An introduction to practical bacteriology for physicians, chemists and students

Front matter

No BOA sce,

Boston Public Library

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Preface

Tuts little work on Practical Bacteriology was written by me to serve as a short text-book for my bacteriological course at the ‘Technical High School, in order to diminish the amount of verbal teaching, and to avoid the continual repetition. of necessary directions. The excellent works of Hiippe, Friinkel, and Giinther are too voluminous for this purpose, being liable to - confuse the beginner from their very fulness of detail. If, however, the student wishes to go more deeply into the subject, he will find these works indispensable.

In consequence, I have, in the following pages, only inentioned the most important methods, and amongst the innumerable ways of staining preparations have only described the most convenient for use. In addition, I have restricted the amount of material to be used as much as possible, so that the subject-matter contained in Chapters I.-XIII., at any rate, should be mastered in one term. ‘lhe subsequent chapters are meant rather to be used in special individual cases, and on that account those. things which are of practical use to the physician or chemist have been treated of lll iv PREFACE.

with some detail, whilst other subjects have been only shortly dealt with. ‘Thus the book is also intended to be a short text-book for physicians and chemists in practice, if they wish to occupy themselves in their short periods of leisure with such bacteriological - experiments as arise out of their work.

The name Practical Bacteriology was suggested by Strasburger’s Practical. Botany, although this work is much more limited in scope.

As to illustrations of apparatus, I have made these as few as possible, in order to keep down the price of the book, having aimed rather at indicating the special form which I consider most suitable than at carefully representing the instruments. This is less important, since all price lists are now so fully illustrated.

On the other hand, I considered it desirable to insert' photographs of the most important pathogenic bacteria, because good drawings as a rule are much more distinct than the pictures under the microscope, so that it is frequently enough said by my pupils, ‘If only the bacteria were as distinctly to be seen as in the picture!” The difference between the microscopical picture and the drawing is often so great, that the beginner can hardly make out the resemblance between them. W. Micuta.

' Careful drawings of these photographs have been inserted in place of the photographs themselves. ,

TRANSLATORS’ PREFACE.

In preparing our translation of this little work, which has been made by arrangement with Dr. Migula, we have endeavoured to convey the author’s meaning as accurately as possible. In only a few instances has new matter been added, and then invariably in the form of footnotes. ‘These footnotes will be found on pages 7, 21, 100, 212, and 236. In addition the titles of some English works and translations and the names of some English manufacturers have been added in the introduction.

We trust that this book may prove useful to English students; the painstaking way in which the minutest practical details have been described will render it, without doubt, especially serviceable to those who have not had the opportunity of going through a systematic course on Bacteriology.

M. anp H. J. Campse tt.

Lonvon, 1893.

CHAPTER

By

XII.

Contents

PREFACE, , : ; ae TRANSLATORS’ PREFACE.

INTRODUCTION . ? : ;

EXAMINATION OF LIVING BACTERIA — THEIR STRUCTURE AND Form—How To OBTAIN MATERIAL FOR EXAMINATION

Introduction

oo

BacTERioLoay is a very wide subject; however, only a small portion of it need be dealt with in a practical course which is not intended for the training of specialists. It frequently happens to both physician and chemist that a few bacteriological experiments are necessary in the course of his ordinary work, and hence that he requires to understand thoroughly the most important methods of manipulation. This book is only intended to supply this need, and the subject is, I hope, treated in a manner which is easily comprehensible by the non-specialist. It only remains to mention the best way of furnishing the laboratory, the places where the necessary appliances can be obtained, and the most important literature on the subject.

Necessary APPARATUS.

1. The first and most important instrument is the microscope. If the student intends to devote himself with some assiduity later on to accurate bacteriological work, it is best for him to choose at the outset an in-

: B strument, to which subsequently it may be possible to add whatever is necessary, as the need arises. In the first place it is absolutely necessary that the stand should be jointed, and that it should be provided with coarse and fine adjustments, with rack and pinion, with a micrometer screw, with an Abbé’s condenser and an iris diaphragm, and above all, with as large an object stage as possible. If later on it is desired to take microphotographs, it is best to get an apochromatic lens, as far better results can be obtained by its means. However, these lenses are very dear, and cannot be afforded by every one.

The best microscopes are furnished by Zeiss in Jena,* but they are much more expensive than those manufactured by other firms. Seibert, in Wetzlar, supplies instruments which are very similar to those of Zeiss, and are much cheaper. It would be a good thing if this firm of manufacturers would arrange to have the micrometer screw above as in Zeiss’ instruments, as when it is underneath it is inconvenient to use. The following firms also supply good microscopes :—

Paul Wachter, Berlin.

Otto Himmler, Berlin.

* London Agent, R. Kanthack, 21, Golden Square, Regent Street, W.

INTRODUCTION.

K. Hartnack, Potsdam.

C. Reichert, Vienna.

EK. Leitz, Wetzlar.

L. Bénéche, Berlin, S.W., Grossbeeren Straasse, 19. J. Klénne & Miiller, Berlin.

Schmidt & Hansch, Berlin, 8.

R. Winkel, Gottingen.

IF. Plossl & Co., Vienna.

A. Nachet, Paris, rue St. Severin, 17.

It is sufficient for all ordinary bacteriological work to have a simple microscope, with six different objectives, magnifying about 50, 100, 200, 500, 700, 1,000 diameters ; it is also well to have an illuminating apparatus, and, if possible, an oil immersion lens. From my own experience, I can recommend the following as good combinations :—

Zeiss, Stand 6. Illuminating apparatus No. 19, "Lenses A, D, and 7; homogeneous immersion. Hye pieces 2, 3, and 4, price £16 2s.

Seibert, Stand 4. Small illuminating apparatus, Lenses I., V.,and X. LHye-pieces I., II., III., price £16 16s. 6d.

Other manufacturers supply similar instruments at somewhat lower prices; however, their instruments scarcely reach the excellence of those supplied by the above well-known firms. Unless the

Ae PRACTICAL BACTERIOLOGY.

student is himself a judge of microscopes, and so is in a position to discover any defect in them, it is best for him not to choose his instrument himself, but to leave the selection of it to some experienced person.

It will be taken for granted in the following pages that the student understands the use of the microscope, and hence any one who does not possess such knowledge is advised to make himself acquainted with general microscopical technique in some work which treats of this subject ; such as—

Frank J. Weruerep, Medical Microscopy. <A guide to the use of the microscope in Medical Practice (Lewis), London, 1892,

Henri vAN Heurcx, the Microscope, its Construction and Management, translated by Baxter (Crosby, Lockwood & Son), London, 1893.

SrrasBurRGER, Practical Botany, translated by Hillhouse. London (Sonnenschein) and New York (Macmillan), 1888. Third Editi n, 1893.

2. Slides made of white glass preferably with

INTRODUCTION. 5

| ground edges, as they are thus less likely to cut the _ hand when being cleaned.

3. Hollow slides and slides on to which glass cells have been cemented. The cement ought not to be destroyed or liquefied by heat.

4. Cover-glasses. For ordinary use, 15 mm. in diameter; for cover-glass cultures, however, they should be 22 mm. in diameter; round cover-glasses are preferable, because their edges are not so likely to catch in the cloth when they are being cleaned as the square ones, and because they are more suitable for permanent preparations.

®. Watch-glasses of about 5 cm. in diameter, with flat bottoms, for holding the staining fluids, etc. Embryo cups, which consist of blocks of compressed glass, in which depressions have been ground, are very useful, only they are rather dear.

6. Glass rods, thin and thick. It is best to fuse the platinum wire into the latter, as it is apt to become loosened from the thin rod on cooling.

7. Glass tubing of various sizes, for various purposes; amongst others, for making cultivations of anaérobic bacteria. |

8. Flasks. A few large ones for preparing nutrient gelatine, etc., and smaller ones for cultivations.

9. Small Hrlenmeyer’s flasks, very useful for bouillon cultures, etc.

10. Glass funnels; large for filtering the nutrient media, and small for filtering staining solutions. A few quite small ones, about 2 cm. in diameter, are also required for filtering the staining solution direct upon the cover-glass.

11. Small glass benches, with their ends bent over, upon which to stand cultures (Fig. 8).

12. Test tubes, of at least 15 mm. in diameter and 15 cm. in length. For anaérobic cultivations longer ones, and for Esmarch’s tubes wider ones are required. A considerable number of tubes are needed, and in procuring them it is unwise to be too economical.

13. Beakers. These are needed for various purposes; I myself use them for boiling the nutrient gelatine. It is also convenient to sterilise small glass tubes, pipettes, etc., in beakers in the hot-air sterilising apparatus.

14, A wash-bottle of about 250 grms. capacity, with a finely drawn out tube.

15. Two graduated measures for measuring out the quantity of water in preparing the nutrient media— one of 1 litre in capacity divided into ten parts, and another of 100 cm. divided into twenty parts.

16. Measuring pipettes, several of 1 ccm. in capacity divided into tenths, and others of 2, 5, 10, 20, 25, 50, and 100 cem. capacity.

17. Glass dishes with double covers, for the so-called plate cultures, of about 10 cm. diameter; the lids should fit as well as possible.

INTRODUCTION. *

18. Glass shades with glass knobs, of about 22-24 em. in diameter, and 5-6 cm. in height. These are made use of as moist chambers for potato cultivations, etc. (Fig. 1). =

19. Porcelain dishes of about 5 cm. in diameter for heating staining solutions, and one larger one which is used for heating the nutrient agar-agar in the salt water bath.

20. A Kipp’s apparatus for the preparation of hydrogen for anaérobic cultivations is not indispensable. In case of need, hydrogen can be prepared in a simple gas generating flask, although of course the operations cannot be as accurately carried on as if a Kipp’s apparatus is used. A small apparatus costs about 10s., and is to be strongly recommended.

21. Thermometer. ‘This need only indicate up to 150° C. <A thermometer with a long stem divided into tenths of degrees, indicating up to 50° C., is very convenient for use in the incubator.

22. A Reichert’s thermo-regulator* is necessary for cultivations of tubercle bacilli, if gas is used, and if the incubator is good.

23. Gas burners with one and four flames, the latter for the hot air steriliser and the steam steriliser. The ones most to be recommended are those of Desaga

* Page’s thermo-regulator is, in some respects, more convenient.

(Heidelberg), which have an arrangement for regulating simultaneously the supplies of gas and air.

24. A micro-burner for warming the incubator. Koch’s safety burners are the best for this purpose, but they are unreasonably dear. See page 89.

25. An incubator. However, unless great uniformity of temperature is necessary, this can be replaced by the very simple apparatus described on p. 88.

26. A steam steriliser (Fig. 4).

27. A hot air steriliser (Fig. 3).

28. A balance. Should a delicate chemical balance be unobtainable, an ordinary pair of scales for weighing gelatine, peptone, staining reagents, etc., will answer the purpose. Great exactness is only essential in a few cases ; for example, in the preparation of normal solutions, such as the caustic soda solution used for staining flagella. In such cases, however, the reagents can always be obtained ready-made from a reliable chemical manufacturer, or they may be ordered at a chemist’s or from a chemical laboratory. The need for a delicate balance so seldom arises that it is hardly worth while to procure one.

29. A filter stand.

30. Filter paper, somewhat thick for filtering gelatine and agar-agar, and a thinner kind, not very permeable, for filtering the staining solutions. Folded filter papers are used for gelatine: these can be obtained ready-made. For staining reagents it is best

INTRODUCTION. 9 to use the circular filter papers, which are prepared with hydrochloric and fluoric acid. In addition, some soft absorbent blotting paper is constantly required for taking up excess of moisture from slides, cover-glasses, etc.

31. Litmus paper, blue and red. This is absorbent paper, coloured with litmus solution; a drop of the solution to be tested is allowed to fall upon it from a glass rod. ‘The more sensitive the Jitmus paper is, the more useful is it, but of course the more difficult is it also to work with. ‘The blue and red papers should be kept separate, and should be protected from the light, preferably in large bottles with smooth stoppers, which have been smeared over with vaseline, the bottles themselves having been blackened with varnish.

32. Cotton wool. Clean cotton wool free from grease, such as can be bought from pharmaceutical chemists.

33. India-rubber stoppers and caps for test-tubes and india-rubber tubing. Some of the stoppers should be perforated.

34. Platinum wire of about ‘8~1 mm. in thickness. For most purposes only short lengths of the wire are needed; but as long pieces are required, as for anaérobic cultivations, it is as well to have it in pieces about 12 cm. long. A few mm. of the wire are fused on to the ends of thick glass rods; some fe) PRACTICAL GACTERTIOLVCG..

of these should have their free ends filed into smooth, short points, and others should have them bent over to form small, closed, oval loops, of about 2 mm. in their greatest diameter.

30. Forceps, which must not have too strong a spring.

36. Needles. The most convenient arrangement is to have nickel-plated metal needle-holders, in which ordinary sewing needles of different sizes can be fixed : as this apparatus is convenient to sterilize.

37. Scalpels. One should be of platinum; it may be prepared in the manner described in a note on p. 123.

38. Razors and strops. It is important that the razor should have a broad blade, and should be flat upon one side. The strop should be made of tightly stretched leather. A glued leather strop is not to be recommended.

39. Various bottles. For alcoholic staining solutions the most convenient kind of bottle is one provided with a cork through whicha glass tube with a drawn out lower end passes. The tube should be long enough to reach the bottom of the bottle, and it also ought to fit loosely in the cork, so that it can be easily drawn out. Then when liquid is wanted for use the tube is drawn out of the bottle, the upper end being closed with the finger. By this means a sufficiently small quantity of the staiming solution is ob-

LNTRODUCTION. II tained. As to the other kinds of bottles which are needed, it will be seen later what is required.

40. Several short glass tubes supplied with well fitting india-rubber caps at one end, the other end being drawn out so as only to leave narrow openings. In the absence of ready-made india-rubber caps, one can use pieces of thin india-rubber tubing tied up tightly at one end. These latter are very useful for taking up staining solutions and dropping them upon the cover-glasses.

41. Distilled water in several bottles of one litre capacity.

42. Absolute alcohol.

43. Xylol.

44, Canada balsam.

45. Cedar-wood oil.

46. Aniline oil.

47. Sulphuric acid.

48. Nitric acid.

49. Hydrochloric acid.

50. Acetic acid.

51. Glycerine.

52. Concentrated solution of soda.

53. Sodic hydrate. 54, Potassic hydrate. 55. Potassic iodide. 56. Paraffin.

57. Vaseline.

58. Lodine.

59. Carbolic acid.

60. Corrosive sublimate.

61. Gelatine.

62. Agar-agar.

63. Peptone.

64. Liebig’s extract of meat.

65. Ordinary salt.

66. Grape sugar.

67. Fuchsine.

68. Methylene blue.

69. Methyl violet or gentian violet.

70. Bismarck brown.

71. Various horn spoons, spatule, ordinary knives, cloths for wiping, etc. |

72. Wolfhiigel’s counting apparatus (Fig. 9).

The following firms may be recommended :—

For BacrerroLogicAL APPARATUS.

F. & M. Lautenschliiger, Berlin.*

F. HK. Becker & Co., 83, Hatton Wall, Hatton Garden, London, H.C.

A. Gallenkamp & Co., 6, Cross St., Finsbury, London, H.C.

* London agent, Kanthack.

INTRODUCTION. 13

Dr. Robert Muencke, Berlin.

Dr. Hermann Rohrbeck, Berlin.

Klénne & Miiller, Berlin.

For MicroscoricAL APPLIANCES.

Stanley, Railway Approach, London Bridge, 8.H. R. & J. Beck, 68, Cornhill, London, H.C. Theodor Schroéter, Leipzig.

R. Jung, Heidelberg.

Klénne & Miiller, Berlin.

Dr. G. Schreiber, Chemnitz i. S.

Marpmann & Schurig, Leipzig.

Adolph Veit, Heidelberg.

For CHEMICAL AND STAINING REAGENTS.

Baird & Tatlock, 14, Cross St., Hatton Garden, London, E.C.

Hopkin & Williams, 16, Cross St., Hatton Garden, London, E.C.

Townson & Mercer, 89, Bishopsgate St. Within, London, E.C.

EK. Merck, Darmstadt.

14 PRACTICAL SACTERIGL UGC.

Dr. Griibler, Leipzig.

Klonne & Miller, Berlin.

Dr. G. Schreiber, Chemnitz.

Th. Schuchhardt, Gorlitz.

Trommsdorf, Erfurt.

Dr. G. Miinder, Géttingen.

Hfeinrich Sohncke, Halle.

Dr. K. Roth, Berlin N., Strassburgerstr. 18.

lor MicroscopicaAL PREPARATIONS.

Kloénne & Miiller, Berlin.

Heinrich Bécker, Wetzlar.

For APppaRATus FoR MicrRopHoToGRAPHY.

Otto Perutz, Miinich.

Albert Glock, Karlsruhe i. B.

Schippang & Wehenkel, Berlin.

Dr. Burstert and Fiirstenberg make excellent microphotographs of preparations which are sent to them. In addition, the author has found their glass plates very convenient, especially for demonstrating.

More Derattep Works on Practica, BACTERIOLOGY.

INTRODOCTION. 15

Crooxsuank, Manual of Bacteriology (Lewis), London.

Grorce M. Sterneere, A Manual of Bacteriology, New York (Wood & Co.), 1892.

Imporrant Works on BactTERIOLOGY.

Fitece, Micro-organisms, trans. by Watson Cheyne, M.B., New Sydenham Soc., London, 1890.

Hisensera, Bacteriological Diagnosis, trans. by Norval H. Pierce, M.D., Philadelphia and London.

Annales de lVinstitut Pasteur, Paris.

All additional literature on the subject is mentioned in these works, which are quite indispensable to any student who wishes to study Bacteriology thoroughly.

Pure cultures can be obtained from Lautenschlager, Klénne und Miller, or from Krall, (II. Opatowitzer Gasse, 9, Prague, Austria.)

CHAPTER; I.

EXAMINATION OF LIVING BACTERIA.—THEIR STRUCTURE AND FORM.—HOW TO OBTAIN MATERIAL FOR EXAMINATION.

Bacrerta are distributed everywhere in nature; they cling to the surface of every substance ; they are to be found in fewer or greater numbers in dust, in water, and in air. We only perceive their presence under ordinary circumstances, however, when they find conditions favourable for their rapid growth and development. Generally they are detected by the odour they produce ; sometimes, too, they impart a colour to the substance upon which they are growing, or sometimes they themselves acquire a coloration of their own, when they are spread over the objects in thick, more or less slimy masses. Although itis easy enough to find bacteria in such masses, yet it is both simpler and more convenient to grow the species to be examined artificially, especially as this can be very easily accomplished in several different ways. Moreover, different forms are obtained, according to the method adopted ; J *nd this, of course, is very useful in the study of bacie rs / 7

18 FRACTICAL BACTERIOLOGY.

If certain plants, preferably some green Algz, such as Spirogyra, Vaucheria, or Cladophora, are left for a short time in a small quantity of water, so that ' they become decomposed, one very soon perceives, by the unpleasant odour produced, that bacteria have begun their destructive processes. The water becomes turbid, and the sides and bottom of the vessel become covered with a slimy film, which grows thicker and thicker, till at last here and there small flakes become detached from the rest. Sooner or later, according to the temperature, the surface becomes covered with a dirty white skin, which at first ~ is very delicate, but which frequently, later on, becomes of a gelatinous consistency. The turbidity of the water, the slimy covering of the vessel’s walls, and the skin on the surface (the bacterium skin), all indicate the presence of bacteria, which have multiplied in enormous quantities at the expense of the dying Alge. The bacteria were originally present in proportionately a small quantity, both in the water and attached to the Algz themselves and to the walls of the vessel.

Similarly bacteria can easily be obtained for examination by cutting up carrots or potatoes, boiling them, - and allowing the resulting liquid to stand in broad open vessels. LHssentially the same appearances mani. fest themselves as with the decomposing Alge, ony as a rule not nearly so many species are obtained: |

Generally quite different ones make their appearance when finely pounded meat is mixed with ditch-water, and left to decompose.

In all these ways a mixture of bacteria is obtained ; that is to say, bacteria of very different forms and kinds are found mixed together. It is, moreover, impossible to say beforehand which forms will be developed, for this depends on the kind of germs which happen to be present, and also on the varying chem1- cal constitution of the special nutrient fluids prepared as above.

By the following method, a pure culture—that is to Say, one in which only one kind of bacterium develops itself—may be obtained. Hay is left in water for twenty-four hours; the resultant liquid is then strained through a linen cloth, and diluted, until a volume, ten centimetres in depth, is of a light chestnut-brown colour.

The success of this experiment depends upon the fact that the spores of the hay-bacillus (Bacillus subtilis Hhrenbery) can endure the heat of boiling water for an hour without being killed. The liquid is poured into a Florence flask, until the latter is three-quarters full. The mouth of the flask is then stopped up with a cotton-wool plug, and its contents are kept simmering gently —only a small amount of steam being formed,—for an hour. In this manner all other organisms and their germs are killed, the spores of the hay-bacillus alone being able to withstand the heat. These now begin to develop in a most vigorous fashion, for the hay-infusion, which contains the spores, is in itself a most favourable nutrient medium for the hay-bacillus. As all other organisms have been killed, and the cotton-wool stopper prevents the entrance of new ones, a pure culture of the hay-bacillus is obtained in this manner. To be sure, there is nolonger any doubt that, under the name of hay-bacillus, there are included many different kinds of bacteria, which for the most part are very closely allied. This, however, for the present, need not be considered, as in the following experiment we shall occupy ourselves with the form and development of the different kinds, rather than with their rigid classification, which will be treated at some length in later chapters. | Bisa a comparatively pure original material for acteriological experiments may be obtained in the following manner :—Slices about one centimetre thick of cooked turnips and potatoes are placed for a time in different places—in the open air, in a cellar, on the ground, or ina sitting-room. ‘They are left in these places for two or three hours freely in contact with the air, and are then further cultivated in moist chambers. The moist chamber (Fig. 1), which has been successfully used up to now, consists of two glass vessels, the upper and smaller one, which is supplied with a handle, fitting closely into the under one. Before use, these vessels should be carefully cleansed. This is most successfully accomplished with a solution of corrosive subhimate, 1: 1000 (it is advisable to have a solution of this strength always handy, as it is in constant use).' A piece of filter-paper, soaked in this solution, is then placed at the bottom of the dish, and the piece of turnip or potato is placed upon it, the same side as before being uppermost. The upper glass is then put over, and the vessel is kept at the temperature of an ordinary sitting-room. After a few days s

=

Pras:

small variously shaped masses appear on the slice in the form of little droplets, dry flat scales, or more

1 In the use of corrosive sublimate as a sterilising agent, it is of importance to remember not to use more of the solution than is absolutely necessary, lest so much of the salt should be left adhering to the sides of the dish, after the water has evaporated, that it may fall on the culture and kill the bacteria. It is further necessary to bear in mind the fact, that, if albuminous substances come into contact with the corrosive sublimate solution, the mercury is precipitated, and thus the sterilising properties of the solution are more or less destroyed. For these reasons it is advisable to sterilise by means of heat whenever possible.

22 PRACTICAL BACTERICOLOGE.

or less slimy masses with irregular outlines. These masses are generally whitish or yellowish, being rarely of any other colour. They are colonies of bacteria, which, if they are situated sufficiently far apart from each other, present small pure cultures of the different species. While the slices of turnip were lying in contact with the air, bacteria germs fell upon them. These, if the soil suits them, develop at the spots, where they fall, into small colonies, which remain isolated upon the firm medium, and only can become mixed with other bacteria, if neighbouring colonies, in consequence of their continued growth, touch eac other. V4 Occasionally, however, it may happen that two germs of different kinds fall upon the same spot, and that thus the resulting colony is impure from the outset. As a rule, however, it may be taken for granted, that — colonies which are pure at the beginning are obtained by this method, and these afford us the original material for the pure cultures to be described later. Generally colonies of moulds (especially of the Penicillium glaucum, or of species of the genera Aspergillus and Mucor) develop amongst the bacteria colonies ; these are easily to be recognised, as 16 can be readily seen with the magnifying glass, or indeed with the naked eye, that they are composed of long threadlike hyphe. Later on, these hyphzx raise themselves above the surface, and develop reproductive organs, by means of which they can immediately be distinguished from the others.

If we now take with a needle a small portion of such a bacterium colony from the surface of a potato, and spread it out in a drop of water upon a slide, cover it with a cover-glass, and examine it with a magnifying power of about 500 diameters, and with a small opening in the diaphragm, we shall find in most cases a specimen of the genus Micrococcus, or more rarely, a rod-bacterium. The Micrococci are small round cells, which on an average are 77/95 mm. in diameter ; sometimes they are a little larger, sometimes a little smaller, according to the species which happens to be present. They are almost always motionless. Up to now we know very few kinds of Micrococci which, like the Mi- _ erococcus agilis Cohen, move of their own accord. The trembling movements exhibited by most Micrococct are not manifestations of their own activity, for they are also exhibited by non-living bodies suspended in fluid, such as small particles of carmine. This phenomenon has not yet been fully explained, but apparently it is caused by the neutralization of the electrical tension of very small bodies. ‘The peculiarity of these so-called Brownian movements consists in this, that the particles, in spite of their continual.trembling and dancing, never move from place to place, but remain practically at one point.

Presently, however, very disturbing movements are to be seen. ‘The water at the edge of the cover-glass evaporates, and thus different currents are continually being created. The bacteria, which are in the field of vision, are swept along by these currents, and thus the difficulties of observation are much increased. In consequence the following plan has been successfully adopted for such observations :—A slide is chosen, in the middle of which either a round hollow or depression has been ground, or better still, on to which a glass ring has been cemented. A small drop of water is now placed ona cover-glass, and the bacteria are spread out in it; the cover-glass is then turned over and placed over the depression in the slide, so that the drop of water hangs freely in the hollow space, and nowhere touches the bottom or the sides. A small drop of olive-oil is then placed at the edge of the cover glass, and this spreads itself out in a circle between it and the slide, making the little hollow quite air-tight. The drop of water is in this manner prevented from evaporating, and in addition is protected from the entrance of new germs from the outside, which, in later experiments, is of great importance. In this moist chamber the development of the bacteria can be very well observed ; and this method of culture, when the drop of water has added to it a drop of nutrient fluid, is called cultivation in the hanging drop.