation (which, especially through Pasteur, have taken a form easy of mastery), and by the disinfection experiments of Koch, the still remaining principles of sterilization have been reduced to a reliable and convenient form. 26 BAOTEUIOLOOICAL INVESTIGATION. Metal objects — scissors, knives, pincettes, plati- num-needles — are first mechanically cleaned, then are heated in the flame, and for cooling are laid upon a sterilized glass plate and protected from dnst by a bell-jar. Glass objects — glass plates, slides, flasks, test-tubes — are first mechanically cleaned, then, if they are very dirty, greasy, or have been used for other purposes, are dipped in concentrated sulphuric acid or hydro- chloric acid, and finally repeatedly washed in dis- tilled water until every particle of the acid has been removed. The water is first allowed to run off, and the articles thoroughly dried in the dry-oven, or it is removed by means of alcohol, in the following manner : they are dipped into alcohol, and the last particles of this are removed by ether, which is al- lowed to evaporate. According to the degree of un- cleanliness of the vessels in the beginning, either this whole procedure must be gone through with, with the greatest care, or it is sufficient to wash them in distilled water. The chemically clean vessels must then be freed from germs. For this purpose the vessels, having their necks stopped with a closely fitting mass of cotton, are immediately placed in the double- walled dry- oven (Fig. 3). This is provided with a thermo-regulator (f) and a thermometer (f). The test- tubes are more conveniently placed in a basket {cC) made of wire, which wlU hold a large ~ number. Catheters, syringes, pipettes, capillary- tubes, and other glass tubes are Fio. 3. SPONTANEOUS GENERATION. 27 placed in a clean glass and then put in the dry-oven. All such glass objects should remain at least two hours* at a temperature of 150° or 160° C, the time required to raise the temperature to this point not being included. The objects are allowed to cool in the oven, so that they can be removed directly before use, or at least care should be taken to protect them from dust. Cork stoppers are to be avoided. Eubber corks, caps, and bands, etc., are sterilized in the steam- cylinder for from three ciuarters of an hour to one hour. Most vessels with their contents should be steril- ized once more; after which it is advantageous to bind over the tops two layers of filter-paper, since the cotton stoppers are proof against bacteria, but not al- ways against fungi. Previous experience has shown that germs from the air are more seldom the cause of failure than the unintentional infection through unclean or insuf- ficiently sterilized vessels, and the manipulation with hand and instruments not certainly sterilized. * Practically I have foand that exposure to a temperature of 150° or 160° 0. for from fifteen to twenty minutes answers exactly the same purpose, and it has been repeatedly shown that neither germs nor their spores wiU withstand exposure to so high a temperature for this length o£ time. — Te. II. FORMS OF BACTERIA AND MICROSCOPICAL TECH- NIQUE. In the examination, under the microscope, of a substance containing bacteria, different forms present themselves, whose general morphological peculiari^ ties, on the one hand, and whose differences or like- nesses, on the other, must be determined. The forms are determined by the use of the gen- eral microscopical technique, in its peculiar applica- tion to bacteria, while the second question can only be solved after obtaining pure cultures. The forms of bacteria (Fig. 4) are round (1, 3, 4, " ooo ^^ In part from Kocli and Frazmowski. BACTERIA AND MIGROSOOPIGAL TECHNIQUE. 29 and 5), oval (2), shorter (6) or longer (7) rod-form cells ; and, in addition, curved rods (11, 12) and spiral-formed organisms (13-16) are observed. These forms appear sometimes isolated, sometimes united in a definite manner (3, 4, 5). The next object to be attained is the separation of these forms and the grouping of them as naturally as possible, so that a general examination of the mor- phology of the bacteria may be made. A. The Teue Ewdospoee Bacteria (Fig. 4). — These multiply sometimes by division, sometimes also by the endogenous formation of spores. 1. Coooi. \ "• 5°™^- [ Cells. ) Tendency to the for- ( S. Oval. J >• ^- J! .. 1 C mation of zoogloea Might rods, f«- fort rods bacilli. j*-^°°Srods l c. Olostndiui Olostridium-forms, 3. Curved rods, vibriones. mation of zoogloea. Tendency to the for- mation of fila- ments, desmo-bao- teria ; the filaments show no variation from end to end. 4 True spirilla forms, spirilla, and spirochfetsB. B. Aethrospoee Bacteria. — These forms also multiply by division, bat they do not produce endoge- nous spores. On the other hand, single individuals may separate themselves from the colonies and form nevp- generations. These individuals appear almost constantly, as round cells, gonidia, or arthrospores. 1. Arthro-Cocci. — These consist only of cocci, and, by the union of the cocci, form torula. 2. ArtJiro-Bucteria. — These form round cells simi- lar to cocci, but also short rods, long rods, and fila- ments, which show no variation from end to end. 3. LeptotJirix.—ThesQ form cells similar to cocci, rods, spirals, and filamehts, which show a variation from end to end. 30 BACTEBIOLOGICAL INYESTIQATION. 4. Cladothrix (Fig. 5), Fig. 5. Cladothrix diohotoma ; after Zopf. plant with Blightly and d( branches. B, spiril, one end of wMol plant with Blightly and branches. B, spiril, one «- — more winding than the other. D, branch SCOpiCal These form cocci, rods, filaments, and spi- rilla. The fila- ments form false ramifications (A). The arthro-coc- ci and arthro-bac- teria have as yet / ) been very little / studied, and are 4j^ perhaps to be ^^Qo classed with the true bacteria. These two groups, so far as they have been studied, seem to stand nearer to the fission algae on account of their form. Zopf * class- es them directly with the bacteria, because of the ap- pearance of forms similar to these in their development. The next ques- tion in the micro- investi- A, hranehinff decidedly spir^ ■ ion wiwi narrow aua uruuu wiuuxu^H. .&, tspii- , . .. , /!+•*» ils ; a, undivided, 6, divided into rodSj and gatlOU IS tO U.eter- at c into Zopf a cocci. .F", epiroohaeta form ; yniTiP whir-h of thp at a, undivided, at 4, schematic division into miUB wnicn OX lUB long rods, at c, into short rods, and at d in cocci, described f OrmS are, in general, present. After obtaining a pure cult- * Oompare my criticism of the work of Zopf in the "Fortsohrit- ten der Medizin," 1883, No. 6. BACTERIA AND MICROSCOPICAL TECHNIQUE. 31 ure, it must then be determined what is the typi- cal form; whether it remains the same under all conditions, so that it not only has the value of growth-form, but also constitues a form-species or form-genus ; or whether it corresponds to external conditions, being sometimes larger and broader, some- times smaller and thinner ; or whether, finally, other forms may appear in their development. Further, it ought to be ascertained whether, in the develop- ment of a species, a single form or a form-cycle ap- pears or can appear. In regard to the individual peculiarities, there re- main yet the following points to be noted, although those already referred to seem to be numerous. In the spiral forms. Fig. 4 (13, 14, 15, 16), the number and the arrangements of the typical spirUs should be observed ; then it is to be noted whether, in divis- ion, the spirils separate iato curved rods similar to the vibriones, and whether these fissions-products again develop iuto typical spirils, or whether the division extends on to the production of forms which are similar to the rod-bacteria and cocci, which finally origiaate new generations. If re- agents are used, it is first necessary to note the stage of development, since, either during the pro- cess of division or immediately before it, the re- agents will make visible the lines of division in place of the apparently homogeneous character of the filar ments. On the other hand, the value of such chem- ical action should not be overestimated, since, in the height of their development, that most susceptible reagent, photography, shows not the slightest trace of union. In the case of the vibriones. Fig. 4 (11, 12) — the smallest forms of which (11) Koch, on account of 8 32 BACTERIOLOGICAL INVESTIQATION. their characteristic appearance with the usual mag- nifying power, called comma-bacilli — it should be ■ noticed whether they divide into smaller vibriones, or whether they can form spherical and rod-shaped forms. Through the union of a number of vibriones, S-forms and long, slender filaments similar to spirilla (11) may be produced which the inexperienced ob- server can easily confound with the true spirilla (13). These spiral filaments, according to the degree of curvature of the individual vibriones, form sometimes slightly wavy lines, sometimes spirils with narrow windings. The spiral forms of the vibriones are to be considered as a kind of thread-formation, which, as contrasted vsdth the true spirilla, appear only un- der certain conditions, as, for example, the partial or total exhaustion of the culture-medium, and never show the regularity of the latter. In the rod-formed bacteria it should be observed whether the division of long rods into short ones, or into oval and round cells, predominates. Some rod- forms, by their union, produce long filaments, while otherS' show a tendency to the formation of zoogloea. On account of the greater tendency of the short-rod bacteria, lik^ the cocci, to form zoogloea, Cohn sepa- rated them from the vibriones and long-rod bacteria, which he classifies as filament-bacteria. It is to be noted whether the character of the culture-medium (solid or fluid) has any influence upon this. In the cocci it should be -determined whether the oval cocci produce, by their division, smaller oval cells or round cells, and whether the round cocci, be- fore their division, become oval, and, finally, whether the oval cocci become short rods previous to their division. The appearance of straight rods may be produced BACTERIA AND MIOROSOOPIGAL TEOENIQUR 33 in the curved rods, when the concavity or convexity of the rod is turned upward. A straight rod stand- ing upright may present the appearance of a round cell, and a long rod placed at an angle to the plane of vision may appear like a short rod. It is only possible to decide such questions after many single observations and with the use of pure cultures. Many hundred observations of mixtures of bacteria do not serve at all for the decision of morphological questions. The consideration of spore-formation, Fig. 4 (55, 8, 9, 10, 12), and germination shows two different types. The rods may develop into filaments before the forma- tion of spores (8), or the spores may appear in the mobile rods (9) ; sometimes they are formed in the interior (8, 9), sometimes near the end (12). The rods often become whetstone- or club-shaped before the formation of the spores (Clostridium, 10). Under other conditions, quite abnormal, so-called involution forms are observed (17). The work of Cohn * is fundamental as regards the value of form. In this he first clearly described the relations of the growth-form, the form-genus, form- species, true varieties, form-varieties, and physiologi- cal varieties. This work is not only often entirely misunderstood by his opponents, but also construed in an opposite sense by his own zealous supporters, who have repeatedly interpreted his arguments for the existence of true species among the bacteria in the sense of a belief in constancy in species and form,t which was accepted before the Darwinian theory. * " UntersTichnngen tlber Bakterien." " Beitrage zur Biologie der Pflanzen," Bd. I, 2. Heft, S. 127, 1872. 2. Abdruck, 1881. t For the further investigation of these questions, consult Naegeli, Si BACTEEIOLOaiCAL INVESTIOATION. Deteemination op the Peesence of Bacteeia UirSTAIKED. The oldest metliod of examining unstained bac- teria consisted in mingling a small drop of fluid or a particle of matter containing bacteria, with a drop of indifferent fluid, placing it upon a slide, putting over it a cover-glass, and then examining it accord- ing to the method of histological procedure. The picture presented in these cases is of the same nature as in unstained tissue-preparations, especially in this : that the objects, because of their different power of refracting light from the inclosing media, or, according to Koch, "on account of the refraction of the rays of light passing through, present a picture of lines and shadows — viz., the structure-picture." In these cases a diaphragm is used, as in other histological work in which it is desired to make out the structure-picture.* The diflEused daylight will "Die niederen Pilze in ihren Beziehungen zu den Inf ectionskrankheiten tind der Gesundheitspflege," 1877. Naegeli und Buchner, in Naegeli's " Untersuohnngen ilber niedere Pilze," 1882. Koch, " Zur Aetiologie des Milzbrandes," " MittheUungen aus dem kaiserlichen Gesundheits- amte," Bd. I, 1881, S. 49. GafEky, " Experimentell erzengte Septi- caemie mit Riloksicht auf progressive Virulenz und accommodative Zuchtung," ibid., S. 80. Fliigge, "Fermente und Mikroparasiten," 1883, und " Deutsche med. Wochenschrift," 1884, No. 46. Zopf, " Die Spaltpiize," 2. Aufl., 1884. De Bary, " Vergleichende Morphologic und Biologie der Pilze," 1884. Hiippe, "Portschritte der Medi- zin," 1883, No. 6, und 1884, No. 6 (Kritik der Ansichten von Zopf), und " TJeber die Zersetzungen der Milch und die biologischen Grund- lagen der Gahrungsphysiologie," "Deutsche med. "Wochenschrift," 1884, Nos. 48-50. * Zur weiteren Orientirung fiber das Mikroskop und die mikro- skopische Technik : Dippel, " Das Mikroskop," 2. Aufl., I, 1882-83 ; Frey, " The Microscope and the Microscopical Technique " ; Strass- BACTERIA AND MICROSCOPICAL TECHNIQUE. 35 not suffice to illuminate the object when the higher powers are used, so that it is necessary to employ a condenser, which does not define the structure-pict- ure, but serves to increase the illumination. For this kind of microscopical work the achromatic condens- ers of the larger English instruments answer the pur- pose better than any others. The dry system of objectives does not suffice for most bacteria ; in order to make out only approxi- mately the correct forms, one must use in this inves- tigation the immersion system, in which, according to Amici, the stratum of light, by passing through a stronger refracting medium, compensates as much as possible for the error caused by the dispersion of the rays by the cover-glass. Since the cover-glass and the front lens of the ob- jective consist of crown-glass, and water, on account of its low refractive power, does not quite correct the error, iu the water-immersion systems correction-ad- justers and cover-glasses of a certain thickness are necessary. But the correction is obtained if the immersion- fluid has the same exponent of refraction as crown- glass. Such a fluid, according to Abb§,* presents an optical homogeneous union between the preparation and the objective, which prevents all refraction of the rays in front of the convex surface of the optical sys- tem. By this means the loss of light through reflec- tion at the natural joints of the different optical media is avoided, and at the same time a very con- burger, " Das botanisohe Praoticum," 1884 ; . Friedlander, " Micro- scopical Technique." * " Ueber Stephenson's System der homogenen Immersion." " Sitz- nngsberichte der Jenaiscben Gesellsohaft f. Med. und Naturw.," 1879, 10. Jannar. 36 BACTHmOLOGIGAL INYE8TI0ATI0N. siderable amount of spherical aberration is prevented. On account ol this, the correction-adjusters necessary in the water-immersion systems can be dispensed with, and the thickness of the cover-glass is of no great moment, since, as soon as the intervening medi- um has the same index of refraction and dispersion as the cover-glass, the same result for optical pur- poses is obtained whether a thick layer of glass and a proportionally thin layer of fluid or the reverse is interposed between the object and the lens sys- tem. Anise-oil was used by Amici for the purpose of increasing the exponent of refraction ; by Spencer glycerin was employed. Stephenson* desired an en- largement of the aperture of the lens, not only to avoid the necessity of the correction of the cover- glass, but also' to increase the power of difEerentia- tion. The union of these two postulates by Stephenson, their settlement by Abb6, the construction of lenses by Zeiss, and the introduction of this system for homogeneous immersion by Koch,t marked a new era for the microscopic side of bacteria investiga- tion. The best fluid is the ethereal oil of cedar, the index of whose refraction is the same as crown-glass, and its index of dispersion differs only in a slight degree from crown-glass. When somewhat inspissated, it is more convenient for use for most optical purposes. By mixing other stronger-refracting ethereal oils — such as oil of cloves, phenol, and anise — with olive- or * On a large-angled immersion objective. " Journal of the Eoyal Microscopical Society," 1878, p. 51. + " Untersucliungen tiber die Aetiologie der Wundinfectionskrank- heiten," 1878. BAGTERIA AND MICROSCOPICAL TECHNIQUE. 37 castpr-oil, an immersion-fluid can be made, wMch is similar in its refracting power to oil of cedar, or which diJBfers from it to a certain degree. In consequence of the removal of the troublesome cover-glass correction (attained by difllerent lengths of the draw-tubes) which allows, in a delicate man- ner, a compensation for the influence upon the aber- ration of the varying distance of the picture, the ob- jectives for homogeneous immersion are always ad- justed for a definite length of the draw-tube. On this account it is to be noted that lengthening the draw-tube beyond this normal length acts in the way of a spherical over-correction, shortening in the way of an under-correction. In using an immersion-lens, one places a drop of oil on the cover-glass, screws down the draw-tube with the coarse adjustment, or, if this is lacking, brings it down with a rotary motion by the hand, so far that the front lens of the objective touches the oil and the picture begins to be visible. Then the fine adjustment with micrometer-screw is used. Some place a drop of oil on the front lens of the objective. Others put a drop not only upon the objective, but also upon the cover-glass. After use, the oil is carefully removed from the lens with a fine linen cloth, less satisfactorily with blotting-paper, and the system is returned to its case. If the cover-glass preparation is to be preserved, then the oil is soaked up with filter- paper, and what remains is finally removed by chloroform or • ben- zine. According to histological tradition, which influ- enced the earlier use of the microscope, the magnify- ing power should be increased rather through the use of a more powerful lens than a more powerful 38 BAGTEBIOLOGIOAL INVESTIGATION. ocular. But, according to a purely physical princi- ple, the strength of an ocular, which an objective will allow with advantage, depends upon the angle of aperture of the latter. The larger the angle of aper- ture, so much the stronger, other things being equal, can the ocular be. Our best homogeneous systems correspond to this requirement, namely, that one can use the same objective and at the same time employ the most powerful oculars. In this way, bacteria without any special prepara- tion may be observed between the slide and cover- glass. One cause of uncertainty is here noticed, i. e., almost all bacteria are in motion. This in part seems to be spontaneous motion ; in part a simple Brownian molecular movement, such as occurs in all fine particles suspended in a fluid. In proportion to the minuteness of the objects, these movements ren- der exact observation difficult. For this reason one should early eliminate this, and fix the forms of micro-organisms by narcotizing them.* For this pur- pose, a particle of spirituous or dilute alcoholic tinct- ure of opium can be added with the point of a needle to a drop of water. Moreover, von Recklinghausen f has shown that small, round, granular tissue-detritus, which is so easily confounded with bacteria, can be sharply dif- ferentiated by the fact that bacteria are pre-eminent- ly homogeneous granules, and are totally unaffected by the action of acetic acid, glycerine, or even caustic soda. Baumgarten :j: succeeded in making the tubercle * Perty, "Zur Kenntniss kleinster Lebensformen," 1862, S. 13. t " Verhandlungen der Physikal-Medizin. Gresellschaft in Wflrz- bxirg," N. F., ir. Bd., Heft 4, 18Y2. " Sitzungsberichte," S. XII. X " Oentralblatt f. d. med. Wissenschaft," 1882, No. 15. BACTERIA AND MIOBOSGOPIOAL TECHNIQUE. 39 bacilli visible by their resistance to a diluted solution of caustic soda, though, he could not recognize them by staining according to the methods in use at that time. We possess now more convenient means, both for fixation and difEerentiation. But it would be a seri- ous error not to examine bacteria unfixed and un- stained. It is, on the contrary, necessary throughout that the bacteria should be observed under the most natural conditions possible in order to study their motion, to follow the formation of spores and their germination, and to control the forms according to other treatment. For this purpose we do not use the previously described form of investigation, but employ the moist chamber. For this the hoUow slides A and B (Fig. 6) serve, A small drop (c) of the bacteria- containing Fig. 6. 1" 0^ 1 A fluid is placed upon a cover-glass (5). The cover-glass is quickly reversed, and, with the drop now hanging underneath (c, B), is laid over the hollow {a) va. the slide, and its edges are surrounded by vaseline, wax, paraffine, or balsam, in order to prevent evaporation of the fluid. Another and a better form is represent- ed in Fig. 7. Upon a slide, A or 5, a glass plate (5) 40 BA CTEEIOL QIGAL INYESTIOA TIOK is cemented, which, has a central circular opening. A chamber is formed by laying a cover-glass (a) over Fig. 1. the opening. This room, in place of the almost half- circular cavity in Fig. 6, is bounded by parallel walls. The drop is prepared in a similar manner, and in the same way hangs within the chamber. These chambers can be improvised if, instead of cementing a piece of glass, a thin piece of paper of corresponding size, with a circular aperture, is fastened upon an ordi- nary slide. The warm stage may be employed for di- rect observation at higher temperatures. Staining Bacteria. In examining unstained bacteria the diaphragm is used in order to make the structure-picture clear ; but small objects and particles, the size of bacteria, imbedded in the tissue made visible in the struct- ure-picture, are hidden by the shadows of the struct- ure-picture. If these particles are stained, and if they are of a certain size, they will be visible in spite of the shadow; but under this size, notwith- standing their color, they are concealed by the BAOTMRIA AND MICROSOOPIGAL TEOHNIQUE. 41 shadows. Therefore it is desirable to stain the bacteria and to so arrange the light that the struct- ure-picture does not further interfere, and that the color-picture, as pure as possible, be presented to the observer. Koch (foe. cit.) succeeded in pro- ducing this isolation of the color-picture by re- moval of the diaphragm. In this way so weak a structure-picture was produced that the minutest particles of the color-picture became distinct. In the stronger structure-pictures, after the remov- al of the diaphragm, he used a condenser which threw so intense a cone of rays upon the ob- ject that the diffraction appearances were entirely avoided. With such a method of illumination, in which the preparation is permeated in all directions by the penetrating rays, only those elements remain visible which produce an absorption of the rays on account of their staining. Further, Abbe in this way has shown that, although the illumination in name re- mains central, yet the important advantages of the oblique illumination are obtained . through the co- operation of the rays passing at a greater inclination to the axis of the microscope. On account of this co-operation of the oblique rays for the isolation of the color-picture, and for the complete development of the capacity for differentiation of the oil-immer- sion objective necessary for this, the condenser must furnish a cone of light of a size at least equal to the aperture of the objective, which is made, according to Stephenson, with a large angle of aperture. This is as yet attained, iu a manner that fulfills all the re- quirements, only by an Abb6 condenser. In order that the structure-picture can be brought out in spite of this condenser, an arrangement for interposing a 42 BACTERIOLOGICAL INVESTIGATIOK diaphragm is added, which is furnished with open- ings of different sizes. An Abb§ condenser belongs to the system for homogeneous immersion in bacteria investigation. Now and then it is useful to place a drop of water or immersion-fluid between the condenser and the under- side of the slide, so that below and above a continuous union is formed. These form immersion-condensers which were formerly used before the Abb§ condenser was constructed. GEWEEAL PEINCIPLES OE STAINIKa. Since Hartig, in 1854, and Gerlach, in 1858, showed, by the systematic use of carmine in histo- logical work, that in employing coloring-matters cer- tain elements of the tissues become more distinct and can be differentiated from other elements, staining has been recognized as equivalent to a chemical re- action. Weigert * first succeeded in staining the zoogloea masses of the micrococci with the nuclei, by the use of the nuclei-staining ammoniac-carmine solution, and the subsequent treatment with hydrochloric - acid glycerine. This staining, it is true, was first used by Weigert as a staining of cement substance ; but later it was corrected by him, and it was in this way shown that the bacteria can be brought into view by other characteristics than their greater resistance to acids and alkalies. In the following year Eberth and Wagner suc- ceeded in staining micrococci, but not bacilli, with hsematoxylin. * " Ueber Bakterien in der Pookenhaut." " Centralblatt f. d. med. Wissenschaft," 1871, No. 49. BACTERIA AND MIGBOSOOPICAL TEOHFIQUE. 43 Then Weigert * showed that the micrococci, espe- cially in zooglcea masses, can be stained by different nnclei-staining materials. For this purpose he used at that time methyl-violet, an aniline-dye which stains nuclei. By the subsequent treatment of prep- arations stained with hsematoxylin (in which the micrococci and nuclei are stained blue), with diluted caustic potash and strong acetic acid, he first suc- ceeded in procuring an isolated staining of the mi- crococci. Weigert f further observed that the larger bacilli, which were not stained by haematoxylin, could be made visible by certain aniline-dyes. Soon after this, Koch:): found that the bacteria take up aniline-dyes with such certainty, and so quickly and completely, that "these dyes can be used as reagents for differentiation of bacteria from crystalline and amorphous precipitates, or from the smallest fat- drop or other minute bodies." Koch* then succeeded ia obtaiaing the isolated staining of the bacteria by washing out the section with a solution of carbonate of potassium, by which all the elements except the bacteria were decolorized. Finally Weigert || obtained a double staining, when he subsequently treated with picro-carmine prepara- tions that had been stained with a blue anLLLae-dye, whereby the bacteria appear blue and the nuclei red ; * " Sitzung der Schlesisohen Gesellschaft far vaterlandische Cul- tur," vom 10. December, 1875. t "Berl. klin. Wochenschrift," 1877, Nos. 18-19; nnd "Beriohte fiber die Manohener Naturforscherversammlung," 1877, S. 283. X "Verfahren zur TJntersuchung, znm Oonserviren nnd Pboto- graphiren der Bakterien." "Beitrage zur Biologie der Pflanzen," Bd. II, 3. Heft, 1877, 8. 399. » " Wnndinfectionskrankheiten," 1878, S. 39. II "Zur Technik der mikroskopischen Bakterienuntersuohungen," Vircliow's " Arcbiv," 1881, Bd. LXXXIV, S. 275. ii BAOTERIOLOQIGAL INVESTIGATION. and Kocli * observed that certain forms of bacteria stained differently from the nuclei and from other bac- teria which were present in the same preparations. What coloring-matters ought to be used for stain- ing bacteria ? Weigert's observation that the micrococci, but not the bacilli, are stained by carmine, and a similar ob- servation by Eberth and Wagner concerning hsema^ toxylin, seemed to show, according to Weigert, that there are essential chemical differences between the single-group forms of Cohn. Safranine, one of the best reagents for the staining of nuclei, is also of more value for micrococci than for other forms of bacteria. Further, Obermeyerf observed that the spirilla are less resistant to the action of acids and alkalies than other bacteria. But these differences are not essential, since some micrococci and bacilli are less resistant to the action of alkalies and acids than others, and since some bacilli stain as well with hsematoxylin as micrococci, while others take this dye badly. However, the basic aniline-dyes have shown them- selves to be available staining materials, under aU conditions, both for the dried cover-glass prepara- tions and for sections, so that we assign to them a first rank among materials for staining bacteria, and give to other dyes a second place. Ehrlich,:]: partly in conjunction with his pupils Schwarze and Wesfcphal,* undertook to classify the * "Berl. klin. Wochenschrift," 1882, No. 15. t "Berl. klin. Wochensohrift," 1873, S. 391. I "Zeitschrift fur klin. Med.," .Bd. I, 1880, S. 553; und kleinere gelegentliche Mittheilnngen. , * Schwarze, "Ueber eosinophile Zellen," Dissert., Berl., 1880. "Westphal, "Ueber Mastzellen," Dissert., Berl., 1880. BAOTEEIA AND MI0R08G0PI0AL TEOENIQUE. 45 dyes used in microscopic work. The principle under- lying tMs theoretical study of dyes rests on the ob- servation that the different elements of the tissues and cells possess the capacity of taking certain dyes only, or of holding them with a greater tenacity than other elements. This "election," this affinity of the dyes for certain elements, lends to staining the value of a chemical reaction ; or, more correctly (in the want of a precise chemical reaction), shows to the eye the presence of differences otherwise impercep- tible or distinguishable only with difficulty. Many stainiag materials color at first many ele- ments of a tissue quite diffusely, so that the individual elements are not recognizable. If, then, certain de- colorizing agents are used, some of these individual elements give up their color, while, on the other hand, other elements retain it persistently. In this way, by an indirect method, it is possible to attain a maximum staining of certain elements, while others remain as much as possible uncolored. Ehrlich called this method, which was first used in another way by Friedlander, * " the principle of the maximal decolorization." Histologically, one must differentiate in every col- oring-material two peculiarities : first, the affinity to certain elements ; and, secondly, the staining power. In respect to the election or affinity for certain ele- ments, Ehrlich divides the aniline colors into two groups — {a) the acid, (6) the basic aniline-dyes — ac- cording as the coloring principle is the acid or base- color. In this histological sense it is of equal import whether the acid in its use acts as a free acid or a salt ; also whether the base acts as such or as a salt. * " Studien liber automisohe Herzbewegimg," in " Untersuchungen aus dem physiologiscLen Institut zu Wtlrzburg," I, 1867. 46 BACTERIOLOGICAL INYESTIOATIOK The acid aniline colors are divided into four classes : 1. Fluorescin — e. g., fluorescin and eosin. 3. Nitrogenous bodies — e. g., martins yellow, pic- ric acid, and aurantia. 3. Sulphuric acid— e. g., tropseolin. 4. Primary dye-acids — e. g., rosol acid, alizarin, and purpurin. Of the basic aniline-dyes, the following are found to have the most value : Fuchsin (muriate of rose- anilin), methyl-violet (muriate of trimethyl rose-ani- lin), gentian-violet, methyl-blue, and vesuvin. Of less value are methyl-green, cyanin, safranin, magdala, and dahlia. Of these, especially the violets (methyl- violet, gentian-violet, iodine-violet, and dahlia) have sometimes the valuable capacity of double staining ; that is, they stain certain elements in a color varying from the fundamental color — e. g., methyl- violet does not stain the amyloid substance violet, as it does the bacteria and nuclei, but red ; methyl-green stains the nuclei green, the amyloid substance violet. The members of the first group show altogether the same elective peculiarities — i. e., they act as acid dyes and stain all the accessible elements, but in a differing degree. The basic amline-dyes also show the same elective peculiarities, since they stain the accessible elements in a basic color ; but also in these there is a difference in the intensity of the staining. This intensity of the staining is dependent upon the coloring power, and the coloring power is conditional on the fact that the different coloring-matters are re- tained in different degrees of intensity in the tissues or cell-elements, in the presence of the individual groups of decolorizers, such as alcohol, acetic acid, and glyceriue. For example, methyl-green in a short BACTERIA AND MIOROSOOPICAL TECENIQUE. 47 time is completely extracted from a preparation by alcohol, while vesuvin is scarcely at all affected. In respect to this coloring power, the basic aniline-dyes arrange themselves in the following order : Vesuvin, bismarck-brown, and aniline-brown theoretically take the first place, because these coloring-matters are not extracted by glycerine, and they are at the same time suitable colors for photography. After these follow in a descending scale fuchsin, methyl-violet, gentian- violet, and methyl-blue ; but these should be placed generally before the brown, as they are to most per- sons more agreeable and satisfactory colors. The re- maining dyes have as yet found no general use. This scale, as I will remark, to avoid misunder- standing, has a conditional value, since certain colors are produced only when certain fluids are used for solution and when the preparations are subsequently treated in a special manner, as will be described later more in detail. At the same time it is not impos- sible that some one of the remaining dyes may prove to be more valuable than these. The basic aniline colors are soluble in water, and for the most part in one or all of the decolorizing agents. In use, a weak watery solution colors at first the intercellular substance and the cell-body, whUe the nuclei remain unstained. Through the subse- quent treatment with alcohol, glycerine, or acetic acid an inversion of the staining takes place, by which the elements previously colored become colorless, while the previously colorless nuclei are stained. In the use of the stronger solutions the staining follows (without any discernible inversion) directly and quick- ly ; and, in general, its intensity is in proportion to the concentration of the solution. In a quite concen- trated watery solution overstaining may occur, which 48 BACTERIOLOGICAL INVESTIGATIOK iu sections can be reduced to the proper degree by subsequent decolorization. Methyl-blue alone does not overstain, according to Ehrlich,* even after a long action, and it is conse- quently to be used if for a special reason no decolor- izing agents should be employed. If the dyes are dissolved in the decolorizing agents — such as absolute alcohol, acetic acid, or thick gly- cerine — they stain slightly or not at all. Instead of using some decolorizing agent subsequently, to re- duce the intensity of the staining to the proper degree, in preparations which have been overstained in watery solutions, in many cases a solution of the dye-stuff in a mixture of water with alcohol (Herrmann), gly- cerine (Schaefer), or acetic acid (Ehrlich) may be used. PEEPAEATIOJSr OP STAINIISTQ PLTJIDS. The basic aniline-dyes are used in the following solutions : 1. Concentrated watery solutions. These are either used directly or after dilution to the desired degree with distilled water. The solu- tions are prepared with distilled water (which has been previously boiled), so that an excess of the col- oring-matter remains undissolved. They must be often filtered. Only a small quantity of these watery solutions should be made at a time. 2. Concentrated alcoholic solutions. The solution of an excess of the coloring-material is brought about in the best way by absolute alcohol, or, in want of this, by the officinal 90 per cent spirit of the Pharmacopoeia. In general, one can calculate about 20 to 25 grammes of the dye-stuff to 100 grammes of the spirit ♦ " Zeitschrift f. klin Med.," Bd. II, 1881, S. TIO. BACTERIA AND MIGROSOOPIOAL TEOENIQUE. 49 or alcoliol. These solutions are kept prepared, and are not used directly for staining, but are mixed with. a certain amount of distilled water. In place of con- centrated watery solutions, these can be used if five or six drops are added to a small watch-glass of dis- tilled water. This mixture I shall briefly designate in the future as the dUuted alcoholic solution. 3. Vesuvin, bismarck-brown, and aniline-brown can not be used in alcoholic solution, nor in a watery solution, even if filtered each time ; so that a concen- trated solution in equal parts of glycerine and water is prepared.* 4. Alkaline solutions. a. Weak. Koch.t Cdncentrated alcoholic solution methyl-blue. . 1 c. cm. Aq. destU 200 c. cm. 10 per cent solution caustic potash 0-2 c. cm. h. Strong.:]: Concentrated alcoholic solution methyl-blue . 30 c. cm. Solution caustic potash, 1 to 10,000 100 c. cm. 5. Aniline- water solution (according to Ehrlich). * Pure aniline-oil in excess is shaken with distilled water for one half to one minute (about 5 c. cm. of oU with 100 c. cm. of water). Then, after allowing it to stand five minutes, the mixture is filtered through a filter which has been previously moistened with dis- tilled water. The filtrate must be perfectly clear, and serves in place of water as a menstruum. Since this saturated aniline solution very quickly becomes un- * Kooh, " Verfahren znr Untersuchung." " Beitrage zur Biologie der Pflanzen," 1877, Bd. II, 3. Heft, S. 5. t "Berl. klin. Woohenschrift," 1882, No. 15 ; " Mittheilungen aus d. k. Gesundlieitsanit," 1884, Bd. II, S. 5. X " Mittheilungen," 1884, Bd. II, S. 439. « " Deutsche med. "Woohenschrift," 1882, No. 19. 50 BAOTEBIOLOOIGAL INVESTIOATION. Stable, it is better to prepare it fresh each time that it is used. If it is desired to make this permanent, according to B. Fraenkel, 5 to 10 per cent of alcohol is added, or 3 c. cm. of aniline-oil is dissolved in 7 c. cm. of alcohol and 90 c. cm. of distilled water is added. Fuchsin, methyl-violet, and gentian-violet are the best dyes for use in this menstruum. In most cases, according to EhrUch, it is more con- venient to add a saturated alcoholic solution of fuch- sin or methyl- violet to the clear aniline- water until a distinct cloudiness of the fluid is present, which indi- cates that the fluid is saturated with the coloring- matter. For certain purposes the following modifi- cation of the Ehrlich solution, according to Weigert and Koch,* recommends itself for common use, but this must be renewed after ten or twelve days, be- cause its coloring power is generally diminished : Saturated aniline- water 100 c. cm. Concentrated alcoholic solution, methyl- violet, or fuchsin 11 c. cm. Absolute alcohol 10 c. cm. 6. In place of aniline, toludin, prepared in the same manner, can be used as a menstruum (B. Fraen- kel). f Also turpentine (Prior);:]: and a five per cent watery solution of carbolic acid (Ziehl),* or one half per cent ammonia (Weigert) || [Liq. ammon. caust., 0'5 c. cm. ; aq. destil., 90 c. cm. ; alcohol abs., 10 c. cm. ; gentian- violet, 2 grm.J. For double staining, the nuclei-staining carmine * " Mittheilungen aug dem kaiserlichen Gesundheitsamt," Bd. II, 1884, S. 6. + " Berl. k. ■Wochenschrifl," 1884, No. 13. X " Berl. k. Woohenschrift," 1883, No. 33. •"Deutsche med. Woohenschrift," 1882, S. 451; 1883, S. 12 tind 247'. II " Deutsche med. Woohenschrift," S. 351. BACTERIA AND MICROHCOPIOAL TEGHNIQUE. 51 and hsematoxylin* can also be used; the first for blue or violet, the latter for red-stained bacteria. In place of the ordinary nuclei-staining carmine, picro- carmine can be used for preparations of bacteria stained blue, which stains the nuclei an intense red, the fibrillar substance of the connective tissue pink, and the protoplasmic substance a more or less yellovc, so that a threefold staining results. Hsematoxylin is best used in the following solution : Hsematoxylin 2 parts Alcohol 100 " Aq. destil 100 " Glycerine 100 " Alum, sulph 2 " This hsematoxylin stains micrococci, many bacUli, and at the same time the zooglcea masses, with the in- tercellular substance. The staining of the bacteria is paler than that produced by the blue or violet basic aniline-dyes, which, on the other hand, do not stain the zooglcea masses. In order to obtain the threefold staining after the bacteria are stained red by fuchsin, it is better to stain the nuclei with hsematoxylin, and then afterward stain the protoplasm with a satu- rated solution of picric acid or eosin. The rose-red eosin can be added to the above solution of hsema- toxylin in the proportion of one half per cent. (To retain the' yellow color of the picric acid, picric -alco- hol and damar resin must be used.) OTHEE EEAGENTS AKD APPARATUS. Other solutions are often required : {a) Iodine 1 part Potas. iodid 2 parts Aq. destil 100 " * Of. die citirten Handbucher, besonders Friedlander. 52 BACTERIOLOOIGAE INVESTIGATION. (5) In the use of the basic aniline-dyes it is sel- dom necessary to extract the fat. If it is desirable to do this, the sections are first washed thorough- ly in absolute alcohol (three to ten minutes), then are transferred to a watch-glass containing ether and chloroform for a few minutes ; after this they are again placed in alcohol, cleared up in acetic acid (for the solution of the coagulated albumen), and are then examined immediately or after previous staining. (c) Nitric acid, diluted in the proportion of one part of the officinal acid to three or four parts of wa- ter,' is sometimes used. {d) For the removal of the lime salts, according to von Ebner, the following solution may be used, but must be often renewed : Ac. muriat 5 parts Alcohol. 100 " Aq. destU 20 " Sodii chlorid 5 " (e) Acetic acid is employed in from one half to one per cent solution for obtaining the maximal decol- orization and for the examination of unstained bac- teria. (/) Chromic acid is used in a one half per cent solution, or as Miiller's fluid : Potas. bichrom 2 parts Sodii sulphat 1 part Aq. destil , 100 parts {g) The hydrates of potassium or sodium in one to three per cent solutions, or a solution made by adding one to two drops of the thirty-three per cent solution (so much used in histology) to a watch-glass of water, may be employed for rendering the un- stained bacteria visible. BACTERIA AND MICBOSCOPIGAL TEOENIQUE. 53 (A) Grlycerine and alcoliol are always to be used only in pure form completely free from acid. («■) The distilled water which is used in bacterio- logical work should always be sterilized by boiling for an hour over a flame or in a sterilization appa- ratus, since the ordinary distilled water always con- tains bacteria and their germs. AH the solutions used in bacteriology should be made with sterilized distil led. water, and all solutions and reagents must be tested for the possible presence of bacteria. For the preservation of the solutions, flasks with ground stoppers are necessary, and for daily use small glass bottles with hoUow-ground stoppers, having above a rubber cap and below a capillary tube, are very con- venient. These capillary pipettes serve to draw up many or few drops as is desired. For the preservation of the bacteria preparations, glycerine can be used only with the brown dyes, since it more or less rapidly extracts the other aniline col- ors. The glycerine gelatin of Klebs can be used for the brown colors. The saturated solution of potas. acetat. (1-2) can be often advantageously used for the preservation of the stained and unstained bacteria ; but the most univer- sally valuable material for mounting is Canada bal- sam, which is used most conveniently from a tube ifhe artists ordinary paint-tub^ after having been dissolved. For diluting Canada balsam, turpentine or xylol must be used, because chloroform extracts the basic aniline-dyes. For the same reason, the precaution should be taken not to warm the balsam. The much- loved oil of cloves should not be employed for clearing up on account of the same objection ; but in place of it oil of turpentine, cedar, or bergamot must be used. 54 BACTERIOLOGICAL INVESTIGATION. In the way of apparatus the following articles are necessary: good slides and cover - glasses ; watch- glasses ; ordinary porcelain dishes, and those with plain ground bottoms ; crystallization-glasses of differ- ent sizes, preferably two sizes, of which the greater can be used at the same time as a cover for the smaller ; beakers of different sizes ; small, square wire baskets for holding test - tubes ; test - tubes ; wash-bottles ; graduates and pipettes; a plate of black glass or porcelain to place under white or unstained objects ; a plate of white glass or porcelain to place under stained objects ; glass tubes, some drawn out to a capillary-point ; glass rods, some having from 3 to 6 cm. of platinum wire of different sizes melted into one end (to be used straight or bent into a loop) ; spider-forceps ; and many slide-pincettes. On one of these latter instruments the ends should be bent out somewhat, and small pieces of fiat cork fastened to them for taking hold of cover-glasses. The slide pincettes can be improvised by placing over ordinary pincettes the narrowest possible ring of cork. Of ftietal instruments, it is necessary to have some teasing-needles, scissors, knives, and a wide spatula of nickel, steel, or platinum, on which the sections can be spread out in transferring them from one fluid to another. Wew slides and cover-glasses are first cleaned with warm water and dried with a linen cloth. But this is not sufficient, and they must then be carefully rubbed with a cloth which has been dipped in spirit. For the preparation of the flat hanging-drop it is often necessary to allow the apparently clean cover-glass to lie some hours in absolute alcohol ; to remove the remains of the alcohol by ether, and then, finally, to dry by evaporation. Slides and cover-glasses which BACTERIA AND MI0R08G0FICAL TEGENIQUE. 55 have been used are laid in concentrated muriatic, ni- tric, or sulphuric acid, and, after cleaning and removal of the acid, are treated with water until aU acid reac- tion has disappeared, and are then further treated as the new. COVEE-GLASS PkEPAEATIONS.*^ After it was observed that the morphological ele- ments in the blood, dried in a thin layer, were not materially altered by drying, Koch * first employed these casual observations methodically in bacteria investigation. He spread out upon a cover-glass, in a very thin layer, a drop of fluid containing bacteria, so that the individual elements were brought very nearly on the same plane. This thin layer was then fixed by simply drying in the ^ir. In order to elimi- nate the slight alteration produced by this, it is nec- essary afterward to cause again a swelling up of the bacteria. If the layer which has been dried in the air remains too long in the water or glycerine used for this purpose, it is entirely dissolved, instead of only partially swelling. If the cover-glass with the dried layer is laid in absolute alcohol, or a one half per cent solution of chromic acid, the layer is rendered insoluble in water and glycerine, and no longer swells up. But if the layer which has been made insoluble is put into po- tassium acetate, it swells sufficiently without being entirely dissolved, and aU the forms seem to be in a natural condition. The solutions of the aniline-dye have the same action and cause the same swelling without removing the layer, and at the same time stain the bacteria. * " Verfahren ziir Untefsnchting," etc. " Beitrage znr Biologie der Pflanzen," 1877, II, 3. Heft, S. 899. 56 BACTERIOLOGICAL INYESTIQATION. In tlie use of this method in the investigation of the blood, Ehrlich * found that the rapid drying pre- vented coagulation of the cell-albumen, and retained the natural staining capacity of the elements. Only the haemoglobin was extracted by aqueous and gly- cerine solutions of the dyes. But if the prepara- tions were kept for a few hours at a temperature of 115°-135° 0., the elements of the blood, without any important alteration and without the appearance of artificial products, retained their elective affinities for dyes. In following up these observations, Koch f discovered that, in place of the fixation by alcohol, the application of heat for only a few minutes an- swered the same purpose. A drop of the fluid containing bacteria, either un- diluted or after the addition of a drop of distiUed water (according to the amount of its morphological elements), is spread out in a thin layer upon the cover-glass, by means of a pointed scalpel or platinum wire, and the excess of fluid soaked up with filter- paper ; or a drop is placed upon one cover- glass and a second is applied to this, which, through its press- ure, spreads out the drop in an even layer. If, then, the two cover-glasses are drawn apart with pincettes, we have two similar preparations. The cover-glasses, protected from dust, are allowed to remain untU com- pletely dry, or they can be dried in a dry-oven some- what more rapidly. The drying can also be hastened by holding the cover-glass with the prepared side upward high above the gas-flame, and moving it to and fro to prevent the direct action of the flame. Upon the dried preparation a drop of the Staining * " Zeitsohrift f. Min. Med.," Bd. I, 8. 563. t " Ziir Untersuohung von pathogenen Organismen." " Mitthei- lungen aus dero kaiserlichen Gesundlieitsamte," 1881, Bd. I, S. 1. BACTERIA AND MIOROSGOPIOAL TECHNIQUE. 57 solution can then be placed to stain the elements, but only in case the fluid is free from albumen and the staining foUows quickly, since, by the prolonged ac- tion of the staining solution, the layer is completely loosened. If the dried layer consists of an albumi- nous substance, such as blood, tissue-fluids, or sputa, on the addition of the staining solution, precipitation occurs. On this account it is especially necessary that the preparation, after drying in the air, should be more securely fixed by heating. Tor this purpose the cover-glass may be placed in a drying-box or upon a copper plate. The copper plate is laid upon a tri- pod, and one end is heated by a gas-flame, so that the different portions, at different distances from the flame, have varying degrees of temperature. A few minutes' exposure to a temperature of 125° C, or ten to twenty minutes at 110° C, is sufficient to thor- oughly dry bacteria preparations. This may be done more convenieutly, and in some cases also more cer- tainly, according to Koch-Loeffler, if the cover-glasses with the dried layer are drawn rather rapidly three times through a gas- or spirit-flame. The reason for heating in exactly this manner, ac- cording to Koch,* is this: because in preparations which have not been heated the above-described pre- cipitation occurs, while in preparations which have been passed through the flame only once or twice, the fixation of the elements, especially in those containing much albumen, is not sufficient for all cases. In those passed through the fiame three times, while the forms themselves are not materially altered, the capacity for staining is retained, and the albuminoid material has become so insoluble that precipitation no longer * " Mittheilungen," 1884, Bd. II, S. 7. 58 BACTERIOLOGICAL INYESTIOATI'ON. takes place. Passing the preparations througli the flame a yet greater number of times destroys the susceptibility of the bacteria to the staining fluid. The want of success in making preparations, which many beginners experience, seems to be especially due to the fact fliat the preparations are generally heated before they have been completely dried in the air. If the preparation still contains any water, coagulation of the albuminoid material occurs when heated, while in those completely free from water this does not hap- pen, and the albumen is rendered homogeneous by heating. The preparations, dried in the air and then drawn three times through the flame, are now stained. The cover-glasses, with the prepared side upward, are laid on a piece of filter-paper, and, by means of a glass rod, a cap pipette, or the glass stopper with the capil- lary-tube, a few drops of a staining solution are placed upon the preparation. The staining fluid should remain about twenty minutes, or until it is seen, by an inclination of the cover-glass, that the preparation has already taken up the color. If the action of the staining solution ought to be prolonged, then it should not be placed drop by drop on the cover-glass, because, in drying, the staining solution forms a ring of color at the edge which it is difficult to remove. In this case a sufficient quantity of the staining solution is placed in a watch-glass or crys- tallization-glass, and the cover-glass is then taken, with the prepared side downward, between the thumb and iadex or middle finger, and allowed to fall flat upon the surface of the staining solution, so that it swims with the prepared side upon the surface of the fluid. To prevent evaporation, the dish is covered with a glass plate. BACTERIA AND MIGR0800PI0AL TECHNIQUE. 59 For the removal of the excess of coloring-matter, a stream from a wash-bottle is thrown obliquely from above upon the cover-glass, taking care not to strike the surface of the preparation directly ; or the cover- glass, held in pincettes, is moved to and fro in a beaker filled with distilled water ; or the excess of the fluid may be soaked up with filter-paper, a few drops of water added to be soaked up anew, and so on until none of the coloring-matter is given up to the filter- paper. Then the cover-glass preparation is examined in a drop of distilled water. The upper side of the cover-glass is freed from every particle of water by soaking it up with filter- paper, because on it must be placed a drop of oil for the homogeneous immersion-lens. If the cover-glass preparations are to be preserved, the oil is removed with filter-paper and chloroform, the water by careful warming or evaporation (pro- tected from dust), and the dried preparation is di- rectly imbedded in Canada balsam. Different dyes are used for each variety of bacteria, since some stain only the bacteria ; others at the same time the fine gelatinous sheath ; others the cap- sule. On this account the correspondtag pictures in all the methods of staining are not absolutely similar ; so that it ought to be self-evident that al- ways, in comparison, only preparations should be used which have been treated in exactly the same manner. These considerations must guide one in the choice of the staining solution. We must therefore distinguish between staining for a special purpose — ^i. e., for the establishment or employment of coloring methods which have been described or proved to be best in particular cases — and the investigation staining used especially to prove the presence of bacteria. 60 BAGTERIOLOGIGAL INVESTIGATION. Since in cover-glass preparations almost ail bac- teria can be stained by watery solutions of the basic aniline-dyes, saturated watery solutions or tlie equal- ly valuable alcoboUc solutions are first employed. The saturated watery solutions have for this testing an advantage, because aU basic aniline-dyes are known to be applicable ; so, with a few preparations, the dif- ferent colors can be tried. If no bacteria come to view in this way, notwith- standing their supposed presence, then aniline-water, with methyl- violet or f uchsin, is used, or the stronger alkaline solution of methyl-blue. The trial examina- tion as to the presence of bacteria resolves itself, in the larger number of cases, into the following pro- cedure : 1. Drying in a thin layer. 2. Fixation by passing the cover-glass three times through the flame. 3. Staining by placing a few drops of a watery or dilute alcoholic solution of a basic aniline-dye upon the preparation. 4. Removal of the excess of the coloring-matter by washing or soaking up with filter-paper. 5. Examination in a drop of distilled water. For the.isolated staining of bacteria in cover-glasa preparations they can be laid for about one minute in a half- saturated solution of potassium carbon- ate ; or, if they are stained in aniline- water-gentian- violet, the remaining elements can be decolorized ac- cording to the method of Gram.* The stained cover- glass preparations are for this purpose laid for about one minute in the solution of potassium iodide {vide page 51), and then placed in absolute alcohol until * " Ueber die isolirte Farbung der Schizomyceten." " FortscLritte der Medizin," 11, 1884, No. 6. BACTERIA AND MIGROSGOPIOAL TEOENIQUK 61 they appear decolorized. The alcohol is soaked out and the preparation examined in water. For double staining the cover-glass preparations, after being decolorized according to the Gram meth- od, they can be taken from the alcohol and placed in a weak watery solution of vesuvin. Then the bac- teria remain blue, often almost blue-black, while the nuclei are stained brown. The preparations stained red or blue can also afterward be stained with car- mine or hsematoxylin ; yet this double staining has much less value in the cover-glass preparations than in sections. BXAMUSTATIOlf FOK TFBEBCLE BACILLI IN SPUTTTM. These preparations can be stained according to the Grram method; but by this both the tubercle bacilli and other bacteria are stained blue in contrast with the brown nuclei. For the differential diagnosis this is not sufficient, and for this purpose the principle established by Koch must be exclusively observed — i. e., that the tubercle baciUi should be stained in a different color from other bacteria and the nuclei. Koch succeeded in doing this, in preparations stained for twenty-four hours in a weak alkaline solution of methyl-blue, and then placed for a short time in a watery solution of vesuvin. In this way the tubercle bacilli (and the bacilli of leprosy) are stained blue ; all other bacteria and nuclei, brown. After this im- portant principle was discovered, Ehrlich showed that aniline- water was stm a better agent for increas- ing the intensity of the color, and that in the prepara- tions stained with aniline-water colors the tubercle bacilli withstood decolorization by nitric acid, while all other bacteria were decolorized by this mineral acid. But the preparations can not be left so long in 62 BAGTEEIOLOGIGAL INVESTIGATION. the acid ttat complete decolorization occurs, because then also many or aU of the tubercle bacilli are de- colorized. They should remain in the acid until the red (fuchsin) or blue (methyl- violet) hue has changed into a yellow-red or greenish blue. At this stage the preparations are placed in water, and again a red or blue color appears. By the action of the acid the simple acid union (red or blue) is changed into a triple acid (yellow-red or blue-green), and, by the addition of the water, the triple acid union is de- stroyed and the red or blue hue reappears. The preparations decolorized by the acid are not washed in water, but in 50 or 60 per cent alcohol ; then they are stained in a dilute solution of methyl-blue (or vesuvin). After washing away the methyl-blue (or vesuvin) the preparations are examined in water, or, after removal of the water, preserved in Canada balsam. After this whole procedure, the tubercle bacilli retain their red or blue color, and are easily recog- nized among the other elements. Aside from this differential diagnostic action of the double staining, the subsequent staining in another color has an ad- vantage by affording an easier examination of the specimens. Concerning the choice of the material containing bacteria, it is to be noted that the cheesy masses are to be spread out thin with a sterilized scalpel. Nod- ules of tubercle must be crushed with a scalpel or between two scalpels, and then be pressed flat upon the cover-glass. The tough, yellowish masses from the sputum are used. One of these particles is taken and spread out in a thin layer on the cover-glass, or flattened by pressing one cover-glass upon another, so that, after separating the two cover-glasses with BACTERIA AND MICROSCOPICAL TECHNIQUE. 63 pincettes, two preparations are obtained. The entire method is, according to Koch (after the adoption of the previously described aniline-water staining of Ehrlich), briefly as follows : 1. Pass the dried cover-glass preparations three times through the flame. 2. Stain with the Weigert-Koch solution of methyl- violet or fuchsin for twelve hours. 3. Treat with dilute nitric acid (1 to 3 or 4) for a few seconds. 4. Wash in a 60 per cent solution of alcohol by a to-and-fro motion. 5. Stain in a dilute solution of vesuvin or methyl- blue. 6. Wash and examine in water or mount in bal- sam. This method is the best thus far discovered, and serves as a control in aU. doubtful cases. For the differential diagnostic decolorization and subsequent staining of preparations of tubercle ba- cilli, the following reagents have been used : 1. Other aniline-dyes (vesuvin by Koch). 3. Acids (nitric by Ehrlich, hydrochloric by Orth, acetic by Petri). 3. Acid alcohol (weak nitric by Eindfleisch, and hydrochloric by Orth). B. Fraenkel combined these three variations by preparing an acid-alcoholic solution of methyl-blue and of vesuvin. a. For blue: Alcohol 50 parts. Water 30 " Nitric acid 20 " Methyl-blue to saturation. To be filtered. 5 64: BAOTERIOLOGIGAL INVESTIGATION: 6. For brown ; Alcohol 70 parts. Nitric acid 30 " YesTivin to saturation. To be filtered. For the use of these solutions the following method of Fraenkel is recommended : About 5 c. cm. of aniline-water are heated in a test-tube to boiling and poured out in a watch-glass ; to this hot aniline- water a, concentrated alcoholic solution of fuchsin or methyl-violet is added, drop by drop, untU the solu- tion of the dye assumes a cloudy appearance. Upon this warm solution the cover-glass preparations are allowed to swim, and even in two or three minutes most of the tubercle bacilli are stained ; but, as a precaution, they should be left five or ten minutes. From this staining solution, the preparations stained red or blue are passed into the blue or brown acid- alcohol solution. After remaining one or two min- utes in the latter, the preparations seem colored and are washed in water or 50 per cent alcohol, to which one half per cent acetic acid has been added, and are then examined in water. Those who prefer the hydrochloric acid of Orth can employ the following method of KaatZer : stain as before, and then decolorize with a mixture of — Alcohol (90 per cent) 100 c. cm. Water 20 c. cm. Concentrated hydrochlor. acid 20 gtt. wash with 90 per cent alcohol to remove the acid,- and then stain with a concentrated watery solution of methyl-blue or vesuvin. In sputum, according to Celli and Guarnieri, some- tipaes very fine fat-crystals are found, which react to the staining almost as the tubercle bacilli, "pseudo- BACTERIA AND MIOROSGOPIOAL TEOHFIQUR 65 bacilli," but which, on careful examination, are not to be confounded with them, on account of their vary- ing size and because they are dissolved by ether and chloroform. The published modifications of the Ehrlich meth- od, founded on the Koch principle,, are so numerous, but without anything having been added, that I must on this account simply refer to some of the compre- hensive descriptions,* and content myself with giving the ■ underlying method of Koch (which is now best employed according to Ehrlich- Weigert-Koch), and two practical modifications of it. In these methods the bacilli of leprosy react as the tubercle bacilli, from which, moreover, they are mbrpholo^caUy not easy to differentiate. The differ- ential diagnosis by staining is founded on this fact : that the bacilli of leprosy do not stain so deeply as the tubercle bacilli, and that, in respect to the ease with which they give up the dye, they stand between these and most other forms of bacteria. According to Baumgarten,f the dried cover-glass preparations are allowed to float for six or seven minutes in a dilute alcoholic solution of fuchsin (^ or 6 drops of a con- centrated alcoholic solution in a watch-glass of dis- tilled water), decolorized fifteen seconds in acid alco- hol (1 part nitric acid to 10 parts alcohol), washed in distilled water, afterward stained in a watery solu- tion of methyl-blue, washed again, and examined in water. * Kaatzer, " Die Technik der Sputumuntersuohung auf Tuberkel- Baoillen," 1884. B. Fraenkel, "Ueber die Farbung des Koch'sohen Bacillus," "Beri. klin. Woohenschrift," 1884, No. 13. Banmgarten, "Beitrage zur Darstellungsmethode der Tuberkel-Baoillen," "Zeit- schrift fur wissensehaftliche Mikroskopie," I, 1884, 8. 61. t " Ueber Untersnohungsmethoden zur Unterscheidung von Lepra- nnd Tuberkel-Baoillen," ibid., 8. 367. ' 66 BACTEBIOLOGIOAL mVESTIQATIOK The bacilli of leprosy seem then as red rods upon a blue ground, while the tubercle bacilli during this time, by this treatment, have as yet taken up no color. The reaction of the tubercle bacilli, both in the method of staining discovered by Koch with its differ- ent modifications, and in the method of Baumgarten (the recognition of these bacteria in unstained condi- tions), seems at first to separate these bacilli qualita- tively from all other bacteria. Further studies, how- ever, have shown that these differences are not quali- tative, but are especially quantitative. The tubercle bacilli are stained with the greatest difficulty; but they retain the color persistently. It has been shown since by Lichtheim,* and especially by the work of Baumgarten, previously cited, that the tubercle bacilli in dried cover-glass preparations are stained in about one hour, both in dUute alcoholic and strong aqueous solutions of methyl-violet, gentian- violet, and fuchsin, or by simultaneous warming, in about five minutes. (For sections the time required is about twelve hours, or about ten minutes with elevation of temperature.) These stainings withstand the decolorization by acids for some time, although after a longer action of the same the tubercle bacilli are also decolorized. The tubercle bacilli remain unchanged after treatment with carbonate of potassium, as do other bacteria. If the stained cover-glass preparations are laid for de- colorization for about one minute (sections, five min- utes) in alcohol, and afterward for five minutes (sec- tions, fifteen to twenty minutes) in a concentrated aqueous solution of vesuvin or methyl-blue, a double staining is obtained. Therefore, neither the addition of the alkali nor the aniliae-water is absolutely essen- * "Znr diagnostischen Verwerthung der Tuberkel-Bacillen." "Fortschritte der Median," 1883, S. 1. BACTERIA AND MIGR08G0PI0AL TECHNIQUE. 67 tial to the staining ; the reaction to acids is not a qualitative differentiation from other bacteria, and the use of the acid is not entirely necessary for ob- taining the double staining. A satisfactory explanation of the theory of stain- ing, which seems to lie so near, has not yet been dis- covered, on account of the varying quantitative re- action of the tubercle bacilli. The possibility of a better understanding is offered by the following fact : The addition of an alkali, as well as the feebly alka- line aniline-oil, renders the staining easier. Other aromatic bodies and ammonia act in a similar man- ner. The addition of an acid to the aniline-water does not arrest its action, so that probably the favor- able action of carbolic acid is reduced to the aromatic element, and is effectual in a similar manner in spite of the acid reaction. In addition, Gibbs * showed that in the simulta- neous action of two aniline-dyes the bacilli are stained differently from the other elements, while by another method these are stained in both of the dyes. A so- lution of 3 c. cm. of aniline-oil in 15 c. cm. of spir- its is added slowly to 2 grammes of fuchsin and 1 gramme of methyl-blue, and, after the solution of the dyes, 15 c. cm. of water are added. This solution is warmed, the cover-glass preparation is laid upon it for five minutes, and then washed in spirits till color is no longer given up. The bacilli then appear red upon a blue ground. This method is, unfortu- nately, not sufficiently reliable. EXAMINATION OY BLOOD FOE BACTEEIA. The examination of blood for bacteria offers very great difficulty, because in the normal blood within * "Lancet," 1883, p. 111. . 68 BACTERIOLOGICAL INVESTIGATION. tlie vessels, and in the normal disintegration of the healthy blood, granular elements are present, or are formed, which, under certain pathological conditions, in anaemic states and in fever, are increased in num- ber, and can be easily mistaken for micrococci. They have already been often confounded with microcoooi, and are almost daily mistaken for them — e. g., the re- nowned syphilitic corpuscle, and the so- called organ- isms of the venom of serpents. Here belongs also much of what has been spoken of as the development of bac- teria from nitrogen molecules, from microzymen, or from the anamorphosis of protoplasm. An exact study of these granules of the blood is on this account an indispensable desideratum in bacteria investigation. These granules form, further, a constituent part of the cellular elements of the blood, and on this ac- count again are of interest in aetiology, because there are parasites which are similar to the amoeboid cells — e. g., those monads found by Lewis in the blood of rats, by Koch in the blood of marmots. The ele- ments of the bloodj which directly or through their granules may be confounded with micro-organisms (with the exception of the red blood-corpuscles, and the products of their disintegration), are divided, ac- cording to Ehrlich,* into — 1. Lymphoid elements. (ds) Small lymph-cells. (5) Larger lymph-cells. 3. Myeloid cells (eosinophile). 3. Undetermined (spleen and [or] marrow). («) Large mononuclear cells. (5) Transitional forms, (o) Polynuclear. * Ofr. the work of Ehrlich, Westphal, Sohwarze, and SpillinR, " TJeber Blutuntersnohungen bei LenkSinie," Dissert., Berlin, 18BC ; and Einhorn, " Ueber das Verhalten der Lymphooyten zu den weis- Ben Blutkorperohen," Dissert., Berlin, 1884. BACTERIA AND MICROSCOPICAL TECHNIQUE. 69 The small lymphoid elements are somewhat small- er than the red blood-corpuscles, possess a very large nucleus, so that there is very i little or no protoplasm to be seen. The large lymph-elements are a further development of the first, and are only to be differen- tiated from them in this, that they possess around the large nuclei a distinct border of protoplasm. The myeloid elements are large, round cells, with large, oblong nuclei. The mononuclear cells are about three times the size of the red blood-corpuscles, and possess round or oval nuclei of large size, and a con- siderable mass of protoplasm. The mononuclear transitional forms are to be differentiated from these cells only in this, that the nuclei are no longer round or oval, but have become indented. The polynuclear elements are somewhat smaller, but stiU are always larger than the red blood-corpuscles, and their nuclei show, as a further differentiation, a polymorphous form. These are the true white blood-corpuscles. The granular elements, or granules, which are pres- ent in the cells, and which become free in the de- struction of the same, are divided with respect to their reaction to anUine-dyes. The a, or eosinophile granule, is coarsely spher- ical, strongly refracting, and can be stained in aU the acid aniline-dyes. It is present in the myeloid ele- ments, seldom in the normal blood, and its number is greatly increased in leucsemio processes. The /8, or amphophile granule, is found especially in the marrow, very often in the leucocytes in the blood of rabbits and guinea-pigs, and can be stained by acid and basic aniline-dyes. The 7, or basophile plasma ceU granule, can be stained, like the bacteria, by the basic aniline-dyes. These granules are coarse, slightly refracting, almost 70 BAOTEBIOLOOIOAL INVESTIGATION. completely wanting in normal human blood, increased in leucsemic processes, and are present normally in the blood of the lower animals, especially the white rat. The S, or basophUe granule, is fine, and can be stained in basic aniline-dyes, and forms a constituent part of the large mononuclear elements. The e, or neutrophile granule, is very fine, and fills the polynuclear elements of the human blood quite thickly, is present sparsely in the transitional forms, and very seldom in the mononuclear ele- ments. It can be stained by the neutral dyes. Without recourse to staining, these granules, as a whole, and also the products of the disintegration of the red blood-corpuscles, may be confounded with micrococci. By systematic staining with the aniline- dyes, the a, /3, and e granules can be excluded. An error is then polsible only with the 7 and S granules, because these are stained in the basic aniline- dyes, the same as the bacteria. These last, on account of their fine grain, can, with comparative ease, be differ- entiated from micrococci, and have not, as yet, been confounded with them. The plasma cell granules, on account of their medium size, come so near to the known forms of cocci, that not only the individual free granules in the blood have been considered as cocci, but even the same so-called plasma cells in the tissues have been described as colonies of cocci. They can, on purely morphological grounds, be dif- ferentiated from them in this way, viz., that they do not aU have the symmetrical appearance of the cocci, but present the greatest differences in the size of the granules. If it is desired to examine the blood for bacteria, a small drop is rapidly spread out in a thin layer, dried, fixed, and then passed three times through the BACTERIA AND MIOROSGOPIGAL TECHNIQUE. 71 flame, arid then stained in the ordinary manner. In such preparations the bacteria are sufficiently stained, but not the granules. The small drop of blood, at most the size of a pin- head, must be taken with the greatest care. In case of a haemorrhage, a small drop is taken with a steril- ized platinum wire, or, for obtaining the blood, the skin may be pricked with a previously sterilized needle, the end of the finger being the best spot. The skin is first cleansed with a brush and soap, and then washed with a solution of corrosive sublimate, 1 to 1,000. The sublimate is removed with alcohol, the alcohol with ether, and the last allowed to evapo- rate. The first drop of blood which weUs up is re- liioved with a sterilized platinum wire, and the fol- lowing drop used, a cover-glass being lightly pressed upon it with the pincettes, without coming in contact with the surrounding skin. Upon this first cover- glass a second is laid, which, by pressure, spreads out the blood in a thin layer, in which the elements are not materially altered. The two cover-glasses are then drawn apart by pincettes, so that two cover-glass preparations are obtained. Some of the cover-glasses are used as above in the examination for bacteria, after the layer has been dried in the air, and heated only for a short time ; but others are heated for one hour at a temperature of 120° C, and then treated with a basic aniline-dye, in order to study more ex- actly the basophile granules. Other preparatioris for the determination of the eosinophile elements are treated with the acid aniline- dyes after heating a short time, and also after an hour's heating. A mixture is made of the yellow, red, and black dye-stuffs of the strongest staining power, each of which alone stains all of the acid- 72 BACTEEIOLOGICAL INVESTIGATION. forming elements, and both the simultaneous action and the elective staining are valuable, since the three eosinophile elements are stained at the same time in difEerent colors. One part of a saturated glycerine solution of aurantia is diluted with two parts of glycerine; then aniline-black (sulphate of indolin) and eosin are added in excess, and, by long .shak- ing, dissolved to saturation. This saturated glyc- erine solution stains all the parts containing hae- moglobin intense orange, the nuclei gray-black to black, and the eosinophile granules red to red- black. For staining the neutrophile granules the neutral dyes are used, which are formed by the union of the basic and acid dyes — e. g., if acid-fuchsin and orange (G) are mixed with the basic methyl-green. Accord- ing to Ehrlich, 125 c. cm. of a saturated aqueous solu- tion of orange are mixed with 125 c. cm. of a saturated solution of acid-fuchsin in 20 per cent alcohol ; 75 c. cm. of absolute alcohol are then added, and after- ward 135 c. cm. of a saturated aqueous solution of methyl-green with shaking. The solution remains standing for some time, and both a precipitation occurs and a film forms on the surface. In order to obtain the solution quite clear, a pipette is introduced into the middle of the solution and a quantity of the clear fluid is withdrawn. The pipette as well as the vessel must be absolutely dry, or otherwise a further cloudiness appears. By this mixt- ure the haemoglobin is stained yellow to orange, the nuclei green, neutrophile granules violet, and the eosinophile dark gray with a'tinge of blue. For the demonstration of the cells in the blood the following mixture is used : * * Ehrlich, " Deutsche med. Wochensohrift," 1883, Ko. 46. BAOTEBIA AND MICROSOOPICAL TEOHNIQUE. 73 Water 100 c. cm. Glycerine 100 " Absolute alcohol 100 " Hsematoxylin 1-2 grm. Eosin 1 " Glacial acetic acid 10 c. cm. Alum to saturation. The red blood-corpuscles show an intense red color, the nuclei of the lymphoid and polynuclear cells are stained intensely blue, and the nuclei of the mono- nuclear cells bluish green. The protoplasm of the large lymphoid and polynuclear cells is reddish, that of the mononuclear cells dark green. The importance of this method of examination for micro-organisms in the blood has been lately pointed out by Koch ; * but it has not as yet received neces- sary attention. The descriptions given here are all the more needful because our good text-books on histological technique do not sufficiently treat this subject, for the reason that the original works are so difficult of access. Methods of Staiwing the Flagella. The flagella (Fig. 4 ; 7, 9, 14, 16), which become visible in the hanging-drop at one or both extremi- ties of the bacteria by f oimtng an eddy, can be best stained in dried cover-glass preparations, according to Kochjt by the addition of a concentrated aqueous solution of campechianum. The flagella are stained brown, but the staining in this manner is not durable. On this account the stained preparations are laid for some time in a 5 per cent solution of chromic acid or * " Mittheilungen," Bd. I, 1881, S. 7. t " Verfahren zur Untersuoliung, etc.'' "Beitrage zuir Biologie der Pflanzen," 1877, Bd. II, 3. Heft, S. 419. 74 BACTERIOLOGICAL INYESTIGATIOK in MuUer's fluid. Then there is formed an insoluble brownish black union of the extract of haematoxylin with the chromic acid. After washing, these prepa- rations can be directly preserved in glycerine, or, after drying, in Canada balsam. Methods op Staininq- Spokes. The spores of bacteria were first observed and de- scribed, but'not rightly understood, by Perty.* Then Pasteur f made a sharp distinction between the biol- ogy of an organism and its spore without quite solv- ing the question morphologically. Cohn:}: was the first to describe biologically and morphologically the formation and germination of spores. Further pecul- iarities were observed by Koch, Brefeld, Buchner, and especially Prazmowski,* who clearly described the different forms of the germination of spores (Fig. 4 ; 18 and 19). By his observations this process of fructification derived a heightened significance. The observation of the spores in the unstained con- dition finally followed (Fig. 4 ; 5 b, 8, 9, 10, 12). They appear, especially in the hanging - drop, as strongly refracting round or oval bodies either within the less refractive bacteria, or free near these. Sometimes they are situated near the middle, sometimes at the end. The cells in which they appear are sometimes unaltered, sometimes peculiarly swollen. Since con- * " Zur Kenntniss kleinster Lebensformen," 1832. Taf. XV, Fig. 26, and following. f "fitudes sur la maladie des vers A 8oie," 1870, I, S. 228. X "Beitrage zur Biologie der Pflanzen," 1876, Bd. II, 2. Heft, S. 263. * " Untersuchtingen fiber die EntwicklungsgeseMchte und Fer- mentwirkung einiger Bakterien-Arten," 1880, und " Ueber den gene- tisohen Zusammenhang der Milzbrand und Heubakterien." "Biolog. Oentralblatt," 1884, No. 13. BACTERIA AND MIOROSOOPIOAL TECHNIQUE. 75 densed bacteriarprotoplasm, according to Prazmow- ski, strongly refracts light, it is to be concluded that a body still more strongly light-refracting is to be re- garded as a spore. Here belong, together with the foregoing morpho- logical changes which regularly appear under certain biological conditions in spores, their great resistance to chemical agents, and especially to high temperature, and the fact that in the use of watery or dilute alco- holic solutions they are not stained, but appear as un- stained refracting spaces within the stained bacteria. An accidental observation showed how the spores could also be brought to view stained. Koch * saw, in staining the tubercle bacilli with aniline-water- methyl-blue, that the spores of a species of large bac- teria were stained blue at the same time, while the bacteria themselves were stained brown by the subse- quent treatment. Gaffky was not able to stain the spores of other bacteria in the same manner. On the contrary, If eisser succeeded in staining the spores red and the bacilli blue when he used warm aniline- water- fuchsia and subsequently stained with' methyl-blue. Bienstock f also used this staining. Further, Buch- ner:]: discovered a means for the isolated staining of spores. Because the staining of the living bac- teria was not successful, but those killed by dry- ing and heatittg were readily colored, Buchner thought that the reason the spores were not stained was because of the greater resistance of the spore- membrane. He endeavored, therefore, to destroy the membrane of the spores of the bacillus subtilis, and * "Mittheilungen," 1884, Bd. II, Tafel V, Fig. 23. t "Zeitsolirift ffir klin. Med.," 1884, 8. 1.^ X "TJeber das Verhalten der Spaltpilzsporen zu den Anilinfar- ben." "Aerztliches InteUigenzblatt," 1884, No. 33, S. 370. 76 BACTERIOLOGICAL INVESTIGATION. tlms make them accessible to the staming fluid. In this manner he succeeded in staining spores in dried cover-glass preparations which had been heated from one half to one hour at a temperature of 210° C. in a dry-oven, or one hour in a steam-kettle at 120° C. A successful result was also obtained when the prepara- tions were dipped in concentrated English sulphuric acid for fifteen seconds and afterward carefully washed, or when they were subjected for a longer time to a concentrated solution of caustic soda. In preparations thus treated, especially in the use of methyl-blue, the spores alone are stained, while the bacteria themselves no longer take up color. Even before the publication of the brief article of Buchner, I had endeavored to bring the spores into view by both isolated and double staining. Although these investigations are not yet in all directions con- cluded, still I shall now describe the general results, because this part of the technique is as yet little known and used. If bacteria are examined in the hanging -drop shortly before the formation of spores, in many, of them refracting bodies are found which have not yet a size equal to that of spores. When the prepa- rations, dried and fixed by passing them through the flame three times, are stained with an aqueous or di- lute alcoholic staining solution, the bacteria in this stage are found to be stained, not so equally as before, but some parts are more deeply colored. This con- densed protoplasm takes the dye more readily than the protoplasm not thus concentrated. (I had pre- viously noticed intimations of this in the involution forms of the bacillus cyanogenus.) Then follows the stage in which the refracting corpuscles become much more equal in size, but they stiU stain well ; and finally BAOTEBIA AND MI0R08G0PICAL TECHNIQUE. 77 a stage in wMch similar refracting corpuscles are pres- ent in the unstained preparations, but these no longer take up the color. Now the spores have first ob- tained an insusceptibility to the dye through the formation of a membrane difficult of penetration, which prevents the absorption of the coloring-matter. In the cover-glass preparations, which have been passed through the flame three times, the bacteria and nuclei are stained weU. If they are passed through the flame more times, say six, the bacteria are stained successively worse, but the nuclei still absorb the dye well, as does also the condensed protoplasm of the bacteria which has not yet formed spores. At this stage, besides the nuclei, granular elements can often be seen which may easily impress one as belonging to the badly stained bacteria. If the preparations are passed through the flame stiU more times, say ten, then both the nuclei and the condensed proto- plasm lose their susceptibility to the dye, and the spores gain it. In the case of some of the bacteria of putrefac- tion, it is sufficient to pass the preparation through the flame only seven times, but in others ten times are required. (In the dry-oven a similar stage is reached in from fifteen to thirty minutes at 180° to 200° 0.) The spores then take up aqueous solutions of red, violet, blue, brown, and green basic aniline- dyes. This isolated staining for the proof of the resist- ance of the spores, as Buchner intended, is perhaps sometimes to be used ; but, since in this way noth- ing is learned concerning the relation of the spores to the bacteria, it is better to use a double staining. The procedure is almost the same, quantitatively in- creased, as that for staining the tubercle bacilli. 78 BAOTERIOLOaiCAL INVESTIGATION. Either the preparations, passed through the flame three times, are stained with a strong alkaline solu- tion from twelve to twenty-four hours (less advan- tageously by warming for one hour), and afterward stained with vesuvin, or the anUine-water-dye solu- tions are used, of which that of Keisser, previously described, has proved to be the most convenient and satisfactory. Stain in hot anUine-water f uchsin, de- colorize with nitric acid, then stain with methyl-blue. Some spores are stained, moreoyer, by saturated aqueous or dilute alcoholic solutions, if they are at the same time heated. The difference of spores, in respect to their susceptibility to dyes, seems to be scarcely less than that of the bacteria themselves. Peepabation oe Sections. Pieces of fresh organ can be cut with the freez- ing microtome. The sections are placed in a one half per cent solution of sodium chloridum, and in part examined fresh, in part stained. For the latter pur- pose, the section, according to Weigert,* is spread out upon a section-lifter in the salt solution with the help of a needle, is then taken out, and the excess of the fluid removed with filter-paper. Then the section is placed in absolute alcohol, introducing it slowly, where it remains at least until the disappearance of all the air-bubbles arising from the thavring of the frozen specimen. Then the section is placed in a staining solution, for which purpose vesuvin is most recommended, because for staining in the other dyes the sections must remain longer in alcohol. Accord- ing to Friedlander,t the section can also be taken out * Virchow's " Archiv fflr pathologische Anatomie," Bd. LXXXIV, 1881, S. 290. t " Mikroskopische Teohnik," 2. Aufl., S. 119. BACTERIA AND MICROSCOPICAL TECHNIQUE. 79 of the salt solution and placed directly in the brown staining fluid, then for a short time in alcohol, and finally in glycerine, or oil of cloves and Canada balsam. For the study of bacteria, it seems to me that the preparation of fresh sections is entirely superfluous work. In the same time in which available sections for this purpose can be made, a dozen dried covers- glass preparations of the tissue-juice can be obtained, which furnish better information as to the presence and import of bacteria. For the exact study of the presence and distribu- tion of bacteria in the tissues, it is necessary to harden the tissues thoroughly, and from the hardened speci- mens to prepare with the microtome a series of deli- cate sections. These sections are partly examined un- stained and partly after staining. Since both the staining itself, as well as the maximal decolorization, is dependent upon the condition of the preparation produced by hardening, it is of the first importance for bacteria specimens to use absolute alcohol as a hardening agent. The susceptibility to dyes, of tis- sues hardened by other agents, as chromic-acid salts, is inconstant ; but possibly these hardening agents are for other parasitic- micro-organisms as valuable as alcohol. (Cf. page 68.) The susceptibility of the albuminates to dyes de- pends very much upon the presence of water ; and since the albumen coagulated by alcohol retains a certain amount of water, which in the course of a few days is entirely removed by the absolute alcohol, the hardening in alcohol must continue until this condi- . tion is constant. For this purpose a small piece of the organ, about the size of a hazel-nut, is allowed to remain at least three days in a large quantity of fre- quently changed absolute alcohol. 80 BACTERIOLOGICAL INVESTIGATIOK In unstained sections from fresh or hardened tis- sue, the bacteria are made visible by their resistance to acids and alkalies.* The sections are fully cleared np by 50 per cent acetic acid or 1 to 3 per cent caustic soda or potash. The bacteria withstand the action of these reagents, as was shown by von Recklinghausen (page 38). Old spirit preparations are heated in these solutions to near the boiling-point. In preparations which have thus been made transparent, the bacteria can be sometimes recognized by the characteristic forms of the individual germs, as was found by Baumgarten (page 38) for the tubercle bacillus, and by Friedlander for the typhoid bacillus. In the case of bacteria not distinguishable by the characteristic form of the single individuals, especially in the cocci, their grouping as diplococci, sarcina, torula, or zo- oglcea is often characteristic. These bodies, of equal size, withstand the action of ether and chloroform, unlike fat-drops, which may be confounded with them. In the vessels the masses of. cocci are characterized, according to von Recklinghausen, by the production in the course of their growth of a varicose condition of the vessels. If we find, in a section of fresh or hardened tissue, masses or chains of small bodies which are of a simi-. lar size, and which withstand both the action of al- cohol and ether, and the treatment with concentrated acetic acid and the alkalies after warming, these are to be considered, according to Friedlander, f as micro- organisms. More important is the determination, by staining, of the presence of bacteria in sections hardened in al- * Friedlander's "Zasammenstellung in der mikroskopischen Teoh- nik," 2. Aufl., S. 45. t " Mikroskopische Technik," 2. Aufl., S. 46. BACTERIA AND MICROSCOPICAL TECHNIQUE. 81 coliol. The necessary duration of the hardening of tissues in alcohol, to produce constant reaction to dyes, diminishes the susceptibility of many elements to the dyes, so that the staining of the hardened sec- tions is as a rale, a quantitative increase of the meth- ods described for the dried cover-glass preparations. The sections are first Overstained, and then, by a sec- ond maximal decolorization, reduced to a proper de- gree of color for the nuclei and bacteria. The sections which have been cut in alcohol and afterward placed in the same fluid are put into a saturated aqueous or dUuted alcoholic solution of the dye, in which they should remain from five to thirty minutes. By warmtag to 40° or 50° C, the time re- quired can be shortened somewhat, and often also the intensity of the staining be increased. Gentian-vio- let, according to Weigert, is used very advantageous- ly in a 1 per cent aqueous solution. The sections are stained diffusely in this. In this method of staining the previously much- used decolorization by acetic acid is omitted, because many bacteria, such as the bacillus of typhoid fever and glanders, give up their color more or less com- pletely in a short time. The sections are placed in distilled water to remove an excess of the color. They are then carefully spread out upon a section-lifter, and with this slowly dipped in absolute alcohol for differentiation of the bacteria and nuclei and for de- hydration. They remain for a few minutes in alco- hol, which must be absolutely free from acid, then are cleared up in oil of turpentine or cedar, and can be immediately examined in this medium. For pre- serving, the oil is removed with filter-paper, and they are then mounted in Canada balsam. Since the bal- sam is soluble in the immersion-oil, it must be suf- 82 BACTEBIOLOOIOAL INVESTIGATION. ficiently hard to prevent solution by tlie shifting of the cover-glas^ If it is desired to examine or send away preparations freshly mounted in balsam, it is advisable to fix the cover-glass upon the slide with shellac or gold-size, which are not soluble in the im- mersion-oil. Many different dyes are also available for use in staining sections. In the use of the method of staining just de- scribed, the typhoid bacillus stains badly ; the spiril- lum of relapsing fever only in brown, and then not weU ; and the bacillus of leprosy badly in brown, but well in red and blue dyes. For these cases the greatest intensity is obtained by the use of the strong alkaline solution (page 49), which, on this account, according to LoflBler, up to the present time has proved the most universally valuable solution for sections. The sections are placed in this solution for a few minutes, then for a few seconds in 4 to 1 per cent acetic acid, and moved to and fro in order to remove the excess of color from the tissue and to differenti- ate the bacteria and nuclei ; then are dehydrated in alcohol, cleared up in oil of cedar, and preserved in Canada balsam. By this treatment many forms of bacteria, otherwise difiicult to stain, are well stained ; the tubercle bacillus as well as by other methods, also the bacillus of glanders (which in the aniline-water solutions is decolorized by the acetic acid) ; the ty- phoid bacillus and the spirillum of relapsing fever, which had been previously stained only very defec- tively in any other manner. In the case of most other bacteria, no great difference has been noticed as to the value of this method. The comparative staining by the use of aqueous and alkaline solutions sometimes allows the recognition of pure microscop- BACTERIA AND MICROSCOPICAL TECHNIQUE. 83 ical differences whicli are available for the differen- tial diagnosis in morphologically similar forms. For the isolated staining of bacteria in sections according to Koch, the sections are brought from the staining solution into a solution of potassium carbon- ate, which is prepared by the mixture of equal parts of distilled water and a saturated solution of the salt. The sections remain in this solution for about five minutes, and then are transferred with a section- lifter to alcohol, cleared up in oil of cedar, and pre- served in balsam. In this method different dyes can be used. If the sections are stained in the aniline- oil-gentian- violet solution, the isolation, according to the Gram method (page 60), is still more beautiful for most bacteria. The sections are taken from the al- cohol in which they are placed after cutting, and put ' from one to three minutes (tubercle bacilli in sections twelve to twenty-four hours) in an aniline-water solu- tion of gentian- violet. Then, either without washing or after gently washing in alcohol, they are trans- ferred to the iodine and potassium iodide solution, in which they remain one to three minutes. In the iodine solution a precipitation occurs, and the sec- tions are stained a blackish purple ; they are then placed in alcohol until completely decolorized, cleared up in oil, and preserved in balsam as usual. The bacteria appear dark blue, the nuclei and tissue pale yellow. The capsule-cocci of pneumonia (at least as a rule), and the typhoid bacilli are decolorized like' the nuclei. Double staining can be obtained in sections which have been treated, according to the Grram method, by placing them in an aqueous solution of vesuvin after the decolorization in alcohol. They are then dehy- drated in alcohol, cleared up in oil, and mounted in 84 BAOTEBIOLOQICAL INVESTIGATION: balsam. The nuclei are stained brown, while the bacteria remain blue. As already stated, carmine and hsematoxylin do not stain all bacteria, and even those stained are not as well stained as by the basic aniline-dyes ; but they are nuclei-staining agents of the first order. Sec- tions in which the bacteria are stained blue, accord- ing to the Koch method, can afterward be placed in a solution of carmine or hsematoxylin for about ten minutes, in order to stain the nuclei. They are then likewise treated with alcohol, oil, and balsam. If the sections are diffusely stained in concentrated aqueous or diluted alcoholic solutions Of dyes, they are first put into alcohol for the differentiation of the bacteria and nuclei ; then, for the removal of the alcohol, for a moment in water, and afterward into a solution of carmine or hgematoxylin, according as the first staining was with a blue or red aniline-dye. For these cases, according to Weigert, a 1 per cent solution of gentian-violet and a subsequent staining with picro-carmine is especially recom- mended. The time required for the ordinary nufclei staining (about ten minutes) must here be somewhat extended, because the carmine must displace the gen- tian-violet from the nuclei. The time required is from a half-hour to an hour. By a still longer action the carmine is also partially substituted for the aniline- dye in cocci. These different elective affinities of the bacteria and nuclei for dyes can be used, perhaps, yet more advantageously if the sections are first placed for ten or fifteen minutes in a solution of carmine or hsema- toxylin. In this time a good and durable staining of the nuclei is obtained, while the bacteria are either unstained or badly stained. After washing in water, BACTERIA AFD MIGROSOOPIGAL TEGENIQUE. 85 the sections are then transferred to a solution of one of the basic aniline-dyes in which the bacteria are stained, while the first dye-stuff is not displaced from the nuclei. They are then treated as usual with alco- hol, oil, and balsam. If picro-carmine is used in place of the ordinary carmine, a threefold staining is ob- tained (page 51). The so-called plasma-ceUs in sections can be easily confounded with cocci. They are spherical or spin- dle-shaped cells with a coarsely granular protoplasm, the granules of which react to the basic aniHne-dyes in the same manner as most cocci. The nuclei of these ceUs are not stained, so that the appearance of colonies of cocci is closely simulated, with which they have often been confounded. These granules do not show the same resistance to acids and alkalies, and are not of quite such equal size as the cocci. Further, they are especially to be recognized by their position, as they are commonly found on the walls of the vessels. A careful study of these cells is in the highest degree essential, and the ear of the white mouse is especially recommended for this purpose. An exact differentiation is often only to be obtained by the isolated staining, of the bacteria, in which the plasmarcell granules remain unstained. For the simultaneous staining of most cocci and the plasma-cells, the f ollovsdng solution of Ehrlich and Westphal is recommended, which also permits of the differentiation of other bacteria from the nuclei : Partsch-Grenacher's carmine (carmine pur., prts. 2; aq., 200 ; alum, 5 ; boil for fifteen minutes ; fil- ter ; add carbolic acid, 1 part) 100 c. cm. Glycerine 100 c. cm. Cone, alcoholic sol. dahlia 100 c. cm. Glacial acetic acid. - 20 c. cm. 86 BACTERIOLOGICAL INVESTIOATION. In this solution the sections remain twenty-four hours, then are transferred for a few minutes to alco- hol, afterward treated with oil and balsam. In this solution the nuclei take a red, the plasma-cell gran- ules and bacteria a blue or violet color. For the demonstration of the capsule cocci of pneumonia, Friedlander now uses : Cone, alcoholic solution gentian-violet 50 c. cm. Aq. destil 100 c. cm. Ac. acetic 10 c. cm. The sections remain in this solution twenty-four hours, and then are placed for differential decoloriza- tion in 1 per cent acetic acid for a few minutes ; after- ward, as usual, in alcohol, oU, and balsam. A contingent differential diagnosis is possible mi- croscopically from the fact that by the use of the Gram method these capsule cocci are decolorized, while the remaining cocci all (?) retain the color. Babes* recommends safranin for the staining of bacteria in sections. The sections are allowed to re- main a half -hour in a mixture composed of equal parts of a concentrated aqueous and a concentrated alcoholic solution of the dye. Then they are put for a short time in water, and a few minutes in alcohol ; after- ward in oil of turpentine and balsam. The bacteria are stained red, and sometimes have almost an iso- lated staining. This method offers no advantage, since the cocci are well stained, but other bacteria partly not at all and partly much worse than by other methods. The typhoid bacilli are ordinarily more difficult to stain than most other bacteria, even if the solution is warmed. According to Graffky,t it is best to allow * '' Arcliiv f. mikroskopische Anatomie," Bd. XXII, S. 359. t "Mittheilungen," 1884, Bd. II, S. 378. BAGTEBIA AND MIGROSGOPICAL TEGHNIQUE. 87 the sections to remain from twenty to twenty-four hours in a deep-blue opaque solution, which is pre- pared fresh each time by the addition of a saturated alcoholic solution of methyl-blue to distilled water. They are then washed in distilled water entirely free from acid, dehydrated in absolute alcohol, cleared up in oil of turpentine, and preserved in balsam. They are also well stained in the alkaline solution of methyl-blue, while by the use of the Gram method they are decolorized. The bacilli of glanders are decolorized, after stain- ing in an aniline aqueous solution, by water contain- ing acetic acid. However, they are well stained in an alkaline solution of methyl-blue. The bacilli of leprosy in sections are stained the same as tubercle bacilli. For differential diagnosis Baumgarten recommends the following (page 65) : The sections are placed for twelve, or at most fifteen, minutes in a dilute alcoholic solution of fuchsin, then thirty seconds in acid alcohol (nitric acid one part, alcohol ten), to decolorize them ; washed in dis- tilled water, dehydrated in alcohol, then treated with oil and balsam. In this time the bacilli of leprosy are well stained, while the tubercle bacilli are not stained at all. The tubercle bacilli can be brought out if the sec- tions are placed for twelve hours in a weak alkaline, or for one hour in a strong alkaline solution of methyl- blue (page 49), then for a few minutes in a concen- trated aqueous solution of vesuvin, and finally in al- cohol. They can also be made visible by the Grram method. For the differential diagnosis the sections are stained with the greatest certainty, according to the previously described principle, in the aniline- water-dye solutions of Ehrlich or Weigert - Koch. 88 BACTERIOLOQIGAL INVESTIOATIOK The sections remain twelve to twenty-four hours in an aniline-water, methyl-violet, or fuchsin solution, then a few minutes in dilute nitric acid (1 to 3 or 4) ; are washed in 60 per cent alcohol for a few minutes, stained in a dilute aqueous solution of vesuvin or methyl-blue, 'ssfg-shed in 60 per cent alcohol, dehy- drated in absolute alcohol, cleared up in. oil of cedar, and finally imbedded in Canada balsam. The vibriOnes of cholera Asiatica can be stained in . sections like most baciUi. The spirilla of relapsing fever are stained in aque- ous and glycerine solutions of vesuvin, but not well in other dyes. On the other hand, they are stained well in strong alkaline solutions of methyl-blue. A few remarks are stUl necessary concerning the epiphytic bacteria and the other epiphytic micro- organisms similar to them. (Whether the parasitic bacteria are to be classed with the epiphytic has not yet been certainly determined.) The chief diflBlculty in this investigation is due to the presence of fat. Balzer* washed the scales of the epidermis with ether and alcohol, and examined them after staining with an alcoholic solution of eosin, or without pre- vious staining, in a 40 per cent solution of caustic soda. According to von Sehlen,f the hairs, after the removal of the fat, are placed for a short time in alcohol, and then in a dilute aniline-oil solution of fuchsin. They are afterward washed in acid alcohol (concentrated hydrochloric acid 1 part, 75 per cent alcohol 99 parts) ; the excess of the acid is removed vnth distilled water, and a double staining is pro- * " Contribution & I'^tnde de I'Irytfeine tricoptytique." " Arch, de physiol.," 3. s6r., 1883, Bd. 1, 8. 171. f " Mikrokokken bei Ai-ea Cilei." " Fortsohritte der Medizin," 1883, No. 23. BAOTEBIA AND MICROSCOPICAL TECHNIQUE. 89 duced by the use of a concentrated aqueous solution of gentian-violet, and then alcohol is used for differ- entiation as before. According to Bizzozero,* the epidermis is touched with a cover-glass, vfhich is afterward passed through a flame three times. The fat is repioved by chloro- form, and the preparation is stained by fuchsin or gentian-violet. For the removal of fat from the epi- dermic scales, they are placed for a few seconds in alcohol, then for a day or two in ether, and afterward again in alcohol. After the fat has been removed there are three methods of procedure : 1. A drop, con- sistiag of equal parts of water and acetic acid, or of a 10 per cent solution of caustic potash, is placed upon a slide. The epidermic scales from which the fat has been removed are placed in this drop and allowed to swell, then a coVer-glass is laid upon them. For the preservation of these preparations, glycerine is intro- duced from the edge. 2. The epidermic scale is spread out with a needle in glycerine, slightly tinged with methyl-blue. In this the fungi are stained blue, while the epidermic cells remain unstained. 3. A drop of 50 per cent acetic acid is put upon a cover-glass, and the epidermic scales, with the fat removed, are placed in it. After fifteen minutes or more, if the scales are well swollen, they are spread out with a needle ; then the acetic acid is evapo- rated by gentle heat. (For this purpose, the cover- glass may be held high over a flame and moved to and fro.) After drjdng, the preparation is passed through the flame three times, and then stained for ten to thirty minutes by adding a drop of an aque- * " Ueber die Mikrophyten der normalen Oberhaut des Menschen." Virchow's " Arohiv," 1884, Bd. XCVIII, S. 441. 90 BACTERIOLOGICAL INVESTIOATION. ous solution of methyl-violet, gentian-violet, vesuvin, methyl-blue, or of a dilute alcoholic solution of fuch- sin. It is then carefully washed, dried, and preserved in balsam. Methyl-blue is preferred by Bizzozero, because only the fungous elements are stained with it. I have used most advantageously the strong alka- line solution of methyl-blue, allowing it to act for about five minutes. With this slight modification, it seems to me that this third method of Bizzozero is at present most serviceable for the determination of bacteria. Presently I will show that in true mycosis the mycelial filaments of the mold fungus are best brought out by the method of Loffler (page 82). The presence of radiating fungi in tissue - sections can generally be determined without special prepara- tion. But in the frequent calcification of the glands in actinomycosis it is often necessary to first decal- cify, by hardening them in alcohol containing hydro- chloric acid, and then to place them in absolute alcohol. The staining of Weigert * is also useful. According to this the sections are placed for one hour in a solu- tion of orchilla {rocTc moss). Pure orchilla, which has previously lain for a long time in the air to allow the escape of the ammonia, is dissolved, according to Wedl, in such quantities, in a mixture of 20 c. cm. absolute alcohol, 5 c. cm. acetic acid, and 40 c. cm. of distilled water, that the fluid becomes dark red, and after filtering appears ruby-red. Then the section is washed in alcohol, placed in a 1 per cent solution of gentian-violet and treated as for the staining of bac- teria. In these sections the nuclei are stained blue- violet, the connective tissue a pale orange, the ihte- rior of the refracting fungi a pale blue, and the outer * VircLow's "Archiv," 1881, Bd. LXXXIV, S. 245. BACTERIA AND MIOROSCOPIOAL TECHNIQUE: 91 parts a ruby-red, often separated by a colorless zone from the central portion. For free amoeba and cells without membranes (page 68) Brass* recommends a solution of 1 part chromic acid, 1 part platinum chloride, 1 part cone, acetic acid, and 400 to 1,000 parts of water. For hard- ening the tissues, in which the cells ought to be as little altered as possible {vide page 79), Brass recom- mends a i to i per cent solution of chromic acid, to which a few drops of a concentrated solution of the same salt is added later. After some time the prepa- ration is placed in 30 per cent alcohol, and generally, for the complete removal of the water, in alcohol gradually made stronger, and finally in absolute al- cohol. The mixture of chromic acid, platinum chlo- ride, and acetic acid also produces excellent harden- ing, especially if four to six drops of 1 per cent osmio acid is added to 100 grammes of the solution. The staining is effected by borax, or ammonio-carmine, or a solution of hsematoxylin. * " Die Methoden bei der TJntersuchung thierisoher Zellen." " Zeit- schrift f. wissenBchaftl. Mikroskopie," 1884, S. 39. III. CULTURE-METHODS; PURE CULTURES. Eheenbeeg first, and later Colin and Schroder, expressed tlie belief that there were true species among bacteria. On the grounds of the investigations of Cagniard-Latour and Schwann, Turpin reached the conclusion concerning fermentation, and Henle concerning the infectious diseases, that specific de- compositions and diseases are produced by specific micro-organisms ; and Pasteur, by inoculation experi- ments, proved experimentally that specific and differr ent micro-organisms underlie specific decompositions; These views, as well as those of their opponents, who argue for inconstancy of form and action among micro-organisms, can only be conclusively proved when they are observed pure and free from all ad- mixtures. For this reason it has been the endeavor of many investigators, for a long time, to establish methods of obtaining pure culture of bacteria, which would be free from all objections. 1. Teakspabebtt Fluid Cultuee-Media. This method is the oldest, and underlies all the experiments of the earlier time on fermentation. It was used for the cultivation of bacteria by Pasteur * * "Memoire snr la fermentation appellee lactique.'' "Compt. rend.," 1857, Bd. XLV, S. 913. CULTURE-METHODS; PURE CULTURES. 93 in Ms first work on the vital fermentation theory. The principle of this method, consists in transferring a particle of the original fluid contataing the micro- organisms of • fermentation, or a particle of yeast (which contains them in itself), to a fluid compounded to resemble as nearly as possible the original one ; from this again a particle is transferred to a third solution, etc. The school of Pasteur has adhered almost exclusively to the use of the transparent fluid media, even up to the present time, often in its origi- nal form ; also often with its modification, the meth- od of dilution, later to be described. The important morphological investigations of Cohn were also joined closely to this method. In fluids which undergo decomposition through the action of bacteria, sometimes a diffuse cloudiness appears ; sometimes precipitation occurs ; more often a mouldy scum is formed. In transfer experiments a particle is transferred to a second solution from each of the portions appearing differently. This transfer- rence to a new solution, briefly spoken of as inocu- lation, is performed as follows : a platinum-needle, previously heated and cooled, straight or bent into a loop, is brought in contact with the mycoderma (the mouldy scum), or is dipped into the cloudy fluid, and then the particle, thus taken up, is introduced into the new solution. In place of the platinum-needle, a drawn-out capiUary-tube can be used, with which a drop of the fluid is transferred. Such a particle or drop consists chiefly of a great mass of bacteria. Following this inoculation, that species develops first which finds in the new solution the best condi- tions for its existence. When the culture-material is exhausted for one species, one or another of the forms, that at first were suppressed, may develop, 94 BACTERIOLOGICAL INVESTIGATION. provided tlie particle or drop transferred contained several species. It is observed, in the course of these experiments, that small differences in conditions, such as placing the inoculated solution at a lovper or higher temperature, favor the development of certain germs. Finally, following out the conditions favorable to the development of a single germ, a more or less pure culture of this organism is obtained in some one of the transfers. But this is not always, in fact it is seldom, the organism which it is desired to isolate. Ordinarily, the result is a pure culture of one of the common septic species which, under the chosen con- ditions, supplanted the other more susceptible forms. On account of this it happened that, ia the use of this method of culture, the bacterium termo and bacillus subtUis often appeared. These play a much greater part in the older bacteria literature than now. ' In this way the dependence of the growth of bac- teria on the culture-media was learned. This led to the choice of such fluids as would offer approximately equally favorable conditions for development to the largest possible number of micro-organisms. Pasteur's* fluid is the oldest of these artificial media for the cultivation of bacteria. This consists of one part tartrate of ammonia, ten parts sugar, and the ashes of one part of yeast, to one hundred parts of water. A. Mayer f used in place of the yeast-ash a solu- tion of the salt contained in it. Then Cohn,:|: after he had used the culture-fluid of Mayer with mineral * " AnnalesdeChimieetde Physique," Bd.LVIII,S. 323. Deutsch von Griessinayer, "Die Alkohol-Gahrung," 1878. t " Unters. tlber die Alkohol-Gabrung," 1869. " LeLrbucli der Gfihrungs-Ohemie," 3. Aufl., 1879. } " Beitrage zur Biologie der Pflanzen," I, 2. Heft, S. 195. CULTURE-METHODS; PURE CULTURES. 95 nutrient salt, omitting tlie sugar, devised the follow- ing normal culture-fluid : Calcii phospliati '5 gramme. Magnesii sulph. (crys.). . . "5 " Calcii phosp. (tribas) "05 " Aq. destU 100 c. cm. In this 1 gramme ammon. tartrate was dissolved. Naegeli * discovered that for the lower fungi and the fission fungi, nitrogen can be best assimilated if it is present as NH„ not as well as NH, still not as well as NO, and not at all if it is in combination with other elements, such as hydrogen and oxygen, so that a de- scending scale may be constructed from the soluble albuminates, to ammonia and nitric acid. For car- bon he arranged the following scale : 1. The forms of sugar. 2. Mannite ; glycerine ; the carbon group ia leu- cin. 3. Tartaric acid ; citric acid ; succinic acid ; the carbon group in asparagin. 4. Acetic acid ; ethyl-alcohol ; quinic acid. 5. Benzoic acid ; salicylic acid ; the carbon group in propylamin. 6. The carbon group in methylamin ; phenol. Naegeli established the following descending scale for the capacity of assimilation of nitrogen and carbon from their compounds : 1. Albumen (peptone) and sugar. 2. Leucin and sugar. 3. Tartrate of ammonia, or sal. ammon., and sugar. 4. Albumen (peptone). 5. Leucin. * "Ernahrung der niederen Pilze durch Kohlenstoff- und Stick- stofEverbindungen," " Untersuohuiigen tlber niedere Pilze," 1882, S. 1. 7 96 BACTERIOLOGICAL INVESTIGATION. 6. Tartrate of ammonia ; succinic ammonia ; as- paragin. 7. Acetate of ammonia. In regard to 1, it is to be noted that the bacteria miist be able to transform the albumen into peptone, and to hydrate the milk and cane-sugar, so that pep- tone and grape-sugar are used to the best advan- tage. Naegeli recommends as the best for the mineral elements — Potassii phos 1 gramme. Magnesii sulph "03 " Calcii chlor -01 " Fluid 100 If the reaction is to be acid, Naegeli uses acid potassi phos. ; for neutral and alkaline solution, po- tassii biphos. The poorer the mitrient elements of the N and C group are, the less concentrated the salt solution should be, while, with good nutrient elements of the N and C group, this normal degree of concen- tration may be exceeded. From this Naegeli devised the following normal fluids for fission fungi : 1. Aqua 100 c. cm. Ammon. tart 1 gramme. Potassii biphos -1 " Magnes. sulph '02 " Calcii chlor Ol " 2. Aqua 100 c. cm. Albumen-peptone 1 gramme. Potassii biphos -2 " Magnes. sulph -04 " Calcii chlor -02 " 3. Aqua 100 c. cm. Cane-sugar 3 grammes. Ammon. tart 1 gramme. CULTURE-METHODS; PURE CULTURES. 97 Potassii biplios "3 gramme. Magnes. sulph "04 " Calciichlor -03 " In place of the tartrate of ammonia, in the 3d for- mula, an equal amount of another of the ammonia salts may be used, or "5 gramme of ammon. nitrat., or '7 gramme of asparagin, or "4 gramme of urea. In place of the nutrient salts, one tenth per cent beef -extract may often be more conveniently used. For the fermentation-experiments, according to Fitz,* solutions may be . employed which contain three per cent sugar, glycerine, or mannite, etc., and one tenth per cent beef-extract, to which a small quantity of calcii carb. has been added. According to previous experiments with cultures in fluids, as was also the case in the experiments of Fitz, it is well to choose the culture-fluid (normal fluid) with special reference to the individual case. In the lack of other data at the beginning, that fluid should be selected (as was the case in the first experi- ments of Pasteur) in which the bacteria are observed to grow spontaneously. For micro-organisms which are observed on solid substances, a decoction or in- fusion of this substance is prepared : fresh soil, sweet dried fruit, hay, roots, etc. "A nutrient fluid which holds in solution such substances as in a solid state furnish naturally a pabulum upon which a fungus develops," according to Brefeld,t "in all probability will also form a suitable fluid for the development of this fungus." Solutions for fungi are kept, as a rule, slightly * " Ueber Spaltpilzgahrung,'' VII. " Beriblite der deutschen ohemischen Gesellsohaft," 1882, XV, S. 867. t "Knlturmethoden znr Untersuchnng der Pilze." "Botanische Untersuchnngen iiber Schimmelpilze," Heft IV, 1881, S. 5 98 BACTERIOLOGICAL INVESTIGATION: acid ; for bacteria they are rendered neutral or slightly alkaline by the addition of ammonia or car- bonate of soda, and then are boiled and filtered. "In the use of a clear nutrient fluid thoroughly sterilized, in which the examination of the fungus by direct observation is made with the same ease as if they lived in clear water, the mycological investiga- tion is turned into an algological — i. e., those condi- tions are artificially prepared in the nutrient solutions for the development of the fungus, under which we naturally find the algae, as they for the most part live in water." These words from Brefeld (Z. c, page 7) also forcibly point out the chief advantages of the nutrient solu- tions for the culture of bacteria. These clear, usually neutral reacting, fluids must be carefully sterilized after filtration. For this pur- pose the sterilized cotton stopper of the sterUized flask or test-tube is removed with clean, previously heated pincettes, and laid upon its side so that it can not be soiled. Then, by the aid of a sterilized funnel or pi- pette, the fluid is transferred into the flask until it is about half filled, or into the test-tube until it is about one third filled. Then the stopper is again replaced. It is often desirable, in addition, to cover the mouth of the vessel with a double layer of thick filter-paper, which is held in place by a rubber band and which prevents the dust from falling directly upon the cot- ton. The cotton plug is proof against the admission of bacteria, but not always proof against fungi, as is to be observed when the sterilized vessels are pre- served for a long time in a moist chamber. The sterilization should be accomplished Record- ing to one of the methods described in Chapter I, preferably in most cases by the use of the steam ster- CULTURE-METHODS; PURE CULTURES. 99 ilizing cylinder. Small volumes of fluid, as those in test-tubes, are sterilized by an exposure for a balf to three quarters of an hour, larger flasks in one or two hours. The time required to bring the water in the cylinder to the boiling-point should not be included. The test-tubes, after being filled, are placed in a wire basket (Fig. 3, d), and then in the steam cylinder. " The manipulations with the fluids should be car- ried on in a place as free as possible from micro-or- ganisms, in order to prevent infection by air-germs. This may be obtained in a laboratory by the prepa- ration of a glass case (similar to those arranged over delicate balances), in which the inoculations and transfers are made. The walls of this case are rubbed with moist cloths, and the air is kept moist by vessels of warm water. The inoculations are made by a platinum-needle, straight or looped, which is previously heated in a flame and again cooled, or by a capillary pipette. By means of one of these a particle or drop of the material or fluid to be used for inoculation is intro- duced as quickly as possible into the sterilized fluid, after removal of the cotton plug. This is then imme- diately replaced. If the solutions have stood for a long time without the mouths of the vessels being pro- tected by filter-paper, so that germs of bacteria and fungi may have collected upon the cotton, it is better to burn the upper layer of cotton in a flame before removal of the plug. A large number of single ex- periments should be made simultaneously. The fur- ther inoculations are made in the same manner. The inoculated tubes are, according to the case, kept at the temperature of the room or exposed to a higher temperature. For the higher temperatures a brood- er culture-oven is used. This is made of metal with 100 BAGTERIOLOOIGAL IFVESTIGATIOK double walls for tlie reception of water (Fig. 8), and is covered with felt or asbestos to prevent the loss of heat ; or the oven is made with a third wall, and the outer chamber is filled with a layer of sand. The dimen- sions vary according to need ; the form, quadrangular or cylindrical, is of little mat- ter. In the quadrangular the height and breadth of the interior is about 25 cm. , ~ '" and the length varies from 50 to 75' cm. A thermometer {t) and thermo-regulator (r) serve for the regulation of the temperature. These last can be dispensed with if a small gas-pressure regulator is interposed between the principal supply- pipe and the culture-oven. It is heated by petro- leum or gas flames, which vary in number, accord- ing to the size of the oven. These are surrounded by glass chimneys to protect them from draughts of air. 2. Feactional Ctjltubes. The inoculations of fluids with pathogenic bacteria were first made by Klebs,* and the particle or drop containing bacteria (" Bakterientropfen " of other au- thors) was designated by him as a "fraction." Klebs proceeded in this manner (J,, c, page 46) : " He intro- duced a recently drawn-out and closed pointed cap- illary-tube to the bottom of the fluid containing bacteria, and then broke off the point. The tube, after withdrawal, was again sealed, washed with strong alcohol, introduced into a sterilized culture-fluid, and * " Beitrage ziir Eenntniss der Mikrokokken." " Archiv f Ur ex- periraentelle Patliologie," Bd. I, 18T3, S. 31. CULTURE-METHODS; PURE CULTURES. 101 "was then again broken. The fluid-medium was con-' tained in a stoppered flask and was covered with a layer of oil." This procedure was often repeated, and "in this manner it is possible to eliminate any im- purities, which may be contained in the original fluid, and to obtaia those organisms pure which are present in this in preponderating number." This method has furnished no important discov- eries, notwithstanding the skiUful working and the sharp emphasis laid upon the development of many germs from the one originally present in preponderat- ing numbers. In this complication of the method of Pasteur it is evident that the objection also holds good that, as a rule, after a series of fractional cul- tivations, the desired pathogenic organism is not present pure. But instead of this almost any form may be present which at the beginning may have been perhaps entirely overlooked on account of their small number ; or else some ordinary species of septic bac- teria, introduced by infection from the air or by manipulation, has been obtained pure, because, un- der the chosen conditions, it thrived better than the more susceptible pathogenic micro-organisms, and quickly supplanted these. By the methods thus far described pure cultures may be finally obtained, vdthout successfully ac- quiring those forms pure which it was desired to obtain. These methods are on this account only now to be used when it is desired to secure an organism in pure or quantity-culture, the source and action of which are of no importance. 3. Opaque Solid Culttjee-Media. Aside from fluids, bacteria are observed develop- ing spontaneously on solid substances. If, for exam- 102 BAOTERIOLOOICAL INVESTIGATION: pie, a slice of cooked potato is allowed to stand in the air, different slimy masses are seen to spread themselves out upon the surface of the potato. Small slimy points of different colors form upon the sur- face, which for some time are quite distinct from each other, but become confluent in their further growth. These observations were first methodically made use of by Schroeder * for the pure cultures of pig- ment bacteria. A particle from one of these slimy points, while it is yet quite isolated, is transferred with a platinum-needle to the middle of the cut sur- face of a freshly cooked potato, placed in a moist chamber in order to prevent infection from the air. The method of cultivation on potato was material- ly improved by Koch. The potatoes were first care- fully cleaned from the coarse dirt by scrubbing, then laid for one half to one hour in a one to five per cent solution of corrosive sublimate, and finally washed in water. The potatoes, thus cleansed, with the germs adhering mostly destroyed by the sublimate, are then heated to secure certain sterilization. This is done in the steam cylinder. After reaching the boiling- point, the potatoes remain at this temperature for about an hour. While the potatoes are cooling, a moist chamber is prepared. Large bell-jars, of the form seen in Fig. 9, are cleansed by rinsing in a one Fig. 9. * "Ueber einige durch Bakterien gebildete Pigmente." Oohn's " Beitrage zur Biologie der Pflanzen," Ed. I, Heft II, 1872 (2. Ab- druck, 1881), S. 109. CULTURE-METHODS; PURE CULTURES. 103 per mille solution of corrosive sublimate. Upon the bottom of the jar a number of layers of filter-paper, moistened with sterilized water or a solution of sub- limate, are placed. Then the potatoes are held in the left hand, between the thumb and index finger, cut with a sterilized knife, and laid in a jar with the cut surface upward. Before this operation the hands should be washed in a one per mille solution of sub- limate. The ordinary kitchen-knife is used for cut- ting, and, previous to use, is sterilized by heating in the flame, and while cooling is protected from the dust. For each potato a fresh knife should be used. Then, by means of a platinum-needle which has been sterilized in the flame, a particle from one of the slimy points which is still entirely isolated, a so- called colony, is taken, and is either placed near the middle of the section of potato, or several lines of inoculation are lightly drawn entirely across the sur- face. In the first case the colonies develop in the middle of the section, and gradually extend toward the edge ; in the other, more or less isolated colonies develop along the lines of inoculation, which later unite and extend throughout the lines. For new inoculation only those colonies are used which are recognized as pure with the naked eye or lens, and from which a particle has been iised to make a cover- glass preparation for microscopical control. Some of the cultures can be kept at the tempera- ture of the room, and others at a higher tempera- ture in a culture-oven. The great advantage of this method, as compared with the previously described methods with trans- parent fluid media, consists ia this : namely, that each germ, whether it has been intentionally inocu- lated upon the surface of the potato, or is the result 104 BACTERIOLOGICAL INVESTIGATION. of air-infection, is isolated at its point of contact with tlie surface, and there develops into a colony, while in fluids the different germs are mingled together. Pure cultures may also be obtained in the fluid media, but that organism is not always present of which pure cultures are desired. In the potato-cul- tures it is possible to transfer the desired micro- organism, and in this way separate it from others, and after several transfers obtain pure cultures. How- ever, the transfers must be made early, while the small colonies developed from a single germ are yet entire- ly isolated, and are solitary and pure, as the result of the isolation. The limitations of the method depend on the fact that potatoes do not farnish favorable conditions for the existence of aU organisms, and, on this account, many species that develop on potato do not grow sufficiently to be visible. This is important, because in the solid opaque media the naked eye or the magnifying-glass can alone be used. The inoculations on potatoes are more useful when it is desired to determine whether pure cultures obtained elsewhere, especially of pathogenic micro- organisms, possess the capacity of developing on vegetable material. Then the sterilized potatoes are inoculated in the same way with these pure cultures. The sections of the potatoes should be kept in the same manner in moist jars, and subjected to different degrees of temperature ; or, as a greater precaution, a potato-section is placed upon a smaU glass plate, which is then lowered (by the help of a strip of nickel bent at a right angle) to the bottom of a cylin- drical glass vessel about 18 cm. high and 6 cm. in diameter. The glass cylinder — previously closed by a cotton plug — the glass plate, and nickel strip should be sterilized in a dry-oven before being used. OULTURE-METHOM ; PURE CULTURES. 105 In place of the sections of potatoes — which, on ac- count of their yellow or white color, are used in prefer- ence to other tubers, as carrots, etc. — ground potato may be used. For this purpose, the boiled and ground potatoes are placed in flasks (preferably in the so-called Erlenmeyer flasks), and suflicient water is added to form a thick broth. This potato-broth is sterilized in the steam apparatus, and is then inocu- lated in the usual way with a platinum-needle. The potato-broth can be made into a very good culture- media for many bacteria by the addition of starch, sugar, peptone, and beef-extract, and can then be very advantageously used to obtain fractional-cul- tures of certain forms of bacteria. 4. The GrELATiif-CuLTUEE OP Klebs and Beefeld. Origin from One Germ. — Moist Chambers. — When Klebs (Z. c.) sought to fix a coccus under the microscope in order to directly observe its division, and so -follow back the entire pure culture to a sin- gle germ, he was unable to do this in a fluid-drop, first, because of the movement which occurred in the drop, and second, because after the admission of air the fluid was altered by the concentration produced by evaporation. This also changed the value of the fluid as a culture-medium. In order to prevent the evaporation of the fluid, or at least to limit it and to avoid' the motion, instead of the ordinary culture - solutions, Klebs used isinglass as a culture-medium, which became stiff on cool- ing. ISTow, in order to fix a single coccus present in the isinglass, Klebs used the chamber of von Keckling- hausen and Geissler. In this (Fig. 10) a tube leads to and a second away from a middle room made of 106 £A CTERIOL GICAL IN VESTIGA TIOK glass of the thickness of cover-glass, the upper and lower sides of which almost touch in the middle, so FiQ. 10. that here is formed a small capillary- chamber. If the chamber is then filled with water, culture-fluid, or liquefied gelatin, and these solutions are again poured out, a capillary-drop remains suspended in the narrow plac'e. If these solutions at the same time contain germs, and in such numbers that eaCh drop holds about one germ, then, in many cases, it will happen that the drop remaining will contain a germ, which can be tolerably well fixed with a high-power dry system and thus observed. Sometimes Klebs filled the entire chamber with gelatin or isinglass, so that the entrance of air to the central portion was prevented ; sometimes he left only one drop in the chamber, but then took care that the air was filtered through cotton before its admission. In other cases Klebs used, in place of the chamber with the capillary - room, a chamber the walls of which ran parallel and whose side-tubes were placed somewhat lower than the upper wall. These cham* bers {b in Pig. 11) (the description of which is after those used by myself) were filled {I. c, page 46) by CULTURE-METHODS; PURE CULTURES. 101 Fig. 11. allowing the excess of the fluid gelatin to run away, so that a thin layer, looking downward, covered the upper side and served for microscopical examination. Any germs fixed in this layer could be directly ob- served with a high dry lens or a low immersion sys- tem, and their development noted. In place of air, different gases may also be admitted. Bref eld * sought at first to procure for fungi an entirely pure culture-medium, in order that, proceed- ing from a single germ, he might follow out without interruption the entire development of the fungus. These studies Pasteur f soon undertook upon yeast, and later, in 1881, Bref eld completed them for' bac- teria, and proved their usefulness in the phases of de- velopment of the bacillus subtilis. "By a mixture of the germs with water in such a proportion that in a certain amount, either none or only one germ is present, the separation of individual germs, even of the smallest forms, may be empirically brought about without direct observation." "There results from this method of separation an equal distribution of the individual germs in the fluid; but this is not always easy to obtain, and, if the work is hot very * (a) " Botam8che Untersuclmngen iiber Schimmelpilze," Bd. I, 1872, S. 10. (J) " Methoden zur Untersnclmng der Pilze." " Ver- handl. der physik. med. Gesellsohaft in Vurzbnrg," W. F., Bd. VIII, 1874-'75, 8. 4.3. (c) " Kulturmethoden znr Untersuohnng der Pilze." " Botanisohe Untersnchungen fiber Schimmelpilze," Bd. IV, 1881, S. 1. t " Etude sur la bi^re." " Compt. rend.," 1873, S. 77. 108 BACTERIOLOGICAL INVESTIGATION: carefully and critically undertaken, numerous foreign germs may be easily included in the culture." Brefeld diluted empirically the fluid containing the spores or germs with water or fluid culture-media {I. c, page 49) "until a drop, removed with the point of a needle and transferred to a slide and examined with a microscope, showed only one or two germs. The slide with the drop containing the germ serves as the origin for the culture, and, on this account, has received the name of 'slide-culture' to distin- guish it from other forms of culture." If now a drop of culture-fluid is added to this one spore, the uninterrupted tracing of the development is made difficult by two factors. On the one hand, the drop of culture-fluid evaporates, and, by this change in its concentration, its value as a culture- medium is diminished ; and, on the other, foreign germs may gain admission. It is essential, on this account, to prevent the evaporation of the culture- drop, and to inclose it so as to prolong the possibility of the observation. "This may be accomplished in two ways : first, by alteration of the culture-fluid, and, second, by the use of a peculiar slide." "In order to prevent evaporation (Z. c, page 15), caraghen or gelatin may be added to the culture-fluid in such amount, that it is still fluid at 30° or 35° C, but becomes solid when cooled to 15° C. In these gelatin solutions the fungi grow as in a thin fluid. Their development is favored rather than retarded. If now the culture can be reversed without danger, in order to prevent foreign germs from falling in it, it can be examined with a strong magnifying power." This can be done by placing it upon a cover-glass. These reversed cultures are placed in a moist Jar (Fig. 9, page 103) upon a support of glass or zinc. CULTURE-METHODS; PURE CULTURES. 109 If it is desirable to avoid the gelatin culture-solu- tions, then a peculiar slide must be used, with which the evaporation of the culture-fluid and the invasion of foreign germs are impossible, without the possibil- ity of a continuous observation being in this way in the least prejudiced. For the observation in the hanging-drop, the slides shown in Figs. 6 and 7 may be used. A cover-glass is thoroughly cleaned by a concentrated mineral acid, alcohol, and ether. Shortly before use it is passed through the flame, cooled, and protected from dust. Then a small, flat drop of the sterilized culture-solu- tion is placed in the center with a sterilized plati- num-loop. This drop is inoculated by the intro- duction on a platinum-needle of a particle from a pure culture. The cover-glass is reversed, laid over the hoUow slide, and a rim of vaseline drawn around it to prevent evaporation. In place of the inoculation of the drop on the cover- glass, a sterilized culture-solution in a test-tube may also be inoculated with a pure culture-solution in such an amount that, after thorough mixture by shaking, a drop of equal size, tested as a dried cover- glass preparation, shows one or two germs. A drop is then taken from this and prepared on a cover-glass in the same manner. Bref eld (Z. c. , page 16) rejected this method. ' ' Only a small culture-drop can be taken, since it becomes globular, and oscillates on the slightest movement ; the germ -spore alters its position, and with the stronger magnifying power is scarcely accessible ; in short, the observation is difficult and incomplete. This is also true if the gelatin culture - solution is used." , With the above-described precautionary measures, 110 BAOTERIOLOOIOAL INVESTIGATION. Fig. 12. after many observations with the hangiag-drop ar- ranged in this way, it may be used with great advan- tage (even for the smallest forms of cocci, both in the fluid and gelatin-drop), to determine whether a colony of bacteria, which vdth the low magnifying power appears isolated, has developed from a single germ. This has been described by Hansen,* in a remarkable manner for colonies of yeast, by the use of a sus- pended gelatin-drop, which was protected from evapo- ration. After some experience, so that this can be used conveniently, the possibility is in this way bet- ter afforded for the study of the individual phases of the development of bacteria from spore to spore (in a drop accessible to the lower systems of homogeneous immersion), and for making parallel experiments with staining for spores, which permits the fixation of these individual phases. Bienstock t even used the staining of spores directly in place of direct ob- servation, which seems to me is going quite too far. Prazmowskil used a slide (Fig. 12) to prevent the ad- mission of air. This slide, 2^ mm. thick, possesses a circular depression {d e) ; the circular surface (c) inclosed by this ring is carefully ground smooth, * " Ueber das Zahlen mikroskopisoher Gegeusfcande in der Bota- nik." " Zeitsclirift f. wissenschaftl. Mikroskopie," 1884, Bd. I, S. 191. t " Zeitschrift fur klin. Med.," 1884, S. 1. I " Untersuohungen fiber die Entwicklungsgeschichte und Fer- mentwirkung einiger Bakterien-Arten," 1880, S. 10. ^ CULTURE-METHODS; PURE CULTURES. HI and its plane lies somewhat lower (for bacteria pref- erably only "5 mm.) than the surface of the slide, so that after laying on the cover-glass (b) between c and b there is a place for a thin layer of fluid. The circular depression extends away at e into a small furrow. A small drop of the sterilized solution is placed upon the surface (c) and is inoculated, or a drop containing an approximately definite number of bacteria is thus used. Then a cover-glass (&), which has previously been passed through the flame, is applied, is sur- rounded by vaseline or wax, so that (to prevent much evaporation) air can only enter by the furrow e, and then flnds access to the layer of fluid inclosed be- tween c and 5 from the entire circle. This layer of fluid is accessible in its entire depth to the strong dry lens or the weak immersion system. Brefeld chose, as did Klebs, the chamber (Fig. 10) of von Recklinghausen and Geissler to flx the germ, but {I. c, c, 1881, page 17) the capiUary-drop is, in his opin- ion, too deep for the smaller forms, and the amount of fluid so great, that movements are produced by the slightest influences, such as can not be prevented during the observation, and which occasion a shifting of the germs out of reach. The germ must be fixed by reducing the amount of the surrounding solu- tion so that the movements are at a minimum ; but yet the amount of the surrounding culture-solution should be sufficient for the germ to complete its full development. This is attained only in a thin layer of fluid. In order to prepare this upon the inner wall of a chamber, so that the highest lenses may penetrate it, smaller chambers must be used, which have a small capillary - room, made of glass, the thickness of the thinnest cover -glass, ground flat upon both sides so that a layer of equal thickness 8 112 BACTERIOLOOIOAL INVESTIOATION. is formed, in wMcli it is possible with the strong dry lens to fix a germ for an indefinite length of time without disturbance. It is wise not to make the chamber-room larger than is necessary to meet the technical requirements. The carefully cleaned chambers are filled full, then the contents allowed to flow out, and the single germs remaining in the thin layer of culture-fluid that adheres on the inner wall are selected. The number of the germs, which are mingled with the culture-fluid, can be far greater in this case. It is not necessary to make a previous test to determine their number, al- though, if they are few, prolonged search is neces- sary to find them on the wall. I was successful without much trouble in observing for an unlimited time the germs of bacilli and other bacteria, and in following thus a complete series of development as with the greater fungi. In this way it is easily pos- sible to determine the length of time which is neces- sary for the process of growth, for division, and final- ly for the entire cycle of development from spore to spore. The value of these chambers for the examination of the fission fungi extends even to the smallest forms, which are still easily accessible to the observer with the strongest dry system. Brefeld used chambers of the form a (Fig. 11), designed by Geissler. I used the form &, also de- signed by Geissler. As was shown by my experi- ments, it is possible to prepare a thin layer of fluid in these chambers, if they have been carefully cleaned with mineral acid, alcohol, and ether, and have been sterilized. In place of the fluid, it is also advantage- ous to prepare a thin layer of gelatin, in similar chambers as was done by Klebs in 1873. In such CULTURE-METHODS; PURE CULTURES. 113 layers of gelatin I have traced, for single species, the formation of colonies, visible to the naked eye, which had developed from one germ. In the chamber chosen by myself, a one twelfth homogeneous immersion-lens with a low ocular could be used. With all these chambers the ordinary warm stage is, as a rule, necessary. Up to this time, the chambers with parallel walls have been used by most observers for the uninterrupted observation of the cycle from spore to spore. For experimental work, I prefer the far more convenient mode of observation by means of the hanging-drop. 5. Method of Dilution. Although Brefeld had positively shown concern- ing mould fungi, and Pasteur concerning yeast, that, by the systematic dilution of a solution containing micro-organisms or spores, a determined quantity of fluid may be prepared so as to contain approximately one germ, with which experiments can be made by transfers tp sterilized media, Naegeli,* in 1877, not- withstanding aU the previous experiments, denied the possibility of obtaining pure cultures of bacteria. "Fission fungi absolutely permit of no pure culture, in part on account of their extraordinary minuteness, in part on account of their general diffusion ia. the water and air." JSTaegeli then gave a method of dilu- tion, not that he wished to make hopeless experi- ments to obtain pure cultures (as was stated in his communication at that time), but in order to prove the capacity for multiplication of the fission fungi, depending upon the degree of alteration of the me- dium. In this method he dUuted a fluid containing * Xaegeli und Sohwendener, " Das Mikroskop," 2. Aufl.^ 1877, S. 644. 114 BAOTERIOLOOIGAL INVESTIGATION. a certaiu number of baeteria with a definite cLuantity of sterilized, water. Later, Waegeli himself came to the conclusion (Z. c, page 646) that the solution of this problem of equalization could only be arrived at by testing. He states that he then conducted such empirical experiments in his studies of the lower fungi,* beginning in 1871. In these experiments, he diluted decomposing urine so greatly with water that each two drops contained one germ. By transfers of a drop to a glass containing a sterilized solution, he then succeeded in obtaining a certain separation of the rods and cocci. After 1877 he denied the possir bility of obtaining pure cultures. I find in Naegeli's investigations, unfortunately, no statements to bring into harmony these later commu- nicated results of 1871, with his complete denial of 1877, and his theoretical views concerning the mor- phology of bacteria and the origin of all bacteria from cocci. Certainly without any knowledge of these experiments of Naegeli in 1871, Lister f in 1878 stated that he had so diluted sour milk that a drop contained one germ. He now inoculated sterilized milk with from two to four drops of the diluted fluid, and constantly obtained a souring and coagula- tion; less certainly, however, if he used only one drop, because in this case not every drop in reality contained a germ. Fitz X also used in his experiments on fermenta- tion the method of dilution which he alone desig- nated as the " one-cell culture." * 1882, S. 13. t " On the Lactic Fermentation and its Bearings on Pathology." "Transactions of the Pathological Society of London," 1878, vol. XXIX. t " Ueber Siraltpilzgahrungen," VIL " Berichte der deatschen chemisohen Gesellschaft," Bd. XV, 1882, S. 867. GULTUBE-METHODS ; PURE CULTURES. 115 "In order to obtain a pure culture of a fission fungus which causes fermentation, it is absolutely- necessary to start out from a single cell as seed. In an ordinary impure culture, with the aid of a count- ing-chamber, the number of fission fungi contained in one drop is approximately determined ; then this drop is so greatly diluted with sterilized distilled water that, in from fi^e to ten drops of the diluted, weU-mixed fluid, only one fission-fungus cell is pres- ent; then a drop is added to each of a series of about fifty flasks, filled with fluid and sterilized, and these are then placed in a thermostat at 37° C. Of the fifty, five or ten, in the course of the next three weeks, show the development of fungi. In each one of these flasks the culture-fungus is solitary and pure, because it has developed from a single cell. Thus the different fission fungi which were contained in the unclean culture are isolated." For pathogenic bacteria, especially for the anthrax bacUli, Buchner* used the method of dilution in which the spleen-pulp is scraped and diluted so much with sterilized water, that a single bacterium is pres- ent in about 10 c. cm. Then the sterilized culture- solutions are infected with 10 c. cm. of the fluid. Han- sen t proceeded in a similar manner. He made the dilution for yeast so that 2 c. cm. contained one cell. For determining also the number of germs in the original fluid as well as in the diluted solution, a defi- nite amount is placed in an apparatus for numbering red blood-corpuscles. For this the chamber of Hay- * " TJeber die experimentelle Erzeugung des Milzbrandoontagiums au8 den Heupilzen." " Untersuohungen tlber niedere Pilze von Naegeli," 1882, S. 147. t " Ueber das Zahlen mikroskopiaoher Gegenstande in der Bota- nik." "Zeitschrift f. wisaensohaftl. Mikroskopie," Bd. 1, 1884, S. 191. 116 BACTERIOLOGICAL INVESTIGATION. em-l^acliet may be used. This (Fig. 7, page 40) con- sists of a slide {A B) Tipon wMcli rests a glass plate (&) provided with, a circular aperture (c). This glass plate is of a definite thickness, 2 mm., or for bacteria stiU better, -1 mm., as is furnished by Zeiss. By application of a very carefully ground cover-glass {d), a room is formed, bounded by parallel walls, the height of which is exactly -1 or -2 mm., and it will contain a definite amount, a unit of vol- ume, of the fluid. The numeration is accomplished by the aid of a crossed ocular micrometer. The fluid must exactly fill the room, and the cover-glass should fit accurately. A modification of this (Pig. 13), which was designed by Thoma,* permits of yet finer work. This is manufactured by Zeiss. Upon a slide {A B) an even polished glass plate {a) is fastened, whose circular * Abb6, " Ueber BlutkdrperzShlung." " Sitznugsberichte der Jenaisohen Gesellsch. f. Med. u. ITaturwissenschaft," 1878, No. 29. Lyon und Thoma, "Ueber die Methode der Blutkorperzahlung." Virchow's " Arcbiv," 1881, Bd. LXXXIV, S. 131. CULTURE-METHODS ; PURE CULTURES. 117 aperture {d) forms the side- walls of the cliainber. In this aperture a circular glass plate (c) is cemented, which is so thick that the room (e) between it and the superimposed cover-glass (b) is exactly "1 mm. high. This is the real counting-room ; a and c are carefully ground parallel to the surface of the slide ; in the same way the cover-glass (6) must be carefully ground into a parallel plane, and so cleaned that, by its join- ing with the polished upper surface of the chamber, it forms Newton's color-rings, which remain af t6r the removal of the pressure. The mixture is made in a mixing-vessel, which is to be also used for the first numbering-chamber. Since exact instructions as to its use are given with the apparatus, these data suf- fice. The advantage of the Thoma chamber is that the ocular micrometer is omitted, because on the plate (c) a crossed division is engraved which has one q. mm. divided into, four hundred quadratic equal fields. In this way the absolute value of a portion is de- termined, which is not possible when this is altered by each objective and each length of the draw-tube, as in an ocular micrometer. But, if the numbering is made ever so exactly, and the mixture also care- fully prepared, still an absolute certainty for the source of a germ is not reached, either in the method of dilution or the "one-cell culture." The error also belongs to this method, as Hansen in his excellent communication frankly states, viz., that it is not certain whether the number of cells are really present in the prepared inoculation-fluid which in the begin- ning are computed to be present. It may also occur that not a single ceU is included, or that several are present, as was desired. The method of dilution also, in its best form, can only be conditionally adapted to the theoretical post- 118 BACTERIOLOOIGAL INVESTIGATION. ulate of the origin from one germ. If, further, only an approximate guarantee.is desired, that the differ- ent forms of bacteria contained in a mixture are mod- erately separated by the method of dilution, large numbers of individual experiments must be made. The number of these was placed at fifty by Fitz, for his individual case ; but there must be hundreds and thousands if decomposing fluids, or a very dirty wa- ter or similar mixtures containing many different bacteria, are employed. AH investigators, who have made use of the method of dilution (on account of the limitations of the method, but without the admission of the prac- tical points causing them), have taken care to pre- viously prepare the cleanest possible inoculation ma- terial by the quantity-culture, with the aid of such secondary means as are dependent, according to Bre- feld {I. c, c, 1881, page 12), on the varying modes of life and certain morphological and physiological pe- culiarities of the different forms. With these restric- tions, the continual dilution, even to a "one-cell cul- ture," is very useful for individual pathogenic organ- isms, still better for ferment bacteria, the conditions of whose existence are already approximately known, or suspected from the manner of their spontaneous presence. The fact first to be determined through pure cultures — viz., the biology of bacteria — ^is in these cases provisory, since it is presupposed that it is already known. Culture-fluids are chosen in which it is certain- ly known, or presumed, that the respective fermen- tation can proceed. These are inoculated with a par- ticle or drop of the original impure material ; then the flasks thus inoculated are placed at a higher or lower temperature ; transfers are made a second time CULTURE-METHODS; PURE CULTURES. 119 after development of the germs has occurred, and so on until an approximately pure culture of an organ- ism is obtained, as in methods 1 and 2. Other flasks are kept free from air, if it is sup- posed that the fermentation under consideration pro- ceeds* better, with a restriction or absence of the oxygen of the air. In the anaerobic experiments this will be more particularly detailed. After a few of these transfers, enumeration and dilution are made, and then the sterilized culture-solutions are inoculat- ed with the unit of volume containing the one germ. METHOD OF ISOLATION BY HEAT. With these measure-cultures belongs a more ex- act description of the "heating method," as it may be briefly designated. In all cases in which bacterial development oc- curs, after a shorter or longer exposure to a boiling temperature, or to a stiU higher temperature (as was first shown by Cohn),t it is always due to the pres- ence of spores forming bacteria, and to the resist- ance of the spores for a varying length of time to the high temperature. Miquel % isolated from urine a micro-organism bacillus urese (that forms ammonia), by heating a glass of water, that contained its spores, to a temperature of 108° C. Brefeld * found that, for * Fitz, "Ueber Spaltpilzgahrungen,'' IX. "Bericlite der deut- Bohen cheraisehen Gesellsohaft," 1884, Bd. XVII, S. 1188. t " Untersuohungen uber Bakterien," Bd. IV. " Die Bakterien nnd die Urzeugung." " Beitrage znr Biologic der Pflangen," Bd. II, Heft 2, 1876, S. 249. Vergl. auch die Literatur im ersten Abschnitt und fiber Sporenfarbung. \ "Balletin de la soci6t6 chimique de Paris," 18T9, Bd. XXXII, S. 127. * " Botanisohe Untersuchungen fiber Schimmelpilze," Bd. IV, 1881, 8. 51. 120 BACTEBIOLOGICAL INVESTIGATION: the destruction of the spores of the bacillus subtilis, exposure to a boiling temperature for three hours was necessary, or to a temperature in the oil-bath of 105° C. for fifteen minutes, or 107° G. for ten minutes, or 110° C. for five minutes. By reducing this time, these bacUli can be obtained in cultures, pure from all less resistant micro-organisms. Prazmowski* boiled fluids containing spores for a longer or shorter time, to gain and retain pure cultures of bacilli and clostridii, which form spores.. Without reference to these investigations. Gunning, f proceeding from a mistaken conception of the method of culture with solid transparent media, has put forward the sys- tematic use of higher temperatures for the isolation of bacteria. Grunning himself in this way also, as all before him had done, isolated spore-forming species. In systematically testing this method by the use of pure cultures of bacteria (both those forming spores and those free from them), I discovered what was to be confidently expected from the biology of bacteria — namely, that, by systematic heating below,, to, and above the boiling point, only the more resist- ant forms can be separated from the less resistant. Consequently the use of this method is limited, for practical purposes, to the separation of spore-contain- ing bacteria from those free from spores. For this, it is to be recommended. If there are two kinds of spore- forming species present having approximately the same resisting power, it is not possible to obtain, by a shorter or longer heating alone, a sufficient separa- tion. The process of obtaining by heat a tolerably * " Untersuchungen tiber die Entwicklungsgeschichte und Fer- mentwirkung elniger Bakterien-Arten," 1880, S. 8. t " Beitrage znr hygienische TJntersuchung des Wassers." " Ar- chly far Hygiene," Ed. I, 1883, S. 385. CULTURE-METHODS; PURE CULTURES. 121 or quite pure culture is, as I have previously stated,* restricted to certain cases ; but for these it is very use- ful — e. g., it is the best method for obtaining from the prepared quantity-cultures a separation of the spore- containing bacteria from the spore-free forms. Often in this way a reaUy pure culture may be obtained. 6. CCTLTTJEES IN CAPILLART-TcTBES, AFTER SaLO- MONSEHr. Salomonsenf observed in spontaneous changes in the blood, caused by putrefaction, that black spots of decomposition appeared (formed by the reduction of the oxy haemoglobin), vrhich, on the bottom of the vessel, possessed a sharp circular contour, and higher up a more club-shaped form. These gradually in- creased in size, became confluent, and then produced a diffuse dark color in the entire mass of blood. While these spots are stiU isolated, according to Salomon- sen, each consists of a single form of bacteria, which by its growth brings about the alterations in the color of the blood. Each such spot is a colony, a genuine pure culture of a single species. If now fresh or defibrinated blood is drawn into a capillary-tube, these isolated decomposing spots also appear in the blood in these tubes. In place of the true capiUary-tube, lymph-tubes or fine glass- tubes can be used, which are drawn out at one end to a capillary-point (Fig. 14). Among the colonies thus developed with a low * " Mittheilungen aus dem kaiserliohen Gesimdheitsamte,'' Bd. II, 1884, S. 330. + " Zur Isolation differentur Bakterienf ormen.'' " Botanisohe Zeitung," 1876, No. 39. , " Studier over Blodets Foraadnilse," 1877. " Eine einfache Methode zur Eeinkiiltur versohiedener Faulnissbak- terien." " Botanisohe Zeitung," 1880, No. 28. 122 BACTERIOLOOIGAL IFVESTIOATIOK magnifying power (sucli as a simple lens), small dif- ferences are observed in the size, Fio. 14. ^jjg rapidity of development, and in the form (&, c, d, e, of the second cut, Fig. 14). Each of these small differences is a visible indication that these different colonies have their origin from different forms of bacteria. If it is now desired to transfer such a pure culture, then the tube is broken near the colony ; a previ- ously sterilized platinum-needle is introduced, and it is rapidly trans- ferred to a sterilized solution. The tubes possess at one end a slight constriction or indentation, at which point (a) the tube is closed 11 with cotton or asbestos, while the end drawn out to a capillary-point is sealed. The tube thus prepared is sterilized by heat. The closed end is first broken in the fluid, then the tube is filled by sucking on the end plugged with cotton (or as- bestos) ; after its withdrawal it is cleaned with alcohol, and then fresh- ly sealed at the capillary end or closed with varnish. The aippearance of isolated de- composing spots, or, in general, of colonies of bac- teria, is similar in substances, which spontaneously solidify or gelatinize (as in blood by coagulation) ; while in true solutions such a sharp isolation of the different colonies does not occur even in the capil- lary-tubes. CULTURE-METHODS ; PURE CULTURES. 123 In regard to this point, then not completely under- stood by Salomonsen, this method was one of the most reliable for producing real pure cultures, until a few years ago. If blood is taken directly from the vessels of sound animals without letting the air come in con- tact with it, such decomposition-spots never occur. According to Salomonsen, it is carried out in the following manner (first part of Fig. 14) : the end of a sterilized glass tube, that has been drawn oat and sealed, is introduced into the vessel which has been previously laid bare, and opened under antiseptic pre- cautions. After the blood in the tube has first flowed out, it is bound fast to the vessel at &. Then the sealed end is broken within the vessel at /, and the tube filled by suction at the opposite end, which has been plugged with sterilized cotton or asbestos. After this the ligatures a, c, and d are tightened, the vessel is cut between a and &, and between c and d, and the tube is sealed in the flame below 5. At this end, at no time during the filling of the tube can air gain admission, and at the other end only filtered air enters. The tube is then placed in a thermostat at the temperature of the blood. The glass tubes are first disinfected with a one per mille solution of sublimate, the sublimate removed by alcohol, the alcohol by ether, and the last evapo- rated by warming. This method I have found to be the most reliable one. This disinfection should be performed before the tubes have received their defi- nite form ; then one end is drawn out and sealed ; the other end receives a slight narrowing or twisting, and here, as at a (second part of Fig. 14), it is securely fiUed with asbestos or cotton. The tubes, thus pre- pared, are finally sterilized by exposure to a tempera- 124 BACTERIOLOGIGAL INVESTIGATION: ture of 150° or 160° C. for one or two hours, and after cooling, immediately before the operation, the capilla- ry end is passed once more quickly through the flame. Zahn,* in place of such glass tubes, uses a pipette that will contain from 50 to 100 c. cm., one end of which is drawn out to a point, and the other has pre- viously received a constriction at one point. Then the balloon of the pipette is heated in the water-bath or in a flame. During the manifold heating the air is displaced by oxygen, carbonic acid, or hydrogen, and then, during this process of heating and the consequent expansion of the gas, both the ends are sealed, the second one at the point of constriction. The sterilization is accomplished by exposure to a high temperature. Since the air in the pipette is ex- panded by the heating, after breaking the point in the vessel the flUing of the pipette follows from the negative pressure. In the subsequent sealing, ac- cording to Zahn, fine cracks sometimes occur, on account of which great care must be taken. I cover over these cracks, immediately after sealing the ends, a layer of sealing-wax. If the blood is taken from sound animals, the clot separates from the blood-se- rum. Upon the latter there forms sometimes a fine scum of the smallest fat-drops and altered blood-cor- puscles. The cellular elements gradually disintegrate by retrograde metamorphosis. The granulations dis- appear, but true bacteria or cocci do not gain an en- trance, and the spots of decomposition do not appear. These blood-granulations, like the bacteria, are im- mediately apparent, and also, like bacteria, become cleared Up, by anamorphosis of the protoplasm. * " Ilntersuohangeii ilber das Vorkommen voh Fauluisskeimen im Blut gesunder Thiere." " Arch. f. pathol. Anatomie," 1884, Bd. OXLV, S. 401. CULTURE-METHODS; PURE CULTURES. 125 If the blood contains bacteria, as is the case in many infectious diseases, pure cultures of these may- be obtained in the tubes in this way. 7. The iNFEOTioiir-METHODS. In the consideration of the strongly obligatory parasites, those referred to in the Introduction, as possessing in the highest possible degree adapta- bility to the parasitic mode of life, only the tissues of the animal or vegetable organism would seem a priori to offer the necessary conditions for their existence, and in many extreme cases only a certain form, or even only a certain variety of these tissues, seems to be able to serve as host to the parasite. These observations lead to the inoculation of sus- ceptible species of healthy animals and plants with the parasites, so that the artificially infected organ- ism may thus contain these parasites in a pure con- dition. I wiU only mention the known experiments concerning trichinosis, and the infection of plants by de Bary, van Tieghem, and especially by Bref eld, who in this way more exactly defined the problems to be solved by the infection-method* — ^i. e., " first, the de- termination of how and where the germs of the fungus gained admission ; then, secondly, the tracing of the development of the fungus, and the continuous ex- tension of the typical disease in the host from the action of the germs that have gained admission." The transmissibUity of many infectious diseases, established both by clinical observation and the facts of epidemiology, as well as by numerous experiments made after we had learned to ascribe many of these diseases to micro-organisms, led to the use of the * " Die kflnstliche Kultur parasitischer Pilze." " Botanische TJii- tersuchungen fiber Hefenpilze," Bd. V, 1883, S. 1. 126 BACTERIOLOGICAL INYESTIGATIOK method of infection in the study of bacteria. We must here forcibly distinguish two things : first, the transfer to animals of pure cultures of bacteria dep- rived elsewhere for the determination of the malig- nant peculiarities of these germs ; and, secondly, the transfer from animal to animal without previously obtaining pure cultures elsewhere. For such infec- tion, almost entirely formerly, and also largely now, the ordinary species of animals have been used, which were ready at hand in the laboratory. Koch* has shown that in the inoculation of field- and house-mice with a decomposing fluid, only the latter died of a certain form of septicaemia caused by a fine bacillus, and that, after several transfers from mouse to mouse, the blood of the last animal presented an absolutely pure culture of this form of bacillus. He demon- strated the same fact for another form of bacillus by the inoculation of rabbits with other decomposing fluids. In these experiments also, after a few trans- fers from animal to animal, all other forms of bacteria originally present in the fluid in large quantity were eliminated, and the blood contained a pure culture of a single form of bacteria. Anthrax bacilli kill mice with such absolute certainty that, by. the inoculation of mice with mixtures of bacteria which contain the anthrax bacilli, after a few transfers these bacteria can be obtained pure. Further, Carter and Koch succeeded in infecting monkeys vplth the blood of relapsing fever, so that the blood of these animals presented pure cultures of the spirochsetse of this disease. * " TJntersuchungen fiber die Aetiologie der Wundinfectionskrank- heiten," 1878. " Ziir Untersuchung von pathogenen. Organismen." " Mittheilnngen aus dem kaiserlichen Gesundheitsamte," Bd. I, 1881, S. 1. CULTURE-METHODS; PURE CULTURES. 127 From these observations Koch, deducted the im- portant principle that for infection-experiments, first of all, such species of animals should be used as are proved to be susceptible to the disease under consid- eration ; and that animals should be chosen of the same species as that in whi(?h the disease occurs. If this is impracticable, the species vrhich resembles most closely the form of animal in which the disease spontaneously appears should be used. The techni- cal rules to be observed are given later in the inocu- lation-experiments. These infection-methods are to be used to obtain pure cultures in those cases where the blood or the artificially infected organism in general presents a pure culture of an infectious micro-organism, which shows in the highest degree parasitic adaptability, and also in the study of all those infectious diseases in which micro-organisms have not yet been found, but which clinically and epidemiologically appear to be purely contagious. But in some of these diseases, in many acute exanthemata, such inoculations seem quite purposeless, because these diseases are most probably confined exclusively to man, an^ because the supposed micro-organisms probably find, in the human body only, the conditions necessary for their existence. In such cases, naturally the solution of aU the questions presented in the Introduction is impos- sible, and it would be unreasonable to ask here, from the bacteriological investigation, the solution of the problems, which are insoluble from the nature of the case. But in these cases those portions of the problems which can be solved must be the more certainly de- termined, and the clinical and epidemiological obser- vations the more critically studied. But bacteriology 9 128 BACTERIOLOGICAL INVESTIGATION. must only first forego the solution of all problems, when all possibilities are really exhausted ; for an improvement in the methods sometimes renders pos- sible the complete solution of problems seemingly insoluble. As a most remarkable example of this, Koch's inquiry into the aetiology of tuberculosis may be cited, which showed that the experimental pure culture of the bacillus , tuberculosis was possible out- side of the animal organism, in spite of the apparently intense degree of its parasitic adaptation. 8. The Culttjees upon Transpaeent Solid Nu- trient Media according to Koch. In the communication upon this method * the means were given, which had been previously ap- proved of for the re-establishment of pure cultures in certain cases. 1. The advantages of the solid opaque media for the isolated culture of the characteristic pigment bac- teria, after Schroeter. 2. The possibility of the isolated development of pure colonies of bacteria in blood, and of the diiier- ential diagnosis of such colonies vrith a low magnify- ing power, after Salomonsen. 3. The advantages of transparent fluid media for many cases, according to Pasteur, Cohn, and Brefeld. 4. The principle of the development from one germ, after Brefeld, Pasteur, Lister, Naegeli, and Pitz. 5. In the use of this principle the necessity of the local separation, in order to give to each individual germ the possibility of development, isolated and pure. * " Zur Untersuchung von pathogenen Organismen." "Mitthei- lungen aus dem kaiserlichen Gesundheitsarate," Bd. I, 1881, 8. 1. CULTURE-METHODS; PURE CULTURES. 129 6. The infroduction of gelatin by Klebs and Bre- feld, to prevent the evaporation of the culture-fluids. The advantages of these different methods had only been attained separately, even by Koch, but he found the connecting link which permitted the union of most of them, and by this combination a most universal method, and at the same time the most simple of all methods, resulted. Upon solid culture-media, germs, which have been' intentionally deposited there or have been derived from the air, develop into isolated colonies. If this solid nutrient medium is opaque, a satisfactory ob- servation is only possible in the case of micro-organ- isms having a peculiarly characteristic growth — as, e.g., pigment-bacteria. But if the culture-medium is not only solid, but likewise transparent, colonies can be differentiated from one another by the help of a low magnifying power, through peculiarities of their growth, which are not perceptible to the naked eye, or a simple lens. Koch united the advantages of the solid culture-media for the separation of differ- ent germs to the advantages which the transparent media possess for direct microscopical observation. This simultaneous emphasis upon the solidity and transparency of the culture-medium distinguishes Koch's method from all others. Koch followed two quite different ways in attain- ing these objects — viz. : he first chose a solid, trans- parent culture-medium which, without any addi- tions, would answer to these requirements; and, second, he produced a solidification of the ordinary clear nutrient solutions by the addition of gelatinizing substances. The addition of these gelatinizing sub- stances to the culture-media led to the discovery of the principle of splidity and transparency, and the 130 BACTEBIOLOGIOAL INVESTIGATION. media thus prepared temporarily preceded the natu- ral solid transparent media. The former were made by the addition to the previously known normal cult- ure-media, and to the approved decoctions and infusions, of sufficient gelatin to convert these solu- tions, at the temperature of the. room, into transpar- ent solid, nutrient substances. Koch found, if he inoculated such a nutrient gela- tin, while it was still liquid, with a drop of fluid con- taining bacteria, that in the subsequent solidification the individual germs were each surrounded by a layer of gelatin. If the fluid used for inoculation did not contain too many germs, these remained sufficiently separated after the gelatin became solid, so that each germ was isolated at the point of its fixation, and could there develop into an isolated colony. If, now, the gelatin solution was allowed to solidify on a transparent glass plate, the development of the colonies from single germs could be observed with the microscope before they were perceptible to the naked eye or the simple lens. Koch did not use the gelatinizing substances, as did Klebs and Brefeld, to prevent evaporation. The better nutrient capacity was not a desirable quality, as Brefeld emphasized, but was often even undesir- able, or at least an indifferent one. The possibility of the reversal of the nutrient gelatin-drop to prevent the air-infection, also advanced by Brefeld as desir- able, was quite unnecessary ; because, upon the solid culture-media, the germs originating from the air were also strictly localized in their development, so that by their position they could be easily distin- guished from the colonies of the germs intentionally inoculated. To this is added the further difference that both Klebs and Brefeld, in order to use the gela- CULTURE-METHODS; PURE CULTURES. 131 tinizing substances in their sense, must previously have a pure culture, which the first obtained by the fractional culture-method, and the second by the method of dilution. Neither in the writings of Klebs nor of Brefeld is there found the slightest intima- tion of an experiment, or even of an. idea of using gelatin to solidify the solutions for separation of dif- ferent germs, or for obtaining pure cultures, as was clearly put forward by Koch.* In a colony composed of very similar organisms, recognized as growiag characteristically, first by the microscope and later by the naked eye, the peculiari- ties of each organism are grouped together. The whole habitat of a colony can in this way be espe- cially valuable for the differential diagnosis of organ- isms, the forms of which are very similar. The study of the morphological differences, which show them- selves in the pure colonies, makes this bacteriological method especially useful for hygienic purposes. The proof that a colony, visible with the naked eye and with a low magnifying power (up to 80 or 120 diameters), which has a representative and char- acteristic growth, has developed from a single germ, was not certainly determined before the discovery of this method, and was later strikingly brought oat by Hansen (page 110). After some practice, the observer learns to determine whether a colony origi- nated from a single germ, or whether it has proceeded from the union of several colonies. * When Zopf, in his work concerning the fission fungi, referred to Koch's methods under Brefeld's methods of gelatin-culture, he paid no regard to the statements of either investigator, since there can scarcely he presented greater differences than exist between these two methods. 132 BACTERIOLOGICAL INVESTIGATION. A. TEANSPAEENT SOLID MEDIA MADE BT THE ADDI- TION OF GELATIKIZING SUBSTANCES — "NUTEIENT GELATIN." Peepaeation of Ntjteient Gelatin. — To one of the tested nutrient solutions, decoction, or infusion, there is added pure, finely cut gelatin, preferably for the test-tube cultures 5 per cent in amount ; but for the slide- and especially the plate-cultures, 10 per cent. This gelatin is allowed to soak for half an hour to one hour, then is completely dissolved by application of heat. Since the reaction of gelatin is acid, and most bacteria require either a neutral or slightly alkaline reaction for their growth, the warm gelatin solution is neutralized, or, still better, for most cases, rendered slightly alkaline with carbonate of sodium. The neutralized gelatin solution is then boiled for about one hour on the water-bath, for the complete separation of the neutralization precipitate and aU other substances coagulable by heat ; and after this, while still hot, is filtered through a moist fluted filter. The filtrate, after renewed boiling, must, when cold, be solid and clear without any cloudiness. A tran- sient cloudiness produced in the cooking by the phos- phates, and which disappears after cooling, has no significance. It is self-evident that no nutrient gelatin offers to all bacteria equally favorable conditions for exist- ence ; but still it is desirable to possess gelatinized solutions, which are as universally valuable as possi- ble, so that they furnish, to the largest possible num- ber of forms of bacteria, conditions sufficiently favor- able, so that the germs can develop into perceptible and separable colonies. The meat-water peptone gela- OnZTUBE-METHOnS; PURE CULTURES. 133 tin, as described by Loffler,* supplies this. It is prepared in the following manner : half a kilogramme of good finely chopped meat is added to one litre of distilled water, well stirred np and allowed to stand in an ice-chest twenty-four hours. Then this is fil- tered through gauze, finally pressed o^t with a pe- culiar meat-press, and the volume of the fluid, by the addition of distilled water, is restored to one litre. To this meat-water is added 10 grammes of dry peptone, 5 grammes of chloride of sodium, and 50 to 100 grammes of the purest gelatin. The swelling and solution of the gelatin, neutralization, boiling, and filtration are performed as described above. I have obtained the same object in a more simple manner with a nutrient gelatin, which consists of peptone 3 per cent, grape- or cane-sugar \ per cent, and beef extract \ per cent, with 5 to 10 per cent of gelatin. The addition of a small quantity of sugar to this otherwise much less advantageous solution makes it available for the same purposes. The boiled and neu- tralized nutrient gelatia must be filtered while hot. A hot-water filter (Fig. 15, T) is most conven- iently used for this purpose, in which the mantel of water between the glass funnel and the outer cop- per wall is kept warm by a flame which is placed under the tube (a), the lumen of which communicates with the water-mantel. It can be done less conveniently by filtering successively small * " Mittheilungen aus dem kaiserlichen Gesnndheitsamte," Bd. I, 1881, S. 169. Fie. 134: BAGTERIOLOOIOAL INVESTIGATION. portions while hot, during which it is advantageous, from time to time, to carefully warm the funnel by a small flame. The neutral, clear, nutrient gelatin is then poured into sterilized test-tubes to about one third their height (equal to about 10 c. cm.), with the aid of a sterilized funnel or pipette, and the sterilized cotton stoppers after this are again replaced. This nutrient gelatin in the test-tubes is sterilized by discontinuous boiling. For this purpose the solidified gelatin in the test-tubes may be caref uUy liquefied directly in the flame, and then boiled, or the gelatin may first be dissolved by placiag the tubes in warm water, and then, after drying the glass, it may be boiled for a short time in the flame, or this may be done in the water-bath. This should be repeated for about ten minutes for four or five successive days. If the gelatin has stood for a long time, so that it has begun to decrease by evaporation, it must be once more liquefied and boiled before using. The nutrient gelatin thus prepared is used for slide-cultures, plate-cultures, and test-tube cultures. a. Slide-Cultures. — The gelatin in the test-tubes is rendered fluid by heating or warming in a water- bath at about 30° C, and the cotton stopper, so far as it projects, is charred in the flame before opening, to destroy whatever germs of fungi or bacteria may have gathered upon it. Slides are thoroughly cleaned with mineral acid, water, alcohol, and ether, and sterilized by expos- ure for one or two hours to a temperature of from 150° to 160° C. in a dry-oyen. For this purpose a number of clean slides are placed in a small beaker, over which a larger one is turned to protect them from dust while cooling. For the reception of the CULTURE-METHODS; PURE CULTURES. 135 gelatin a number of slides are placed, as nearly hori- zontal as possible, on a table or glass plate, and cov- ered with a glass jar to protect them from dust. For this purpose the apparatus (Fig. 16) is most conven- Fio. 16. ient. This consists of a triangle made of wood, pro- vided with leveling-screws, and upon the triangle a ground-glass plate {g) is laid. This plate, with the aid of a level (Z) and the screws, is made horizontal. Under the glass plate there is room to place a dish with ice- water, in order to thoroughly cool the glass plate, and thus the solidification of the gelatin is accelerated. With a sterilized pipette the fluid gelatin is placed upon the slide in the form of a long, thin layer, which is a few millimetres thick, and does not extend at any point to the edge of the slide. If it is not pos- sible to arrange the slide exactly horizontal, then the consequent difficulty can be avoided by dipping the test-tube in cool water, or holding it under a water- jet, so that the gelatin is so far cooled that it is no longer a, thin fluid, but has more consistency. "When these layers of gelatin have so far solidi- fied as to render the gelatin not completely solid, but very consistent, then they are inoculated with a sterilized platinum needle, with which a particle of the infecting substance or fluid has been taken up. With this, from three to five lines of inoculation are 136 BAGTERIOLOOIOAL INVESTIGATION. lightly drawn upon the layer of gelatin. The depth of these lines should not extend to the slide. When the gelatin is completely solidified the germs become fixed, and, if there are not too many present in one line, each is so far separated from others that it may develop into an isolated colony. The inoculated slides are placed in a moist jar (Fig. 9) upon a bench of glass or zinc, across which are arranged from two to four such slides. Over these a second glass bench is placed for support and protection, and in this way several tiers, one above the other, can be prepared (Fig. 17). These glass benches are care- fully cleaned and sterilized by heat before use. Such benches can be prepared from a strip of zinc about 4 cm. broad and 14 cm. long, each end of which, from 1 to 1^ cm. wide, is bent over, or from a strip of glass 14 cm. long and 4 cm. broad, by cementing to the ends small glass supports with Canada balsam. Upon these benches, strips of dry filter^paper of corresponding size are laid, and upon them the slides are placed. An absolute protec- tion against infection from the air is not essential. The germs in the air can only settle upon the surface of the gelatin, and are in this way recognizable, even if by chance they develop into a colony upon a line of inoculation or in the immediate vicinity. The growth of a colony in the line of inoculation is ob- served with a dry lens of low power, magnifying from 80 to 150 diameters. If different forms of colonies develop in the line of inoculation, a second inoculation is made with the aid of a low magnifying power of 15 to 20 diameters for the removal of a colony. This transfer is made with each colony. CULTURE-METHODS; PURE CULTURES. 137 showing differences in growth, to other slides, so that after a few transfers there is only a single form upon each slide. In all gelatin cultures the following should be noted : In respect to their behavior toward the gela- tin, all bacteria can be practically divided into those which leave the gelatin solid and those which liquefy it. The first develop in the interior of the gelatin in a round, oval, or disk form, etc., while on the surface a characteristic superficial growth is produced, some- times in the form of a sharply circumscribed circle, sometimes in leaf- or grape-like arrangement, some- times in the form of concentric rings, etc. Transfers of such bacteria should be made both from the iso- lated colonies in the iaterior and from the edge of the colonies developing superficially. The bacteria producing liquefaction of the gelatin do this in different ways — sometimes rapidly, some- times only slowly, in the form of a funnel. Since, vnth.the advance of the liquefaction, the advantages of the solid culture-media are lost, these forms should be separated from each other as quickly as possible by using the fewest germs possible for inoculation, so that the liquefying colonies do not come in contact with the others until they have developed sufficiently for further inoculation. The inoculation should al- ways be made from the edge of the colony where the liquefaction is extending upon the stiU solid gelatin, because in the interior of the liquefied portion a mixt- ure with other forms may have taken place already. Colonies should be chosen for inoculation which, in the microscopical examination with a dry objective, present a uniform appearance throughout, and in which the characteristic differences are as clearly pronounced as possible. In addition to this, for con- 138 BACTEEIOLOOIGAL INVESTIGATION: trol, it is necessary to make dried cover-glass prepara- tions for the microscope from the colonies transferred. By direct observation with a low magnifying dry objective or an especial preparation-microscope, the colony to be transferred is picked out with a sterilized platinum needle. Some experiiBUce is required for obtaining the manual dexterity necessary to remove only the exact portions to be used for inoculation, and not to touch other portions of the surface with the needle before or after. 6. Plate-Cultures.*— The^a&s plates necessary for use are of the thickness of a slide, and of such width that all points on their surface can be made accessible for microscopical observation. The relation of the width to the length depends upon the width of the stage of the microscope — about 8 x 14 or 10 x 12 cm. These plates are thoroughly cleaned (mineral acid, water, alcohol, ether), then placed in a tin box of cor- responding size, covered over, and sterilized for two hours at a temperature of from 150° to 160° C. After cooling, such a glass plate is placed upon the glass plate of the apparatus (Fig. 16), and is protected from dust by a clean glass jar. The gelatin in a test-tube is then liquefied in a water-bath at 30° C, or by heat- ing, and again so far cooled that it is somewhat con- sistent. The firmly fixed cotton stopper is then loos- ened by twisting with previously heated pincettes, so that its removal can be quickly and easUy accom- plished. It is advantageous to free the upper portions * These were first demonBtrated at the Hygienic Exhibition, and in au address by Koch at the Eleventh German Congress of Physi- cians at Berlin in 1883. More recently directions for these cultures have appeared from Biedert in an article in the " Deutsche Medicinal- Zeitung," 1884, and from Johne, " Ueber die Koch'sohen Eein-Kul- turen," 1885. CULTUBE-METSODS ; PURE CULTURES. 139 of the cotton plug from any possibly deposited germs by charring it in the flame. When the gelatia in the test-tube has been thus prepared, the tube is held with the thumb and forefinger of the left hand in a direction as oblique as possible, without the gelatin coming in contact with the cotton. Then the previ- ously sterilized platinum needle or loop, while held by its glass rod in the right hand, is introduced into the material to be used for inoculation, thus removing a smaU portion ; then the cotton plug is removed with the fourth and fifth fingers of the right hand, and the platinum needle or loop is passed into the lique- fied gelatin, moved to and fro in it, wiped off on -the sides of the test-tube, removed, and the cotton plug is again quickly replaced. After this, the material introduced is mingled as equally as possible with the gelatin by turning, in- cliniug, or gentle shaking ; then the fluid gelatin, containing the germs thus separated, is poured out upon the cooled glass plate, and is spread out upon it with a glass rod or platinum wire, previously sterilized by heat and again cooled. This plate is laid upon a glass bench in a moist chamber, and over it a second glass plate is placed, so that here also several tiers of jdates can be arranged in one jar. While in the slide-cultures not the entire mass of gelatin, but only the lines of inoculation and their immediate surroundings are used, and while on this account a relatively large number of germs appear for separation along a relatively small line of inoculation, in the plate cultures, on the other hand, a relatively small number of germs are distributed in a proportion- ately much larger quantity of gelatin. Further, the division of the germs in the still fluid gelatin is far better, because the individual germs can develop into 140 BAOTEBIOLOOIGAL mVESTIOATIOK colonies, being fixed in the solidifying gelatin and being isolated and more widely separated from other germs. An enormons number of germs can in this way be certainly separated from one another in a single platerculture, in which each colony corresponds to a single isolated germ, and in which at first but few colonies are in contact. All colonies which show differences are first exam- ined microscopically, and then from the same, pure cultures are prepared by a second inoculation. For this purpose, in exactly the same manner, under the control of the microscope, a particle from a pure colo- ny ns introduced into new gelatin, and from this a new plate-culture is prepared, in which then, with the exception of possible air-infection, only this one or- ganism develops. In these second inoculations, pure cultures can. with certainty be obtained. Contamina- tion by the air-germs can not be avoided during the opening, but it is not even approximately to be so much feared as infection by imperfectly sterilized instruments and hands, since these air-germs also de- velop in isolated colonies. If infection from the air has taken place during the opening and inoculation of the test-tubes, these air-germs can naturally de- velop ia the same manner in the gelatin as those in- tentionally introduced ; but their small number in comparison with the other organisms furnishes a sus- picion of their source, and, moreover, not only a single plate-culture, but several should be made from the same material, so that one aids to control the other. If infection from the air has taken place after the solidification of the gelatin, this can be easily recog- nized by the position of the colonies on the surface. In the larger number of cases this procedure suf- fices ; but now and again in decomposing fluids, pus, OULTUBE-METHODS ; PURE CULTURES. 141 f seces, and very dirty water, the number of germs trans- ferred with the drop or particle is so great that a suf- ficient isolation of the individual germs is impossible, because bpf ore the appearance of perceptibly charac- teristic differences in growth the colonies have come in contact with one another. In these cases the dilution must be carried still further, and this can be done in two ways : first, the material taken by the platinum loop can be brought into sterilized distilled water, mingled with it by shaking, and from this greater or less diluted mixture a drop may be used for inoculating the fluid gelatin. A second procedure is dependent on fractional cultures. A tube of sterilized fluid is first inoculated in the described manner, and is " the original." After thorough mixing, a few small drops — e. g., five — are in the same manner transferred from this tube to another for " the first dilution " (erste Verdiinnung). The original tube is held between the thumb and index-finger of the left hand, and the tube to be in- oculated between the index and middle finger ; then the stopper from the original is removed with the pincettes and laid to one side, or it is placed between the fourth and fifth fingers of the left hand, which then holds simultaneously two tubes and the cotton plug. After this the plug from the second tube is removed by the fourth and fifth fingers of the right hand, the platinum loop is introduced into the origi- nal tube, and a small drop is transferred into the second. By a to-and-fro movement the drop is care- fully removed from the loop, and then another drop is added, either with the same or a second platinum loop, previously prepared, and the same process is repeated about five times. Then the plug, held in the right hand, is placed in the newly inoculated 142 BACTERIOLOGICAL INVESTIGATION. tube ; afterward that in the left hand is placed in the original tube. Now the original (equals No. 0) and the first dilution (equals No. 1) are ready. Then a "second dilution" is made, and a third, in exactly the same manner, taking the same number of drops from the culture preceding. Each of these tubes furnishes a plate-culture designated by Nos, 0, 1, 2, and 3, which are placed in the same moist jar in tiers (Fig. 17). Each of these cultures controls the others. If a number of different forms of bacteria have been- separated, a plate-culture is made for each one of these, which, with the exception of possible air- infections, constitutes a pure culture of one of the single forms present in the original mixture. These inoculations of the pure cultivated organisms are repeated often, making peculiarly characteristic colo- nies, as tested microscopically, in order in this way to eliminate every soluble chemical constituent of the first substance. c. Test- Tube Cultures. — On account of their slight protection, after standing a long time, it is scarcely possible to prevent the infection of the slide in plate- cultures by air-germs, which is especially incon- venient if a liquefaction of the gelatin is brought about by them. On this account it is necessary to renew the slide- and plate-cultures often, in order to avoid the accumulation of cultures occupying time and space, but especially to preserve certainly pure a culture once obtained. In order to do this, test- tube cultures are employed, in which, besides, many peculiarities of growth can be better noted. The test-tube, one third filled with solidified, ster- ilized nutrient gelatin, needs no other preparation than the loosening of the cotton stopper by turning with the heated pincettes in order to remove it quickly. CULTURE-METHODS; PURE CULTURES. 143 Fia. Then, witli a sterilized platinum needle, a particle from the pure culture, under control of the micro- scope, is taken. The test-tube is held in the left hand so that the mouth looks downward ; then with the fourth and fifth fingers of the right hand, or with the piacettes, the cotton plug is removed, and, with the platinum needle held by its glass rod be- tween the thumb and index-finger, one or more thrusts are made into the gelatin (Fig. 18). Finally, while the mouth stUl looks downward, the cotton stopper is again securely replaced. The changes in such a tube-culture after the inoculation with the bacteria vary con- siderably. As a general statement, the fol- lowing data serve : Those forms of bacteria which do not liquefy the gelatin produce a characteristic surface-growth, extending out from the point of iaoculation, so that soon a flat or more prominent head is formed, which, in connection with the line of inocu- lation of the culture, presents the form of a nail — "nail-cultures" (" IsTagelculturen " of Friedlander) (Fig. 19, In place of such a growth, others develop on the surface in the form of con- centric rings, or present leaf- or grape-formed appearances. Some S;how an intense surface- growth and a want of develop- ment along the line of inocu- lation ; in others this is exact- ly reversed. The cultures ap- pear sometimes dry, sometimes mucoid ; some are refracting, others transparent. The color of the cult- 10 a). Fio 19. Ui BAOTERIOLOOICAL INVE8TI0ATI0K ures is very variable. The gelatin is sometimes col- ored, sometimes not, and often a peculiar odor is pro- duced. All of these small morphological and biolog- ical differences are to be noted, because they facilitate the differential diagnosis. In the case of the bacteria that liquefy the gela- tin, this is at times quite gradual, so that the gela- tin-culture presents a delicate funnel-formed appear- ance (Fig. 19, &). In other cases it is more rapid ; in these, wider funnels are produced or the lique- faction extends more in the form of layers. Some- times a scum or mycoderma forms on the top ; in others not. Often on the border-line between the liquefied and still solid gelatin (Fig. 19, c) the culture appears in the form of diflerent-shaped • clouds ad- vancing forward. Sometimes the liquefaction seems closely united to the advances of the growth ; many times it precedes it, as if the liquefied material was produced by the bacteria which extend farther than the visible growth reaches. In the case of the bac- teria producing liquefaction also, the cultures may present different colors, and the gelatin may undergo change of color. An odor may be produced. These differences are collectively to be noted. The adaptability of the ordinary gelatinizing sub- stances — as isinglass, caraghen, and gelatin — in re- gard to temperature is limited to about 25° C, be- cause, in the concentration to be used, complete liquefaction takes place at this temperature, and then the advantages of the solid nutrient media are lost. If it is desired to make use of the advantages of the gelattaizing substances, transparency and solid- ity, at the temperature of the culture-oven, the vege- table gelatin called agar-agar, obtained from Graci-, CULTURE-METHODS; PURE CULTURES. 145 laria lichenoides and Gigartina speciosa, is used in place of gelatin. Instead of 5 or 10 per cent of gelatin, 1^ or at most 3 per cent of iinely cut agar-agar is added to the solution. This is allowed to stand in an ice-chest for twenty-four hours to swell, and is then dissolved as completely as possible by boiling. The swelling can be accomplished in a shorter time by a slow heat- ing. This hot solution is neutralized with carbonate of soda, and then cooked two hours on the water- bath, or one hour in the steam cylinder, for the sepa- ration of the substances precipitated by the neutrali- zation and those coagulable by heat. Although the precipitated material may have been previously sepa- rated as much as possible by thick gauze, still these agar-agar solutions filter very badly, even in small quantities and in a hot-water filter. ■ According to Rosenbach, they are better filtered through cotton, while the glass funnel filled with cot- ton or glass-wool, and the vessel for receiving the fil- trate, are placed in the steam apparatus. The filling of the tubes with the agar-agar solution and their sterilization is done in the same way as with gelatin. In thick layers the agar-agar seldom seems so clear as does a layer of gelatin of equal thickness. The inoculation of the plate-cultures is performed as usual ; the solid agar-agar is liquefied at about 42° C. in a water-bath, and the inoculation and distribu- tion of germs in the fluid-agar solution is accom- plished while the tube is in part submerged in warm water, since these solutions under 40° C. lose their fluid state. At a temperature above 40° C, in which the liquefaction is more easily made, the germs are injured, so that the operation must be done between 40° and 42° C. 146 BACTERIOLOGICAL INVESTIGATION. The pouring out of the inoculated fluid upon the plate, on this account, must be done as quickly as possible. A li per cent solution of agar-agar-gelatin is so solid at 37° C. that all the advantages of the transparent and solid nutrient media are stiU pre- served at this high temperature in the culture-oven. Many bacteria do not develop so weU in agar-agar as in gelatin ; but Rosenbach, however, has clearly shown the great superiority of this culture-medium, because most bacteria which liquefy gelatin, and which therefore produce no surface-growth, do not liquefy the agar-agar, but, on the contrary, show on it a very characteristic superficial growth. The cultures upon agar-agar can be used to sup- plement the cultures in gelatin, and in this way the number of characteristics which are presented to the naked eye for differential diagnosis is materially in- creased. In the form of plate-cultures, with gelatin at the temperature of the room, with agar-agar for the culture-oven, Koch's method of the pure culture with the solid and transparent media affords, with the greatest simplicity, the highest guarantee for the certain separation of the different germs, however numerous, from a mixture of bacteria. Impeovised Means. With the great simplicity already arrived at, on the purely technical side of the investigation, not much can bq said in regard to improvised means, since also here complete certainty must be preserved under aU conditions. The experiences, especially in hygiene, with the so-called expedited methods have taught aU unprejudiced observers that only those who have worked out these methods, and those who are thoroughly experienced and practiced investi- CULTURE-METHODS; PURE CULTURES. Ul gators, can use them sometimes with advantage and often without detriment, while beginners and inexpe- rienced workers, for whom they are really intended, are, almost without exception, led by them into error. All who would really study bacteriological inves- tigation, or would found the important facts on a really practical basis, are by all means to be provided with a microscope having an oil-immersion system and an Abb6 condenser, the most necessary apparatus. For a possible series of experiments on a more or less extensive expedition it is advisable not to take too much with you to perform this or that operation without the convenient aid of the laboratory instru- ments. Under these circumstances he only will im- provise proper means who has had experience in laboratory work, and on this account can determine the limits to be reached with safety, or else these small improvised aids must be used under definite guidance as in other laboratory work ; but then they have with improvisation nothing more to do. In this sense, in place of the gas-flame, the flame of the spirit-lamp can be used. The culture-plates may be washed with a 1 per mille solution of subli- mate and spirit, dried in the flame of the spirit-lamp, and sterilized in it by strongly heating the surface in its entire extent while the plate is held with pincettes. Then the plate, with the heated side up, is laid upon a clean piece of paper, which lies upon the table as horizontal as possible, and is covered with a soup- plate which has been cleaned with sublimate and spirit, and so allowed to cool. If gelatin in test-tubes has not been taken, but a larger quantity of sterilized gelatin in a flask, test- tubes may be sterilized in the following manner : They are cleaned with a 1 per mille solution of subli- 148 BACTERIOLOGICAL INVESTIOATIOK mate and spirit ; then tlie spirit is removed, and the tube dried by holding it over the flame of the spirit- lamp. A cotton plug is then shoved a few centime- tres down into the tube with strongly heated pincettes. Now the lower portion of the tube is heated over the flame as far as the cotton plug. After the tube has so far cooled that it can be held by its lower portion, then the upper portion is heated so thoroughly and so long that the cotton is browned ; when cooled, the plug is drawn so far out with the heated pincettes that it can be grasped with the fingers. A tube thus ster- ilized is filled one third fuU with gelatin, licLuefied by placing in hot water ; the plug is replaced, and then the gelatin is again carefully boiled, allowed to cool by dipping in cold water, and while it is yet fluid it is inoculated, and the germs thoroughly mingled with it. The gelatin is not poured on the plate when it is perfectly fluid, because the plate does not lie com- pletely horizontal ; but it is done when the gelatin has become quite consistent, though before the ap- pearance of lumps. As moist chambers', two . plates, laid one above the other, may be used, the lower one of which is covered with moistened filter-paper. Two pieces of wood laid upon this serve to support the culture-plate in place of the glass benches. B. TEAWSPAEENT SOLID MEDIA, WITHOTTT THE ADDI- TION OP GELATINIZING SUBSTANCES — BLOOD-SEEUM. For some pathogenic bacteria the transparent solid media, in the form of gelatin and agar-agar cultures, are not suitable. For these cases Koch devised, while he fully preserved the principle of the solid- ity and transparency of the culture-medium, a quite CULTURE-METHODS; PURE CULTURES. 149 different way. Koch* had observed that sterilized blood-serum, when heated above 65° C, but before reaching coagulation temperature, became solid with- out losing its transparency. Such solid, but trans- parent, blood-serum was then introduced as a nutri- ent medium. For collection of the blood, cylindrical vessels pro- vided with glass stoppers, about 29 cm. high and about 8 to 10 cm. wide, are used. These vessels, after previous mechanical cleaning, are sterilized by wash- ing with 1 per mille sublimate. After pouring away the sublimate, the remainder of it is removed with alcohol ; the alcohol is poured off and the traces of it removed with ether, and this last is evaporated by warming in the dry-oven. After removing the glass stopper, the blood of the slaughtered animal is al- lowed to run into this vessel. The neighborhood of the cut opening must be well cleaned, or, at least, must be thoroughly moistened. The blood flowing out immediately after the cut, which washes away the dirty particles of the skin, the fur, and the cut hair, is not preserved. The vessel is now filled to the brim, closed with the stopper, and, as soon as possible, placed in an ice-chest, in which it remains standing from twenty- four to thirty hours, in order to render possible the formation of a solid blood-clot. If the vessel is moved during the formation of the clot, the blood- corpuscles are mingled with the serum, which pre- vents the latter from becoming completely clear after warming. After the specified rest and time a rich layer of completely transparent, amber-colored serum forms above the blood-clot. This is removed with a * " Berliner klinisclie 'Wochensohrift," 1882, Ko. 15, nnd "Mitthei- lungen aus dem kaiserliehen Gesundheitaamte," II, 1884, S. 48. 150 BACTERIOLOOIGAL mVESTIGATIOK FiQ. 20. sterilized pipette and transferred to sterilized test- tubes, wMch are filled one third full, and then closed with cotton plugs. Sterilization can only be accomplished below the coagulation temperature of albumen by discontinuous heating. For this purpose the test-tubes are placed in the apparatus (Fig. 2). The temperature of the inner room (5) is kept at about 58° C. The blood- serum is exposed to this temperature on five or six successive days for about one hour daily. This can also be accomplished by means of a water-bath, but less conveniently. Upon the fluid, sterilized blood-serum there often forms a sciim of cholesterin, which should not be con- founded with the mycoderma produced by bacteria. This sterilized, fluid blood-serum, which is often used as such, is placed, for bringing about so- lidification, with the test-tubes inclined at a sharp angle, for attain- ing the greatest possi- ble extent of surface. For this purpose a double - walled box, made of zinc, provided with a glass cover, is used. The chamber between the double walls is filled with water. The front side of this box can be lowered by means of set- screws {st in Fig. 20, d in Fig. 21). The box is then OULTUBE-METEODS ; PURE OULTUBES. 151 placed at such an angle that the blood-serum reaches to the upper third of the test-tube ; taut care should be taken that it does not come in contact with the cotton plug. The regulation of the temperature of the air- chamber (&, Fig. 21) is accomplished by a thermome- ter placed between the test-tubes upon the floor. Solidification takes place at 65° C. ; the higher the temperature rises above 65° C, and the more it ap- proaches the coagulation temperature of 75° C, the more rapidly solidification occurs ; but transparency is more surely obtained the lower the temperature is used. The serum always becomes opaqne when the temperature approaches the point of coagulation. On this account it is best to, keep the temperature as nearly as possible at 65° C, and at least not to allow it to rise above 68° C. The blood of different animals solidifies vsdth different rapidity — the blood of- sheep most rapidly, calves' blood most slowly. Grenerally, solidification takes place iu from one half to one hour's time. Blood-serum that has been properly solidified is as solid and firm as hard-cooked white of egg, similar to amber, transparent, and only in the lower third a light milky cloud appears. During the heating, water of condensation formed on the upper waU of the test-tube, which is coolest, collects at the bottom of the test-tub6 when it is placed upright, and forms, by absorption of soluble substances, a nutritive solution, so that the simultane- ous growth upon solid and fluid nutrient media may be observed after the inoculation, if the inoculation extends to the edge of the fluid. By evaporation the serum gradually dries, begin- ning from above, yet the middle and lower portions 162 BAOTERIOLOaiOAL INVESTIGATION. remain available for months. Loffler showed that so- lutions which heighten the nutrient value of the blood-serum, when added in small amounts, do not diminish its capacity for solidifying in a transparent form, so that in place of the pure blood an improved blood-serum can often be advantageously used. Lof- fler* adds to three parts of blood-serum one part of beef-infusion. The beef-infusion is prepared ex- actly after the manner of Loffler's gelatin (page 133) ; 1 per cent of peptone, 1 per cent of grape-sugar, and i per cent of chloride of soda is added ; the solution is boiled, neutralized with carbonate of soda, boiled again on the water-bath until the albumen is com- pletely precipitated, and then filtered. This bouillon is sterilized in the steam-cylinder, and, after cooling, is added to the serum ; and then the serum, with the added bouiUon^ is sterilized by discontinuous heating, and solidification is brought about. It is self-evident that other substances may also be added — e. g., solu- tions of beef-extract with sugar, etc. Since the test- tube cultures can not be directly observed with the microscope, blood-serum may also be allowed to so- lidify in watch-glasses, or in hoUowed-out glass blocks which are covered with glass plates ; but then the greater protection which the cotton stopper of the test-tube affords is lost. For inoculation of the test-tubes, they are held in the deft hand as nearly horizontal as possible ; then a platinum loop is dipped into the substance to be inocu- lated, the previously loosened cotton plug is drawn out with the fourth and fifth fingers of the right hand, and the germs are transferred to the surface of the solidified serum by firmly drawing lines with the * " Mittheilungen aus dem kaiserlichen Gesnndheitsamte," Bd, II, 1884 S. 452 und 461. CULTURE-METHODS; PURE CULTURES. 153 platinum loop upon the same. The cotton stopper is then again replaced. Since in test-tubes, on the one hand, a direct mi- croscopical control, and on the other an isolation of the germs in the solidification, is not possible as in gelatinizing substances on slide- and especially plate- cultures, many individual experiments (say ten) must be made with each substance, and, aside from this, the cleanest possible inoculating material must be obtained. These two facts make the blood-serum cultures, as contrasted with gelatin and agar-agar cultures, seem incomplete. But, by overcoming this technical difficulty, it is possible, on account of the retention of the principle of transparency and solidity, which distinguishes Koch's method so fundamentally from all others, to obtain on solid blood-serum fault- less clean cultures of obligatory parasitic bacteria, which can not be obtained by any other method. The method of obtaining pure original material for the blood-serum cultures needs still some expla- nation, especially for the cases in which, as in tuber- culosis, the looked-for organism develops so slowly that, in the interval, contingent rivals outnumber it. In the preceding microscopical examination of sections of different organs of tissue-fluids and blood, it is to be determined at what places the suspected bacteria are to be found most certainly, and in the purest condition. After observation of these points, it is shown that the removal of the inoculation- ma- terial is best performed when it is taken from an ani- mal recently dead or killed. The removal is proportionately more difficult and uncertain the longer the time that has elapsed after death, because then the possibility of a mixture with the rapidly developing micro-organisms of decomposi- 154: BAGTERIOLOOIGAL INVESTIGATION. tion is always greatly increased. "AU preparation cuts, wMch do not come in contact with the inoculation substance, are to be made, according to Koch,* with hot instruments, but the inoculation mass itself is re- moved with cooled scissors and pincettes, or with the cooled platinum loop. The operation should be al- ways done with heated instruments, which should be changed every time when a new section is to be laid open. This constant change of the instruments is necessary, that the contamination which adheres to them in cutting through the skin and the super- ficial layers may not be introduced into the cultures." With regard to the previous preparation, it should be noted that, after placing the animal upon the dis- secting-table, the skin, so far as the cut is to extend, should be thoroughly moistened throughout its ex- tent with a 1 per mOle solution of sublimate, in order to prevent as much as possible the scattering of dust, hair, etc., in seizing and cutting. A number of knives, scissors, and pincettes are pre- viously heated in the flame, and laid under a jar to protect them against contact and dust. Then the skin along the line for the incision — in the larger animals vdth a hot scalpel, in the smaller with hot pincettes and scissors — is cut through and laid back on both sides, so far that further operations wiU be un- impeded. With a second pair of hot pincettes and scissors, if it is desired to remove the pleura or sur- face of the lung, an opening is made in the wall of the thorax, 1 by 2 cm., and thus the surface of the lung is laid bare. If in this way an infected portion is exposed, for instance, nodules of tubercles, one or more of these nodules is snipped out with the cooled instruments. In order to release the bacilli found in * " Mittheilungen," Bd. II, 1884. OULTUBE-METEODS ; PURE CULTURES. 155 tlie tubercle nodules, a nodule is cut with a cooled knife or scissors, and a particle from the interior is removed with the cooled platinum loop and trans- ferred to the surface of the blood-serum ; or a nodule is crushed between two cooled scalpels, and this crushed mass is taken with a platinum needle and transferred to the blood-serum. If it is less consistent, it is cut into with the cooled knife and the material removed with a platinum loop. K blood is to be taken from the heart, after the skin has been cut in the same way, the wall of the thorax over the heart is opened with hot pincettes and hot knife or scissors : then, in the same way, the pericardium is opened with fresh instruments. After this the apex of the heart is fixed with cooled pincettes and a cavity opened with a scalpel, out of which the blood to be used for inoculation is vrithdrawn on a platinum loop. If an organ from the abdomen is chosen, this is removed with heated instruments after the abdominal cavity has been laid open in the same manner, and is placed upon a clean dish or filter-paper, and then only the connective-tissue capsule is cut into. After this the edges are seized with cooled pincettes and a deep tear made in the organ ; then with a platinum needle, out of the bottom of the rent, the tissue, fluid, or particles are removed. For the superficial lymph-glands the skin is cut through in the same way ; then the gland is fixed with cooled instruments, and is opened in situ for removal of material from the interior, or it is released from its surroundings, laid upon a glass plate previ- ously heated and cooled, cut or torn, and then fluid or a particle of tissue is taken from the interior. If blood is to be taken intra mtam for cultures, this can be done with the capillary-tube of Salomon 156 BACTERIOLOOIGAL INVESTIGATION. sen ; also, after opening a vessel, a drop of blood can be taken from the interior with a platinum loop. It is simplest to free a spot from hair, clean this with brash and soap, wash it with a 1 per riiille solution of sublimate, then with alcohol, and finally remove the last particle of sublimate with ether, after which a sterilized needle is introduced or a small cut is made with a sterilized knife. The first few drops of blood welling forth are removed with a platinum needle, and those coming later are used for inoculation. For removal of a piece of the skin in erysipelas and similar parasitic processes extending in the skin, a spot is chosen in which the process is advancing. The skin is cleaned in the same manner, and then, with heated instruments, a piece is snipped out which finally is still further reduced in size or crushed. If some time has already elapsed since death, the organs must be thoroughly cleaned from the organ- isms of putrefaction adhering to the exterior. Ac- cording to Koch (Z. c), this is accomplished by repeat- edly and thoroughly washing the organ in a 1 per miUe solution of sublimate. Then with hot instruments, changed at each cut, sections are removed from the surface of the organ, and only at a greater depth the tissue-fluid or particle is taken. In the larger organs — e. g. , the spleen — according to Gaflfky,* after thorough washing in a 1 per miUe sub- limate solution, a long cut is made, almost dividing the entire organ ; then, with a second sterilized knife upon the clean-cut surface, a section is made that does not extend at any point to the capsule. Upon this a third is made, and then out of the depth tissue-fluid or a particle is taken. * " Mittheilangen aus dem kaiserliohen Gesundheitsamte," 1884, Bd. II, S. 386. CULTURE-METHODS; PURE CULTURES. 157 According to Loffler,* in order to make even tlie very unclean organs useful, tlie organ is washed for ten minutes, with a continuous motion by a glass rod, in a 5 per cent solution of carbolic acid in order to destroy all the cocci, bacUli, and fungi adhering to the surface. For the destruction of contingent spores of bacilli, the organ is placed for five minutes in a 1 per miUe sublimate solution, and laid upon clean fil- ter-paper. After the surface has become dry, the connective-tissue capsule is cut into with a hot knife, the organ is torn, by seizing the edges of the cut with hot pincettes, and out of the bottom of this rent inocu- lation-material is taken. The course of the culture ought to be the same as in individual cases which f oUow from the spontaneous presence of bacteria. In the case of the septic forms, and the pigment and ferment bacteria, the object is always well at- tained with plate-cultures of gelatin or agar-agar. Hereby the small advantages which a quantity-cult- ure possesses are most favorably employed, es- pecially with regard to the heating and anaero- biosis which may be realized in the plate-cultures also. Among the parasitic bacteria there are, with the greatest probability, some as to which epidemiology furnishes evidence that they are probably optional . parasites or optional septic forms, and can live en- tirely, or, under certain conditions, outside of the animal organism. These can be obtained pure in gelatin or agar-agar cultures. For the obligatory parasitic bacteria — as are, in a greater or less degree, the parasites of the purely contagious diseases — only the blood-serum cultures, * n>id.,&. 461. 158 BAOTEBIOLOOIOAL INVESTIGATION. up to the present time, offer the possibility of ob- taining pure original material. For those bacteria, as spirilla and spirochsetse, which as yet it has been impossible to cultivate upon solid nutrient media, or only badly, and for those more movable parasitic micro-organisms, which possibly may be the cause of one or another dis- ease, it is best to recur to the tested method of dilution — the "one-cell culture." This method is exceedingly minute and not quite so reliable, but surpasses all others and is far preferable, if other means can be previously used in order to secure, if not absolutely, yet approximately, pure quantity- cultures. For other micro-organisms, as mould-fungi and yeast, the solid nutrient media, both in the opaque form of layers of potato, of potato-broth, and a thick bread-broth, are of value, and also the transparent forms of gelatin, agar-agar, and blood-serum cult- ures. Attention must also be given to the reaction, which, as a rule, contrary to the cultures of bacteria, should be kept feebly acid ; and also to the consider- ation of the spontaneous presence of the germs on the nutrient media. While previously the pure cultures had only been considered a means intended for unobjectionable posi- tive iaoculation, Koch has shown, upon the ground of the biological peculiarities manifested in the pure culture, that there was not the slightest reason to be- lieve that the aetiology of anthrax is explained by the gradual adaptation of a quite harmless bacterium to a parasitic mode of life, but that the anthrax bacilli are not at all dependent on the animal body for the retention of their species, and that they find, outside of the organism, at least for a time, all the condi- CULTURE-METHODS; PURE CULTURES. 159 tions necessary for their existence, and that their parasitic existence is only occasional,* Then Koch succeeded for the first time in cultivat- ing, artificially outside of the animal body, the para- site of a purely infectious disease, tuberculosis, f and Bref eld % showed, on the ground of his investigations regarding the septic stage of the parasitic fungus of the cereals, that it was also possible, by the use of the right methods, to bring out from its parasitic life this germ — a parasite ia the narrowest sense. In conclusion to this, I suggested * that each pure culture is nothing else than the septic stage of a para- sitic micro-organism ; and likewise de Bary || per- ceived the capacity of parasitic bacteria of being bred ia dead organic material. * "Zur Etiologie des Milzbrandes." " Mittheilungen aus dem kaiserlichen Gesundheitsamte," Bd. I, 1881, S. 49. t "Berliner klin. Wochensohrift," 1882, No. 15. I " Die kunstliohe Kultur parasitisoher Pilze.'' " Botanisohe TJn- terBuohungen flber Hefenpilze," V, 1883, S. 1. » "Fortscliritte der Medizin," 1884, No. 2, S. 70. II " Vergleiohende Morphologie und Biologie der Pilze," 1884, S. 526. 11 IV. INOOULATIONS FOE THE DETERMINATION OF THE CAUS- AL EELATION OF BACTERIA-GROWTH TO DECOMPO- SITION AND DISEASE. A. Septic Bacteria. Stjbstaitces capable of decomposition, such as so- lutions of sugar, glycerin, etc., are prepared accord- ing to the indications which the spontaneous presence of bacteria suggest. The details of the preparation have been already mentioned in the description of cultures in fluids, as also the methods of sterilization. For the qualitative experiments, test-tubes and small flasks are filled with the solutions ; for the quantita- tive experiments, flasks containing a litre and still larger are used, which have applied, over the cotton stopper, caps made of a double layer of filter-paper. If it is desired to investigate what degree of decom- position the bacteria spontaneously bring forth, then the solutions receive no additions ; but if the sub- stances are to be chemically changed, a corresponding quantity of the purest carbonate of lime is added, in order to neutralize the acid formed in proportion to their development. Inoculations are made by picking out, with a pla- tinum needle, under control of the microscope, a par- ticle from a pure culture in gelatin or agar-agar, and introducing it quickly into the solution. In these SEPTIO BACTERIA. 161 cases not only one germ, but a great number of the same germs are introduced, wMcli in this way are im- mediately transferred into a position to more easily suppress contingent rivals in the favorable solution. If the pure culture was obtained by the method of dilution, as a one-celled culture, then the unit of vol- ume (a drop, a cubic centimetre) with the hypotheti- cal germ is transferred, by means of a sterilized pi- pette, to the sterilized solution. Siace air-infection in a fluid is not immediately recognizable, and the possibility referred to is never entirely excluded from cultures in fluids and frac- tional cultures, and since in some way such an acci- dental germ introduced from the ordinary septic bacteria may outgrow all others, a greater number of individual experiments must be made in all investi- gations with fluids. It is recommended that these transfers be made in a glass case kept moist. An elevation of temperature favors the growth and action of all septic bacteria, but the temperature most favorable to the different forms is limited, and for many ferment bacteria it is about the tempera- ture of the blood. On this account, after the in- oculation of the solutions, some must be kept at the temperature of the room and others at a higher temperature in the culture-oven, in order to ap- proximately determine the favorable temperature in a short time. Control is followed out in three directions : flrst, by exposure of sterilized, uninoculated solutions to the same external conditions ; secondly, by the use of the microscope ; and, thirdly, by pure cultures. If in the inoculated solutions a change becomes visi- ble to the eye by a cloudiness, by formation of a mycoderma, or by the appearance of air-bubbles, the 162 BACTERIOLOGICAL INVESTIGATION. solution is tested microscopically by a dried cover- glass preparation. The drop of fluid must be carefully taken by ster- ilized instruments, a platinum loop or pipette. Care should be taken to see whether only one and the same form is present, and whether the form is iden- tical with that observed in the pure culture. Small differences may appear, because the solutions are more favorable to the bacteria than nutrient gelatin. Then, at the height of the fermentation, and more often toward the end, sometimes involution -forms appear, or there occur, in a more typical manner, in the individual bacteria, especially in the bacUli, pecul- iar enlargements, which produce in the rods whet- stone, spindle, or tadpole forms. If such appear- ances are observed, it is to be determined whether these forms are associated with the activity of the fermentation — i. e., heightened function— or whether, on the contrary, they constitute the first visible indi- cations of the total or partial exhaustion of the nutri- ent medium ; or whether they prepare for the f oima- tion of spores, by which, in the exhaustion of the medium, the preservation of the species is rendered sure. Concerning this the microscope alone gives no in- formation ; consequently, if these forms are seen, cult- ures are made, in which different species develop, if in the inoculation foreign bacteria have been includ- ed ; but only the inoculated forms develop after a really pure transfer. With the determination of the most advantageous temperature, the quantity of oxygen most favorable to the development is also to be especially observed. While many septic bacteria — viz., those producing oxidation decomposition and the ordinary aerobic SEPTIC BACTERIA. 163 forms — ^present no especial peculiarity in respect to this, and, with the admission of air both in fluids and in gelatinized solutions, can be easily obtained pure, other forms show a greater intensity of growth and action if the entrance of air is not quite free. When they appear spontaneously these bacteria have a tend- ency to hydrobiosis — i. e., they develop less on the surface and more in the interior of the fluid. In these cases the entrance of oxygen should be restricted ; but the oxygen in solution is always at the command of these bacteria. It is very doubtful whether the ex- treme ground is tenable which Pasteur takes for the departure of his theoretical observations on decompo- sition — ^viz. ,'that the anaerobic forms are so constituted that the air acts as a poison, since in all these experi- ments still other factors come into play. Also, the anaerobic bacteria can be obtained pure by gelatin and agar-agar cultures, because, in most cases, a restriction of the entrance of air guarantee? a sufficient intensity of the development. This can be better attained in the thick layer in the test-tube cultures, and stUl better if, according to Koch,* upon the gelatin or agar-agar plates a • thin sheet of isinglass or mica is laid which covers at least a third of the surface of the gelatin in the center. This should be applied as the gelatin begins to solidify. " The mica-sheet, on account of its elasticity, adapts itself completely to the surface of the gelatin and excludes the air from the portion covered." An- aerobic bacteria develop into colonies under the mica-plate, while exquisite aerobic forms grow well only about 2 mm. within the edge of the plate, " so far * "Oonferenz zur Erorterung der Oholerafrage.'' "Berl. klin. Woohenschrift," 1884, No. 31, ff. Und "Deutsche med. Woohen- aohrift," 1884, No. 32, ff. 164 BAGTERIOLOOIGAL mVESTIGATIOK as a diffusion of tlie air can take place." Of the aero- bic bacteria only extraordinarily small colonies, not visible to tbe naked eye, are formed under the mica- plate. These probably prolong their existence by means of the oxygen contained in the gelatin, but can not subsequently develop farther. The inocu- lated plate- and test-tube cultures without these mica- plates can be placed under the jar of the air-pump, or in a jar in which the air has been replaced as much as possible by carbonic acid. In this manner it is possible to make available the advantages of the solid and transparent nutrient media for obtaining pure cultures also of the anaero- bic bacteria. Bocperiments concerning AnaeroMosis in Fluids. — It is necessary further to make experiments con- cerning anaerobiosis in fluids in order, on the one hand, to study the decomposition with the absence of air or restriction of its admission ; and on the other, also, to separate, by the aid of anaerobiosis in measure- cultures, the anaerobic bacteria from the purely aero- bic — as was done by Fitz,* with the result of win- ning, by dilution from this original material, a one- cell culture. The experiments concerning anaerobiosis may be divided into two classes : {a) those in which only the air is expelled, and (&) those in which the air is re- placed by other gases. The expulsion of the air can be accomplished by boiling, and then it must be pos- sible to make the inoculation after cooling, so that in the inoculation no air can gain admission. Either the iaoculation of the solution with a pure culture in gelatin, or a one-celled culture, takes place before * " Ueber Spaltpilzgahrungen," IX. " Berichte der dentsohen chemisohen Gesellschaft," Bd. XVII, 1884, S. 1188. SUP TIG BACTERIA. 165 Fig. 22. Via. 23. i the expulsion of the air, and then the air must be driven out at a temperature which can not be injuri- ous to the bacteria. It is advantageous in the first case, necessary in the second, that the solution to be inoculated be previously thoroughly sterilized. The simplest order of experiments for examination and for obtaining quantity-cult- ures consists in filling a fiask {a, Fig. 22), having a long neck, half full with the impure mixt- ure of bacteria which is to be tested as to the presence of anaerobic bacteria, or vsdth the ^ i "' sterilized, inoculated solution ; fl ^ then the neck is drawn out nar- ^Bj row in the gas-flame at &, and the open , ^^ end (c) is joined to an aspirator or air- pump. IDuring the exhaustion of the air the flask {a) is placed in a water-bath at about 38° or 40° C, in which it re- mains boiling strongly for about half an hour ; then the neck at b is sealed in a porated flame, while the suction is continued. In this way a water-hammer quite free from air is obtained, which strikes strongly upon the wall, and in which the fluid boils at the temperature of the body. According to the material, these flasks are kept at the temperature of the room, or in a culture-oven. Nencki * proceeds in the following manner (Fig. 23) : The vessel is filled half full with * "Beitrfige zur Biologie der Spaltpilze," 1880. ] I \ trf- jp n^ 166 BAGTERIOLOGICAL mVESTWATION. the mixture of bacteria or the inoculated solution, and afterward clpsed with the double-perforated rubber stopper. Through one opening of this stopper a glass rod (J) passes, which ends in a ground stopper that fits closely in the constriction at c, so that the con- tents of the ball {A) are completely shut off from the fluid above. In the second opening a tube {d), bent at a right angle, is placed, the end of which is con- nected with an air-pump. During the suction the ball {A) is placed in the water-bath, and the glass rod (6) is withdrawn so that the stopper rests at about a. As soon as the air is removed, which is indicated by the shocks produced by the boUing and striking of the fluid on the waUs of the vessel, the glass rod is pressed downward by careful turning, untU the fluid in the ball {A) is completely cut off by the stopper. Then, during the suction, the tube is sealed at