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The Elements of Bacteriological Technique
by John William Henry Eyre
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THE ELEMENTS OF BACTERIOLOGICAL TECHNIQUE

A Laboratory Guide for Medical, Dental, and Technical Students

by

J. W. H. EYRE, M.D., M.S., F.R.S. (EDIN.)

Director of the Bacteriological Department of Guy's Hospital, London, and Lecturer on Bacteriology in the Medical and Dental Schools; formerly Lecturer on Bacteriology at Charing Cross Hospital Medical School, and Bacteriologist to Charing Cross Hospital; sometime Hunterian Professor, Royal College of Surgeons, England

Second Edition Rewritten and Enlarged



Philadelphia and London W. B. Saunders Company 1913

Copyright, 1902, by W. B. Saunders and Company Revised, entirely reset, reprinted, and recopyrighted July, 1913

Copyright, 1913, by W. B. Saunders Company

Registered at Stationers' Hall, London, England

Printed in America Press of W. B. Saunders Company Philadelphia



TO THE MEMORY OF

JOHN WICHENFORD WASHBOURN, C.M.G., M.D., F.R.C.P.

Physician to Guy's Hospital and Lecturer on Bacteriology in the Medical School, and Physician to the London Fever Hospital

MY TEACHER, FRIEND, AND CO-WORKER



PREFACE TO THE SECOND EDITION

Bacteriology is essentially a practical study, and even the elements of its technique can only be taught by personal instruction in the laboratory. This is a self-evident proposition that needs no emphasis, yet I venture to believe that the former collection of tried and proved methods has already been of some utility, not only to the student in the absence of his teacher, but also to isolated workers in laboratories far removed from centres of instruction, reminding them of forgotten details in methods already acquired. If this assumption is based on fact no further apology is needed for the present revised edition in which the changes are chiefly in the nature of additions—rendered necessary by the introduction of new methods during recent years.

I take this opportunity of expressing my deep sense of obligation to my confrere in the Physiological Department of our medical school—Mr. J. H. Ryffel, B. C., B. Sc.—who has revised those pages dealing with the analysis of the metabolic products of bacterial life; to successive colleagues in the Bacteriological Department of Guy's Hospital, for their ready co-operation in working out or in testing new methods; and finally to my Chief Laboratory Assistant, Mr. J. C. Turner whose assistance and experience have been of the utmost value to me in the preparation of this volume. I have also to thank Mrs. Constant Ponder for many of the new line drawings and for redrawing a number of the original cuts.

JOHN W. H. EYRE.

GUY'S HOSPITAL, S. E. July, 1913.



PREFACE TO THE FIRST EDITION

In the following pages I have endeavoured to arrange briefly and concisely the various methods at present in use for the study of bacteria, and the elucidation of such points in their life-histories as are debatable or still undetermined.

Of these methods, some are new, others are not; but all are reliable, only such having been included as are capable of giving satisfactory results even in the hands of beginners. In fact, the bulk of the matter is simply an elaboration of the typewritten notes distributed to some of my laboratory classes in practical and applied bacteriology; consequently an attempt has been made to present the elements of bacteriological technique in their logical sequence.

I make no apology for the space devoted to illustrations, nearly all of which have been prepared especially for this volume; for a picture, if good, possesses a higher educational value and conveys a more accurate impression than a page of print; and even sketches of apparatus serve a distinct purpose in suggesting to the student those alterations and modifications which may be rendered necessary or advisable by the character of his laboratory equipment.

The excellent and appropriate terminology introduced by Chester in his recent work on "Determinative Bacteriology" I have adopted in its entirety, for I consider it only needs to be used to convince one of its extreme utility, whilst its inclusion in an elementary manual is calculated to induce in the student habits of accurate observation and concise description.

With the exception of Section XVII—"Outlines for the Study of Pathogenic Bacteria"—introduced with the idea of completing the volume from the point of view of the medical and dental student, the work has been arranged to allow of its use as a laboratory guide by the technical student generally, whether of brewing, dairying, or agriculture.

So alive am I to its many inperfections that it appears almost superfluous to state that the book is in no sense intended as a rival to the many and excellent manuals of bacteriology at present in use, but aims only at supplementing the usually scanty details of technique, and at instructing the student how to fit up and adapt apparatus for his daily work, and how to carry out thoroughly and systematically the various bacterioscopical analyses that are daily demanded of the bacteriologist by the hygienist.

Finally, it is with much pleasure that I acknowledge the valuable assistance received from my late assistant, Mr. J. B. Gall, A. I. C., in the preparation of the section dealing with the chemical products of bacterial life, and which has been based upon the work of Lehmann.

JOHN W. H. EYRE.

GUY'S HOSPITAL, S. E.



CONTENTS

PAGE

I. LABORATORY REGULATIONS 1

II. GLASS APPARATUS IN COMMON USE 3

The Selection, Preparation, and Care of Glassware, 8—Cleaning of Glass Apparatus, 18—Plugging Test-tubes and Flasks, 24.

III. METHODS OF STERILISATION 26

Sterilising Agents, 26—Methods of Application, 27—Electric Signal Timing Clock, 38.

IV. THE MICROSCOPE 49

Essentials, 49—Accessories, 57—Methods of Micrometry, 61.

V. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER MICRO-FUNGI 69

Apparatus and Reagents used in Ordinary Microscopical Examination, 69—Methods of Examination, 74.

VI. STAINING METHODS 90

Bacteria Stains, 90—Contrast Stains, 93—Tissue Stains, 95—Blood Stains, 97—Methods of Demonstrating Structure of Bacteria, 99—Differential Methods of Staining, 108.

VII. METHODS OF DEMONSTRATING BACTERIA IN TISSUES 114

Freezing Method, 115—Paraffin Method, 117—Special Staining Methods for Sections, 121.

VIII. CLASSIFICATION OF FUNGI 126

Morphology of the Hyphomycetes, 126—Morphology of the Blastomycetes, 129.

IX. SCHIZOMYCETES 131

Anatomy, 134—Physiology, 136—Biochemistry, 144.

X. NUTRIENT MEDIA 146

Meat Extract, 148—Standardisation of Media, 154—The Filtration of Media, 156—Storing Media in Bulk, 159—Tubing Nutrient Media, 160.

XI. ORDINARY OR STOCK CULTURE MEDIA 163

XII. SPECIAL MEDIA 182

XIII. INCUBATORS 216

XIV. METHODS OF CULTIVATION 221

Aerobic, 222—Anaerobic, 236.

XV. METHODS OF ISOLATION 248

XVI. METHODS OF IDENTIFICATION AND STUDY 259

Scheme of Study, 259—Macroscopical Examination of Cultivations, 261—Microscopical Methods, 272—Biochemical Methods, 276—Physical Methods, 295—Inoculation Methods, 315—Immunisation, 321—Active Immunisation, 322—The Preparation of Haemolytic Serum, 327—The Titration of Haemolytic Serum, 328—Storage of Haemolysin, 331.

XVII. EXPERIMENTAL INOCULATION OF ANIMALS 332

Selection and Care of Animals, 335—Methods of Inoculation, 352.

XVIII. THE STUDY OF EXPERIMENTAL INFECTIONS DURING LIFE 370

General Observations, 371—Blood Examinations, 373—Serological Investigations, 378—Agglutinin, 381—Opsonin, 387—Immune Body, 393.

XIX. POST-MORTEM EXAMINATION OF EXPERIMENTAL ANIMALS 396

XX. THE STUDY OF THE PATHOGENIC BACTERIA 408

XXI. BACTERIOLOGICAL ANALYSES 415

Bacteriological Examination of Water, 416—Examination of Milk, 441—Ice Cream, 457—Examination of Cream and Butter, 457—Examination of Unsound Meats, 460—Examination of Oysters and Other Shellfish, 463—Examination of Sewage and Sewage Effluents, 466—Examination of Air, 468—Examination of Soil, 470—Testing Filters, 478—Testing of Disinfectants, 480.

APPENDIX 492

INDEX 505



BACTERIOLOGICAL TECHNIQUE.



I. LABORATORY REGULATIONS.

The following regulations are laid down for observance in the Bacteriological Laboratories under the direction of the author. Similar regulations should be enforced in all laboratories where pathogenic bacteria are studied.

Guy's Hospital.

BACTERIOLOGICAL DEPARTMENT.

HANDLING OF INFECTIVE MATERIALS.

The following Regulations have been drawn up in the interest of those working in the Laboratory as well as the public at large, and will be strictly enforced.

Their object is to avoid the dangers of infection which may arise from neglect of necessary precautions or from carelessness.

Everyone must note that by neglecting the general rules laid down he not only runs grave risk himself, but is a danger to others.

REGULATIONS.

1. Each worker must wear a gown or overall, provided at his own expense, which must be kept in the Laboratory.

2. The hands must be disinfected with lysol 2 per cent. solution, carbolic acid 5 per cent. solution, or corrosive sublimate 1 per mille solution, after dealing with infectious material, and before using towels.

3. On no account must Laboratory towels or dusters be used for wiping up infectious material, and if such towels or dusters do become soiled, they must be immediately sterilised by boiling.

4. Special pails containing disinfectant are provided to receive any waste material, and nothing must be thrown on the floor.

5. All instruments must be flamed, boiled, or otherwise disinfected immediately after use.

6. Labels must be moistened with water, and not by the mouth.

7. All disused cover-glasses, slides, and pipettes after use in handling infectious material, etc., must be placed in 2 per cent. lysol solution. A vessel is supplied on each bench for this purpose.

8. All plate and tube cultures of pathogenic organisms when done with, must be placed for immediate disinfection in the boxes provided for the purpose.

9. No fluids are to be discharged into sinks or drains unless previously disinfected.

10. Animals are to be dissected only after being nailed out on the wooden boards, and their skin thoroughly washed with disinfectant solution.

11. Immediately after the post-mortem examination is completed each cadaver must be placed in the zinc animal-box—without removing the carcase from the post-mortem board—and the cover of the box replaced, ready for carriage to the destructor.

12. Dead animals, when done with, are cremated in the destructor, and the laboratory attendant must be notified when the bodies are ready for cremation.

13. None of the workers in the laboratory are allowed to enter the animal houses unless accompanied by the special attendant in charge, who must scrupulously observe the same directions regarding personal disinfection as the workers in the laboratories.

14. No cultures are to be taken out of the laboratory without the permission of the head of the Department.

15. All accidents, such as spilling infected material, cutting or pricking the fingers, must be at once reported to the bacteriologist in charge.



II. GLASS APPARATUS IN COMMON USE.

The equipment of the bacteriological laboratory, so far as the glass apparatus is concerned, differs but little from that of a chemical laboratory, and the cleanliness of the apparatus is equally important. The glassware comprised in the following list, in addition to being clean, must be stored in a sterile or germ-free condition.

Test-tubes.—It is convenient to keep several sizes of test-tubes in stock, to meet special requirements, viz.:

1. 18 x 1.5 cm., to contain media for ordinary tube cultivations.

2. 18 x 1.3 cm., to contain media used for pouring plate cultivations, and also for holding sterile "swabs."

3. 18 x 2 cm., to contain wedges of potato, beetroot, or other vegetable media.

4. 13 x 1.5 cm., to contain inspissated blood-serum.

The tubes should be made from the best German potash glass, "blue-lined," stout and heavy, with the edge of the mouth of the tube slightly turned over, but not to such an extent as to form a definite rim. (Cost about $1.50, or 6 shillings per gross.) Such tubes are expensive it is true, but they are sufficiently stout to resist rough handling, do not usually break if accidentally allowed to drop (a point of some moment when dealing with cultures of pathogenic bacteria), can be cleaned, sterilised, and used over and over again, and by their length of life fully justify their initial expense.

A point be noted is that the manufacturers rarely turn out such tubes as these absolutely uniform in calibre, and a batch of 18 by 1.5 cm. tubes usually contains such extreme sizes as 18 by 2 cm. and 18 by 1.3 cm. Consequently, if a set of standard tubes is kept for comparison or callipers are used each new supply of so-called 18 by 1.5 cm. tubes may be easily sorted out into these three sizes, and so simplify ordering.

5. 5 x 0.7 cm., for use in the inverted position inside the tubes containing carbohydrate media, as gas-collecting tubes.

These tubes, "unrimmed," may be of common thin glass as less than two per cent. are fit for use a second time.



Bohemian Flasks (Fig. 1).—These are the ordinary flasks of the chemical laboratory. A good variety, ranging in capacity from 250 to 3000 c.c., should be kept on hand. A modified form, known as the "pear-shaped" (Fig. 2), is preferable for the smaller sizes—i. e., 250 and 500 c.c.

Erlenmeyer's Flasks (Fig. 3).—Erlenmeyer's flasks of 75, 100, and 250 c.c. capacity are extremely useful. For use as culture flasks care should be taken to select only such as have a narrow neck of about 2 cm. in length.

Kolle's Culture Flasks (Fig. 4).—These thin, flat flasks (to contain agar or gelatine, which is allowed to solidify in a layer on one side) are extremely useful on account of the large nutrient surface available for growth. A surface cultivation in one of these will yield as much growth as ten or twelve "oblique" tube cultures. The wide mouth, however, is a disadvantage, and for many purposes thin, flat culture bottles known as Roux's bottles (Fig. 5) are to be preferred.



An even more convenient pattern is that used in the author's laboratory (Fig. 6), as owing to the greater depth of medium which it is possible to obtain in these flasks an exceedingly luxuriant growth is possible; the narrow neck reduces the chance of accidental contamination to a minimum and the general shape permits the flasks to be stacked one upon the other.

Filter Flasks or Kitasato's Serum Flasks (Fig. 7).—Various sizes, from 250 to 2000 c.c. capacity. These must be of stout glass, to resist the pressure to which they are subjected, but at the same time must be thoroughly well annealed, in order to withstand the temperature necessary for sterilisation.

All flasks should be either of Jena glass or the almost equally well-known Resistance or R glass, the extra initial expense being justified by the comparative immunity of the glass from breakage.

Petri's Dishes or "Plates" (Fig. 8, a).—These have now completely replaced the rectangular sheets of glass introduced by Koch for the plate method of cultivation. Each "plate" consists of a pair of circular discs of glass with sharply upturned edges, thus forming shallow dishes, one of slightly greater diameter than the other, and so, when inverted, forming a cover or cap for the smaller. Plates having an outside diameter of 10 cm. and a height of 1.5 cm. are the most generally useful. A batch of eighteen such plates is sterilised and stored in a cylindrical copper box (30 cm. high by 12 cm. diameter) provided with a "pull-off" lid. Inside each box is a copper stirrup with a circular bottom, upon which the plates rest, and by means of which each can be raised in turn to the mouth of the box (Fig. 9) for removal.

Capsules (Fig. 8, b and c).—These are Petri's dishes of smaller diameter but greater depth than those termed plates. Two sizes will be found especially useful—viz., 4 cm. diameter by 2 cm. high, capacity about 14 c.c.; and 5 cm. diameter by 2 cm. high, capacity about 25 c.c. These are stored in copper cylinders of similar construction to those used for plates, but measuring 20 by 6 cm. and 20 by 7 cm., respectively.



Graduated Pipettes.—Several varieties of these are required, viz.:

1. Pipettes of 1 c.c. capacity graduated in 0.1 c.c.

2. Pipettes of 1 c.c. capacity graduated in 0.01 c.c. (Fig. 10, a).

3. Pipettes of 10 c.c. capacity graduated in 0.1 c.c. (Fig. 10, b).

These should be about 30 cm. in length (1 and 2 of fairly narrow bore), graduated to the extreme point, and having at least a 10 cm. length of clear space between the first graduation and the upper end; the open mouth should be plugged with cotton-wool. Each variety should be sterilised and stored in a separate cylindrical copper case some 36 by 6 cm., with "pull-off" lid, upon which is stamped, in plain figures, the capacity of the contained pipettes.



The laboratory should also be provided with a complete set of "Standard" graduated pipettes, each pipette in the set being stamped and authenticated by a certificate from one of the recognised Physical Measurement Laboratories, such as Charlottenburg. These instruments are expensive and should be reserved solely for standardising the pipettes in ordinary use, and for calibrating small pipettes manufactured in the laboratory. Such a set should comprise, at least, pipettes delivering 10 c.c., 5 c.c., 2.5 c.c., 2 c.c., 1 c.c., 0.5 c.c., 0.25 c.c., 0.2 c.c., 0.1 c.c., 0.05 c.c., and 0.01 c.c., respectively.

In the immediately following sections are described small pieces of glass apparatus which should be prepared in the laboratory from glass tubing of various sizes. In their preparation three articles are essential; first a three-square hard-steel file or preferably a glass-worker's knife of hard Thuringian steel for cutting glass tubes etc.; next a blowpipe flame, for although much can be done with the ordinary Bunsen burner, a blowpipe flame makes for rapid work; and lastly a bat's-wing burner.



1. The glass-cutting knife. This article is sold in two forms, a bench knife (Fig. 11) and a pocket knife. The former is provided with a blade some 8 cm. in length and having two cutting edges. The cutting edge when examined in a strong light is seen to be composed of small closely set teeth, similar to those in a saw. The knife should be kept sharp by frequent stroppings on a sandstone hone. The pocket form, about 6-cm. long over all, consists of a small spring blade with one cutting edge mounted in scales like an ordinary pocket knife.

2. For real convenience of work the blowpipe should be mounted on a special table connected up with cylindrical bellows operated by a pedal. That figured (Fig. 12) is made by mounting a teak top 60 cm. square upon the uprights of an enclosed double-action concertina bellows (Enfer's) and provided with a Fletcher's Universal gas blowpipe.

3. An ordinary bat's-wing gas-burner mounted at the far corner of the table top is invaluable in the preparation of tubular apparatus with sharp curves, and for coating newly-made glass apparatus with a layer of soot to prevent too rapid cooling, and its usually associated result—cracking.



6. Sedimentation tubes 5x0.5 cm., for sedimentation reactions, etc., and for containing small quantities of fluid to be centrifugalised in the haematocrit. These are made by taking 14-cm. lengths of stout glass tubing of the requisite diameter and heating the centre in the Bunsen or blowpipe flame. When the central portion is quite soft draw the ends quickly apart and then round off the pointed ends of the two test-tubes thus formed. With the glass-cutting knife cut off whatever may be necessary from the open ends to make the tubes the required length.

A rectangular block of "plasticine" (modelling clay) into which the conical ends can be thrust makes a very convenient stand for these small tubes.

Capillary Pipettes or Pasteur's Pipettes (Fig. 13 a).—These little instruments are invaluable, and a goodly supply should be kept on hand. They are prepared from soft-glass tubing of various-sized calibre (the most generally useful size being 8 mm. diameter) in the following manner: Hold a 10 cm. length of glass tube by each end, and whilst rotating it heat the central portion in the Bunsen flame or the blowpipe blast-flame until the glass is red hot and soft. Now remove it from the flame and steadily pull the ends apart, so drawing the heated portion out into a roomy capillary tube; break the capillary portion at its centre, seal the broken ends in the flame, and round off the edges of the open end of each pipette. A loose plug of cotton-wool in the open mouth completes the capillary pipette. After a number have been prepared, they are sterilised and stored in batches, either in metal cases similar to those used for the graduated pipettes or in large-sized test-tubes—sealed ends downward and plugged ends toward the mouth of the case.



The filling and emptying of the capillary pipette is most satisfactorily accomplished by slipping a small rubber teat (similar to that on a baby's feeding bottle but not perforated) on the upper end, after cutting or snapping off the sealed point of the capillary portion. If pressure is now exerted upon the elastic bulb by a finger and thumb whilst the capillary end is below the surface of the fluid to be taken up, some of the contained air will be driven out, and subsequent relaxation of that pressure (resulting in the formation of a partial vacuum) will cause the fluid to ascend the capillary tube. Subsequent compression of the bulb will naturally result in the complete expulsion of the fluid from the pipette (Fig. 14).



A modification of this pipette, in which a constriction or short length of capillary tube is introduced just below the plugged mouth (Fig. 13, b), will also be found extremely useful in the collection and storage of morbid exudations.

A third form, where the capillary portion is about 4 or 5 cm. long and only forms a small fraction of the entire length of the pipette (Fig. 13, c), will also be found useful.

"Blood" Pipettes (Fig 15).—Special pipettes for the collection of fairly large quantities of blood (as suggested by Pakes) should also be prepared. These are made from soft glass tubing of 1 cm. bore, in a similar manner to the Pasteur pipettes, except that the point of the blowpipe flame must be used in order to obtain the sharp shoulder at either end of the central bulb. The terminal tubes must retain a diameter of at least 1 mm., in order to avoid capillary action during the collection of the fluid.



For sterilisation and storage each pipette is placed inside a test-tube, resting on a wad of cotton-wool, and the tube plugged in the ordinary manner. As these tubes are used almost exclusively for blood work, it is usual to place a lance-headed hare-lip pin or a No. 9 flat Hagedorn needle inside the tube so that the entire outfit may be sterilised at one time.

For the collection of small quantities of blood for agglutination reactions and the like, many prefer a short straight piece of narrow glass tubing drawn out at either extremity to almost capillary dimensions. Such pipettes, about 8 cm. in length over all, are most conveniently sterilized in ordinary metal thermometer cases (Fig. 16).

Graduated Capillary Pipettes (Fig. 17).—These should also be made in the laboratory—from manometer tubing—of simple, convenient shape, and graduated by the aid of "standard" pipettes (in hundredths) to contain such quantities as 10, 50, and 90 c. mm., and carefully marked with a writing diamond. These, previously sterilised in large test-tubes, will be found extremely useful in preparing accurate percentage solutions, when only minute quantities of fluid are available.



Automatic ("Throttle") Pipettes.—These ingenious pipettes, introduced by Wright, can easily be calibrated in the laboratory and are exceedingly useful for graduating small pipettes, for measuring small quantities of fluids, in preparing dilutions of serum for agglutination reactions, etc. They are usually made from the Capillary Pasteur pipettes (Fig. 13, a). The following description of the manufacture of a 5 c. mm. pipette will serve to show how the small automatic pipettes are calibrated.

1. Select a pipette the capillary portion of which is fairly roomy in bore and possesses regular even walls, and remove the cotton-wool plug from the open end.

2. Heat the capillary portion near the free extremity in the by-pass flame of the bunsen burner and draw it out into a very fine hair-like tube and break this across. This hair-like extremity will permit the passage of air but is too fine for metallic mercury to pass.

3. From a standard graduated pipette deliver 5 c. mm. clean mercury into the upper wide portion of the pipette.

4. Adjust a rubber teat to the pipette and by pressure on the bulb gradually drive the mercury in an unbroken column down the capillary tube until it is stopped by the filiform extremity.

5. Cut off the capillary tube exactly at the upper level of the column of mercury, invert it and allow the mercury to run out.

6. Snap off the remainder of the capillary tube from the broad upper portion of the pipette which is now destined to form the covering tube or air chamber, or what we may term the "barrel." This barrel now has the lower end in the form of a truncated cone, the upper end being cut square. Remove the teat.

7. Introduce the capillary tube into this barrel with the filiform extremity uppermost, and the square cut end projecting about 0.5 cm. beyond the tapering end of the barrel.



8. Drop a small pellet of sealing wax into the barrel by the side of the capillary tube and then warm the tube at the gas flame until the wax becomes softened and makes an air-tight joint between the capillary tube and the end of the barrel.

9. Fit a rubber teat to the open end of the barrel, and so complete a pipette which can be depended upon to always aspirate and deliver exactly 5 cm. of fluid.

Slight modification of this procedure is necessary in making tubes to measure larger volumes than say 75 c. mm. Thus to make a throttle pipette to measure 100 c. mm.:

1. Take a short length of quill tubing and draw out one end into a roomy capillary stem, and again draw out the extremity into a fine hair point, thus forming a small Pasteur pipette with a hair-like capillary extremity.

2. With a standard pipette fill 100 c. mm. into the neck of this pipette, and make a scratch with a writing diamond at the upper level (a) of the mercury meniscus (Fig. 19, A).



Now force the mercury down into the capillary stem as far as it will go, so as to leave the upper part of the tube in the region of the diamond scratch empty (Fig. 19, B).

3. Heat the tube in the region of the diamond scratch in the blowpipe flame, and removing the tube from the flame draw it out so that the diamond scratch now occupies a position somewhere near the centre of this new capillary portion (Fig. 19, C).

4. Heat the tube in this position in the peep flame of the Bunsen burner, and draw it out into a hair-like extremity. Snap off the glass tube, leaving about 5 mm. of hair-like extremity attached to the upper capillary portion (Fig. 19, D). Allow the glass to cool.

5. Lift up the bulb by the long capillary stem and allow the mercury to return to its original position—an operation which will be facilitated by snapping off the hair-like extremity from the long piece of capillary tubing.

6. Mark on the capillary stem with a grease pencil the position of the end of the column of mercury (Fig. 19, E.)

7. Warm the capillary tubing at this spot in the peep flame of the Bunsen burner, and draw it out very slightly so that when cut at this position a pointed extremity will be obtained.

8. With a glass-cutting knife cut the capillary tube through at the point "b," and allow the mercury to run out.

9. Now apply a thick layer of sealing wax to the neck of the bulb.

10. Take a piece of 5 mm. bore glass tubing and draw it out as if making an ordinary Pasteur pipette.

11. Break the capillary portion off so as to leave a covering tube similar to that already used for the smaller graduated pipettes. Into this covering tube drop the graduated bulb and draw the capillary stem down through the conical extremity until further progress is stopped by the layer of sealing wax.

12. Warm the pipette in the gas flame so as to melt the sealing wax and make an air-tight joint.

13. Fit an india-rubber teat over the open end of the covering tube, and the automatic pipette is ready for use (Fig. 19, F).

Sedimentation Pipettes (Fig. 20).—These are prepared from 10 cm. lengths of narrow glass tubing by sealing one extremity, blowing a small bulb at the centre, and plugging the open end with cotton-wool; after sterilisation the open end is provided with a short piece of rubber tubing and a glass mouthpiece. When it is necessary to observe sedimentation reactions in very small quantities of fluid, these tubes will be found much more convenient than the 5 by 0.5 cm. test-tubes previously mentioned.



Pasteur pipettes fitted with india-rubber teats will also be found useful for sedimentation tests when dealing with minute quantities of serum, etc.



Fermentation Tubes (Fig. 21).—These are used for the collection and analysis of the gases liberated from the media during the growth of some varieties of bacteria and may be either plain (a) or graduated (b). A simple form (Fig. 21, c) may be made from 14 cm. lengths of soft glass tubing of 1.5 cm. diameter. The Bunsen flame is applied to a spot some 5 cm. from one end of such a piece of tubing and the tube slightly drawn out to form a constriction, the constricted part is bent in the bat's-wing flame, to an acute angle, and the open extremity of the long arm sealed off in the blowpipe flame. The open end of the short arm is rounded off and then plugged with cotton-wool, and the tube is ready for sterilisation.

CLEANING OF GLASS APPARATUS.

All glassware used in the bacteriological laboratory must be thoroughly cleaned before use, and this rule applies as forcibly to new as to old apparatus, although the methods employed may vary slightly.

To Clean New Test-tubes.

1. Place the tubes in a bucket or other convenient receptacle, fill with water and add a handful of "Sapon" or other soap powder. See that the tubes are full and submerged.

2. Fix the bucket over a large Bunsen flame and boil for thirty minutes—or boil in the autoclave for a similar period.

3. Cleanse the interior of the tubes with the aid of test-tube brushes, and rinse thoroughly in cold water.

4. Invert the tubes and allow them to drain completely.

5. Dry the tubes and polish the glass inside and out with a soft cloth, such as selvyt.

New flasks, plates, and capsules must be cleaned in a similar manner.

To Clean New Graduated Pipettes.

1. Place the pipettes in a convenient receptacle, filled with water to which soap powder has been added.

2. Boil the water vigorously for twenty minutes over a Bunsen flame.

3. Rinse the pipettes in running water and drain.

4. Run distilled water through the pipettes and drain.

5. Run rectified spirits through the pipette and drain as completely as possible.

6. Place the pipettes in the hot-air oven (vide page 31), close the door, open the ventilating slide, and run the temperature slowly up to about 80 deg. C. Turn off the gas and allow the oven to cool.

Or 6a. Attach each pipette in turn to the rubber tube of the foot bellows, or blowpipe air-blast, and blow air through the pipette until the interior is dry.

Glassware that has already been used is regarded as infected, and is treated in a slightly different manner.

Infected Test-tubes.

1. Pack the tubes in the wire basket of the autoclave (having previously removed the cotton-wool plugs, caps, etc.), in the vertical position, and before replacing the basket see that there is a sufficiency of water in the bottom of the boiler. Now attach a piece of rubber tubing to the nearest water tap, and by means of this fill each tube with water.

2. Disinfect completely by exposing the tubes, etc., to a temperature of 120 deg. C. for twenty minutes (vide page 37).

(If an autoclave is not available, the tubes must be placed in a digester, or even a large pan or pail with a tightly fitting cover, and boiled vigorously for some thirty to forty-five minutes to ensure disinfection.)

3. Whilst still hot, empty each tube in turn and roughly clean its interior with a stiff test-tube brush.

4. Place the tubes in a bucket or other convenient receptacle, fill with water and add a handful of Sapon or other soap powder. See that the tubes are full and submerged.

5. Fix the bucket over a large Bunsen flame and boil for thirty minutes.

6. Cleanse the interior of the tubes with the aid of test-tube brushes, and rinse thoroughly in cold water.

7. Drain off the water and immerse tubes in a large jar containing water acidulated with 2 to 5 per cent. hydrochloric acid. Allow them to remain there for about fifteen minutes.

8. Remove from the acid jar, drain, rinse thoroughly in running water, then with distilled water.

9. Invert the tubes and allow them to drain completely.

Dry the tubes and polish the glass inside and out with a soft cloth, such as selvyt.

Infected flasks, plates, and capsules must be treated in a similar manner.

Flasks which have been used only in the preparation of media must be cleaned immediately they are finished with. Fill each flask with water to which some soap powder and a few crystals of potassium permanganate have been added, and let boil over the naked flame. The interior of the flask can then usually be perfectly cleaned with the aid of a flask brush, but in some cases water acidulated with 5 per cent. nitric acid, or a large wad of wet cotton-wool previously rolled in silver sand, must be shaken around the interior of the flask, after which rinse thoroughly with clean water, dry, and polish.

Infected Pipettes.

1. Plunge infected pipettes immediately after use into tall glass cylinders containing a 2 per cent. solution of lysol, and allow them to remain therein for some days.

2. Remove from the jar and drain. Boil in water to which a little soap has been added, for thirty minutes.

3. Rinse thoroughly in cold water.

4. Immerse in 5 per cent. nitric acid for an hour or two.

5. Rinse again in running water to remove all traces of acid.

6. Complete the cleaning as described under "new pipettes."

When dealing with graduated capillary pipettes employed for blood or serum work (whether new or infected), much time is consumed in the various steps from 5 onward, and the cleansing process can be materially hastened if the following device is adopted.

Fit up a large-sized Kitasato's filter flask to a Sprengel's suction pump or a Geryk air pump (see page 43). To the side tubulure of the filter flask attach a 20 cm. length of rubber pressure tubing having a calibre sufficiently large to admit the ends of the pipettes.

Next fill a small beaker with distilled water. Attach the first pipette to the free end of the rubber tubing, place the pipette point downward in the beaker of water and start the pump (Fig. 22).



When all the water has been aspirated through the pipette into the filter flask, fill the beaker with rectified spirit and when this is exhausted refill with ether. Detach the pipette and dry in the hot-air oven.

Slides and cover-slips (Fig. 23), when first purchased, have "greasy" surfaces, upon which water gathers in minute drops and effectually prevents the spreading of thin, even films.

Microscopical Slides.—The slides in general use are those known as "three by one" slips (measuring 3 inches by 1 inch, or 76 by 26 mm.), and should be of good white crown glass, with ground edges.

New slides should be allowed to remain in alcohol acidulated with 5 per cent. hydrochloric acid for some hours, rinsed in running water, roughly drained on a towel, dried, and finally polished with a selvyt cloth.



If only a few slides are required for immediate use a good plan is to rub the surface with jeweler's emery paper (Hubert's 00). A piece of hard wood 76x26x26 mm. with a piece of this emery paper gummed tightly around it is an exceedingly useful article on the microscope bench.

Cover-slips.—The most useful sizes are the 19 mm. squares for ordinary cover-glass film preparations, and 38 by 19 mm. rectangles for blood films and serial sections; both varieties must be of "No. 1" thickness, which varies between 0.15 and 0.22 mm., that they may be available for use with the high-power immersion lenses.

Cover-slips should be cleaned in the following manner:

1. Drop the cover-slips one by one into an enamelled iron pot or tall glass beaker, containing a 10 per cent. solution of chromic acid.

2. Heat over a Bunsen flame and allow the acid to boil gently for twenty minutes.

NOTE.—A few pieces of pipe-clay or pumice may be placed in the beaker to prevent the "spurting" of the chromic acid.

3. Turn the cover-slips out into a flat glass dish and wash in running water under the tap until all trace of yellow colour has disappeared. During the washing keep the cover-slips in motion by imparting a rotatory movement to the dish.

4. Wash in distilled water in a similar manner.

5. Wash in rectified spirit.

6. Transfer the cover-slips, by means of a pair of clean forceps, previously heated in the Bunsen flame to destroy any trace of grease, to a small beaker of absolute alcohol.

Drain off the alcohol and transfer the cover-slips, by means of the forceps, to a wide-mouthed glass pot, containing absolute alcohol, in which they are to be stored, and stopper tightly.

NOTE.—After once being placed in the chromic acid, the cover-slips must on no account be touched by the fingers.

Used Slides and Cover-slips.—Used slides with the mounted cover-slip preparations, and cover-slips used for hanging-drop mounts, should, when discarded, be thrown into a pot containing a 2 per cent. solution of lysol.

After immersion therein for a week or so, even the cover-slips mounted with Canada balsam can be readily detached from their slides.

Slides.

1. Wash the slides thoroughly in running water.

2. Boil the slides in water to which "sapon" has been added, for half an hour.

3. Rinse thoroughly in cold water.

4. Dry and polish with a dry cloth.

Cover-slips.

1. Wash the cover-slips thoroughly in running water.

2. Boil the cover-slips in 10 per cent. solution of chromic acid, as for new cover-slips.

3. Wash thoroughly in running water.

4. Pick out those cover-slips which show much adherent dirty matter, and rub them between thumb and forefinger under the water tap. The dirt usually rubs off easily, as it has become friable from contact with the chromic acid.

5. Return all the cover-slips to the beaker, fill in fresh chromic acid solution, and treat as new cover-slips.

NOTE.—Test-tubes, plates, capsules, etc., which, from long use, have become scratched and hazy, or which cannot be cleaned in any other way, may be dealt with by immersing them in an enamelled iron bath, containing water acidulated to 1 per cent. with hydrofluoric acid, for ten minutes, rinsing thoroughly in water, drying, and polishing.

PLUGGING TEST-TUBES AND FLASKS.

Before sterilisation all test-tubes and flasks must be carefully plugged with cotton-wool, and for this purpose best absorbent cotton-wool (preferably that put up in cylindrical one-pound packets and interleaved with tissue paper—known as surgeons' wool) should be employed.

1. For a test-tube or a small flask, tear a strip of cotton-wool some 10 cm. long by 2 cm. wide from the roll.

2. Turn in the ends neatly and roll the strip of wool lightly between the thumb and fingers of both hands to form a long cylinder.

3. Double this at the centre and introduce the now rounded end into the open mouth of the tube or flask.

4. Now, whilst supporting the wool between the thumb and fingers of the right hand, rotate the test-tube between those of the left, and gradually screw the plug of wool into its mouth for a distance of about 2.5 cm., leaving about the same length of wool projecting.



The plug must be firm and fit the tube or flask fairly tightly, sufficiently tightly in fact to bear the weight of the glass plus the amount of medium the vessel is intended to contain, but not so tightly as to prevent it from being easily removed by a screwing motion when grasped between the fourth, or third and fourth, fingers, and the palm of the hand.

For a large flask a similar but larger strip of wool must be taken; the method of making and inserting the plug is identical.



III. METHODS OF STERILISATION.

STERILISING AGENTS.

Sterilisation—i. e., the removal or the destruction of germ life—may be effected by the use of various agents. As applied to the practical requirements of the bacteriological laboratory, many of these agents, such as electricity, sunlight, etc., are of little value, others are limited in their applications; others again are so well suited to particular purposes that their use is almost entirely restricted to such.

The sterilising agents in common use are:

Chemical Reagents.Disinfectants (for the disinfection of glass and metal apparatus and of morbid tissues).

Physical Agents. HEAT.—(a) Dry Heat:

1. Naked flame (for the sterilisation of platinum needles, etc.).

2. Muffle furnace (for the sterilisation of filter candles, and for the destruction of morbid tissues).

3. Hot air (for the sterilisation of all glassware and of metal apparatus).

(b) Moist Heat:

1. Water at 56 deg. C. (for the sterilisation of certain albuminous fluids).

2. Water at 100 deg. C. (for the sterilisation of surgical instruments, rubber tubing, and stoppers, etc.).

3. Streaming steam at 100 deg. C. (for the sterilisation of media).

4. Superheated steam at 115 deg. C. or 120 deg. C. (for the disinfection of contaminated articles and the destruction of old cultivations of bacteria).

FILTRATION.—

1. Cotton-wool filters (for the sterilisation of air and gases).

2. Porcelain filters (for the sterilisation of various liquids).

METHODS OF APPLICATION.

Chemical Reagents, such as belong to the class known as antiseptics (i. e., substances which inhibit the growth of, but do not destroy, bacterial life), are obviously useless. Disinfectants or germicides (i. e., substances which destroy bacterial life), on the other hand, are of value in the disinfection of morbid material, and also of various pieces of apparatus, such as pipettes, pending their cleansing and complete sterilisation by other processes. To this class (in order of general utility) belong:

Lysol, 2 per cent. solution; Perchloride of mercury, 0.1 per cent. solution; Carbolic acid, 5 per cent. solution; Absolute alcohol; Ether; Chloroform; Camphor; Thymol; Toluol; Volatile oils, such as oil of mustard, oil of garlic.

Formaldehyde is a powerful germicide, but its penetrating vapor restricts its use. These disinfectants are but little used in the final sterilisation of apparatus, chiefly on account of the difficulty of effecting their complete removal, for the presence of even traces of these chemicals is sufficient to so inhibit or alter the growth of bacteria as to vitiate subsequent experiments conducted by the aid of apparatus sterilised in this manner.

NOTE.—Tubes, flasks, filter flasks, pipettes, glass tubing, etc., may be rapidly sterilised, in case of emergency, by washing, in turn, with distilled water, perchloride of mercury solution, alcohol, and ether, draining, and finally gently heating over a gas flame to completely drive off the ether vapor. Chloroform or other volatile disinfectants may be added to various fluids in order to effect the destruction of contained bacteria, and when this has been done, may be completely driven off from the fluid by the application of gentle heat.

Dry Heat.—The naked flame of the Bunsen burner is invariably used for sterilising the platinum needles (which are heated to redness) and may be employed for sterilising the points of forceps, or other small instruments, cover-glasses, pipettes, etc., a very short exposure to this heat being sufficient.

Ether Flame.—In an emergency small instruments, needles, etc., may be sterilised by dipping them in ether and after removal lighting the adherent fluid and allowing it to burn off the surface of the instruments. Repeat the process twice. It may then be safely assumed that the apparatus so treated is sterile.



Muffle Furnace (Fig. 25).—Although this form of heat is chiefly used for the destruction of the dead bodies of small infected animals, morbid tissues, etc., it is also employed for the sterilisation of porcelain filter candles (vide p. 42).

Filter candles are disinfected immediately after use by boiling in a beaker of water for some fifteen or twenty minutes. This treatment, however, leaves the dead bodies of the bacteria upon the surface and blocking the interstices of the filter.

To destroy the organic matter and prepare the filter candle for further use proceed as follows:

1. Roll each bougie up in a piece of asbestos cloth, secure the ends of the cloth with a few turns of copper wire, and place inside the muffle (a small muffle 76x88x163 mm. will hold perhaps four small filter candles).

2. Light the gas and raise the contents of the muffle to a white heat; maintain this temperature for five minutes.

3. Extinguish the gas, and when the muffle has become quite cold remove the filter candles, and store them (without removing the asbestos wrappings) in sterile metal boxes.

NOTE.—The too rapid cooling of the candles, such as takes place if they are removed from the muffle before it has cooled down to the room temperature, may give rise to microscopic cracks and flaws which will effectually destroy their efficiency.

Hot Air.—Hot air at 150 deg. C. destroys all bacteria, spores, etc:, in about thirty minutes; a momentary exposure to a temperature of 175 deg. to 180 deg. C. will effect the same result and offers the more convenient method of sterilisation. This method is only applicable to glass and metallic substances, and the small bulk of cotton-wool comprised in the test-tube plugs, etc. Large masses of fabric are not effectually sterilised by dry heat—short of charring—as its power of penetration is not great.

Sterilisation by hot air is effected in the hot-air oven (Fig. 18). This is a rectangular, double-walled metal box, mounted on a stand and heated from below by a large Bunsen burner. The interior of the oven is provided with loose shelves upon which the articles to be sterilised are arranged, either singly or packed in square wire baskets or crates, kept specially for this purpose. One of the sides is hinged to form a door. The central portion of the metal bottom, on which the Bunsen flame would play, is cut away, and replaced by firebrick plates, which slide in metal grooves and are easily replaced when broken or worn out. The top of the oven is provided with a perforated ventilator slide and two tubulures, the one for the reception of a centigrade thermometer graduated to 200 deg. or 250 deg. C., the other for a thermo-regulator. An ordinary mercurial thermo-regulator may be used but it is preferable to employ a regulating capsule of the Hearson type (see p. 219) with a spring arm adjusted to the lever so that when the boiling-point of the capsule (e. g., 175 deg. C.) is reached the gas supply is absolutely cut off and the jet cannot again be lighted until the spring-arm has been readjusted by hand. The thermo-regulator is by no means a necessity, and may be replaced by a large bore thermometer with a sliding platinum point, connected with an electric bell, which can be easily adjusted to ring at any given temperature. Even if the steriliser is provided with the capsule regulator above described the contact thermometer should also be fitted.



TO USE THE HOT-AIR OVEN.—

1. Place the crates of test-tubes, metal cases containing plates and pipettes, loose apparatus, etc., inside the oven, taking particular care that none of the cotton-wool plugs are in contact with the walls, otherwise the heat transmitted by the metal will char or even flame them.

To prepare a wire crate for the reception of test-tubes, etc., cover the bottom with a layer of thick asbestos cloth; or take some asbestos fibre, moisten it with a little water and knead it into a paste; plaster the paste over the bottom of the crate, working it into the meshes and smoothing the surface by means of a pestle. When several crates have been thus treated, place them inside the hot-air oven, close the door, open the ventilating slide, light the gas, and run the temperature of the interior up to about 160 deg. C. After an interval of ten minutes extinguish the gas, open the oven door, and allow the contents to cool. The asbestos now forms a smooth, dry, spongy layer over the bottom, which will last many months before needing renewal, and will considerably diminish the loss of tubes from breakage.

Copper cylinders and large test-tubes intended for the reception of pipettes are prepared in a similar manner, in order to protect the points of these articles from injury.

2. Close the oven door, and open the ventilating slide, in order that any moisture left in the tubes, etc., may escape; light the gas below; set the electric alarm to ring at 100 deg. C.

3. When the temperature of the oven has reached 100 deg. C., close the ventilating slide; reset the alarm to ring at 175 deg. C.

4. Run the temperature up to 175 deg. C.

5. Extinguish the gas at once, and allow the apparatus to cool.

6. When the temperature of the interior, as recorded by the thermometer, has fallen to 60 deg. C.—but not before—the door may be opened and the sterile articles removed and stored away.

NOTE.—Neglect of this precautionary cooling of the oven to 60 deg. C. will result in numerous cracked and broken tubes.

On removal from the oven, the cotton-wool plugs will probably be slightly brown in colour.

Metal instruments, such as knives, scissors, and forceps, may be sterilised in the hot-air oven as described above, but exposure to 175 deg. C. is likely to seriously affect the temper of the steel and certainly blunts the cutting edges. If, however, it is desired to sterilise surgical instruments by hot air, they should be packed in a metal box, or boxes, and heated to 130 deg. C. and retained at that temperature for about thirty minutes.

Moist Heat.Water at 56 deg. C.—This temperature, if maintained for thirty minutes, is sufficient to destroy the vegetative forms of bacteria, but has practically no effect on spores. Its use is limited to the sterilisation of such albuminous "fluid" media as would coagulate at a higher temperature.

METHOD.—

1. Fit up a water-bath, heated by a Bunsen flame which is controlled by a thermo-regulator, so that the temperature of the water remains at 56 deg. C.

2. Immerse the tubes or flasks containing the albuminous fluid in the water-bath so that the upper level of such fluid is at least 2 cm. below the level of the water. (The temperature of the bath will now fall somewhat, but after a few minutes will again rise to 56 deg. C).

3. After thirty minutes' exposure to 56 deg. C, extinguish the gas, remove the tubes or flasks from the bath, and subject them to the action of running water so that their contents are rapidly cooled.

4. The vegetative forms of bacteria present in the liquid being killed, stand it for twenty-four hours in a cool, dark place; at the end of that time some at least of such spores as may be present will have germinated and assumed the vegetative form.

5. Destroy these new vegetative forms by a similar exposure to 56 deg. C. on the second day, whilst others, of slower germination, may be caught on the third day, and so on.

6. In order to ensure thorough sterilisation, repeat the process on each of six successive days.

This method of exposing liquids to a temperature of 56 deg. C. in a water-bath for half an hour on each of six successive days is termed fractional sterilisation.

Water at 100 deg. C. destroys the vegetative forms of bacteria almost instantaneously, and spores in from five to fifteen minutes. This method of sterilisation is applicable to the metal instruments, such as knives, forceps, etc., used in animal experiments; syringes, rubber corks, rubber and glass tubing, and other small apparatus, and is effected in what is usually spoken of as the "water steriliser" (Fig. 27).



This is a rectangular copper box, 26 cm. long, 18 cm. wide, and 12 cm. deep, mounted on legs, heated from below by a Bunsen or radial gas burner, and containing a movable copper wire tray, 2 cm. smaller in every dimension than the steriliser itself, and provided with handles. The top of the steriliser is hinged to form a lid.

METHOD.—

1. Place the instruments, etc., to be sterilised inside the copper basket, and replace the basket in the steriliser.

2. Pour a sufficient quantity of water into the steriliser, shut down the lid, and light the gas below.



3. After the water has boiled and steam has been issuing from beneath the lid for at least ten minutes, extinguish the gas, open the lid, and lift out the wire basket by its handles and rest it diagonally on the walls of the steriliser; the contained instruments, etc., are now sterile and ready for use.

4. After use, or when accidentally contaminated, replace the instruments in the basket and return that to the steriliser; completely disinfect by a further boiling for fifteen minutes.

5. After disinfection, and whilst still hot, take out the instruments, dry carefully and at once, and return them to their store cases.

Streaming steam—i. e., steam at 100 deg. C.—destroys the vegetative forms of bacteria in from fifteen to twenty minutes, and the sporing forms in from one to two hours. This method is chiefly used for the sterilisation of the various nutrient media intended for the cultivation of bacteria, and is carried out in a steam kettle of special construction, known as Koch's steam steriliser (Fig. 28) or in one of its many modifications, the most efficient of which is Arnold's (Fig. 29).

The steam steriliser in its simplest form consists of a tall tinned-iron or copper cylindrical vessel, divided into two unequal parts by a movable perforated metal diaphragm, the lower, smaller portion serving for a water reservoir, and the upper part for the reception of wire baskets containing the articles to be sterilised. The vessel is closed by a loose conical lid, provided with handles, and perforated at its apex by a tubulure; it is mounted on a tripod stand and heated from below by a Bunsen burner. The more elaborate steriliser is cased with felt or asbestos board, and provided with a water gauge, also a tap for emptying the water compartment.

TO USE THE STEAM STERILISER.—

1. Fill the water compartment to the level of the perforated diaphragm, place the lid in position, and light the Bunsen burner.

2. After the water has boiled, allow sufficient time to elapse for steam to replace the air in the sterilising compartment, as shown by the steam issuing in a steady, continuous stream from the tubulure in the lid.

3. Remove the lid, quickly lower the wire basket containing media tubes, etc., into the sterilising compartment until it rests on the diaphragm, and replace the lid.

4. After an interval of twenty minutes in the case of fluid media, or thirty minutes in the case of solid media, take off the lid and remove the basket with its contents.

5. Now, but not before, extinguish the gas.

NOTE.—After removing tubes, flasks, etc., from the steam steriliser, they should be at once separated freely in order to prevent moisture condensing upon the cotton-wool plugs and soaking through into the interior of the tubes.

This treatment will destroy any vegetative forms of bacteria; during the hours of cooling any spores present will germinate, and the young organisms will be destroyed by repeating the process twenty-four hours later; a third sterilisation after a similar interval makes assurance doubly sure.

The method of sterilising by exposure to streaming steam at 100 deg. C. for twenty minutes on each of three consecutive days is termed discontinuous or intermittent sterilisation.

Exposure to steam at 100 deg. C. for a period of one or two hours, or continuous sterilisation, cannot always be depended upon and is therefore not to be recommended.

Superheated steam—i. e., steam under pressure (see Pressure-temperature table, Appendix, page 500) in sealed vessels at a temperature of 115 deg. C.—will destroy both the vegetative and the sporing forms of bacteria within fifteen minutes; if the pressure is increased, and the temperature raised to 120 deg. C., the same end is attained in ten minutes. This method was formerly employed for the sterilisation of media (and indeed is so used in some laboratories still), but most workers now realise that media subjected to this high temperature undergo hydrolytic changes which render them unsuitable for the cultivation of the more delicate micro-organisms. The use of superheated steam should be restricted almost entirely to the disinfection of such contaminated articles, old cultivations, etc., as cannot be dealt with by dry heat or the actual furnace. Sterilisation by means of superheated steam is carried out in a special boiler—Chamberland's autoclave (Fig. 30). The autoclave consists of a stout copper cylinder, provided with a copper or gun-metal lid, which is secured in place by means of bolts and thumbscrews, the joint between the cylinder and its lid being hermetically sealed by the interposition of a rubber washer. The cover is perforated for a branched tube carrying a vent cock, a manometer, and a safety valve. The copper boiler is mounted in the upper half of a cylindrical sheet-iron case—two concentric circular rows of Bunsen burners, each circle having an independent gas-supply, occupying the lower half. In the interior of the boiler is a large movable wire basket, mounted on legs, for the reception of the articles to be sterilised.

TO USE THE AUTOCLAVE.—

1. Pack the articles to be sterilised in the wire basket.

2. Run water into the boiler to the level of the bottom of the basket; also fill the contained flasks and tubes with water.

3. See that the rubber washer is in position, then replace the cover and fasten it tightly on to the autoclave by means of the thumbscrews.

4. Open the vent cock and light both rings of burners.

5. When steam is issuing in a steady, continuous stream from the vent tube, shut off the vent cock and extinguish the outer ring of gas burners.

6. Wait until the index of the manometer records a temperature of 120 deg. C., then regulate the gas and the spring safety valve in such a manner that this temperature is just maintained, and leave it thus for twenty minutes. In the more expensive patterns of autoclave this regulation of the safety valve is carried out automatically, the manometer being fitted with an adjustable pointer which can be set to any required pressure-temperature and so arranged that when the index of the manometer coincides with the adjustable hand the safety valve is opened.

7. Extinguish the gas and allow the manometer index to fall to zero.



8. Now open the vent cock slowly, and allow the internal pressure to adjust itself to that of the atmosphere.

9. Remove the cover and take out the sterilised contents.

Sterilisation Periods.—An exceedingly useful device for the timing of sterilisation periods (and indeed for many other operations in the laboratory) is the

ELECTRIC SIGNAL TIMING CLOCK.

This is a clock of American type in which the face is surrounded by a metal plate having a series of 60 holes at equal distances apart, corresponding to the minutes on the dial. This plate is connected with one of the poles of a dry battery, the other pole of which is connected to the metal case of the clock for the purpose of actuating an ordinary magnet alarm bell. In the centre of each of the holes in the plate a metal rod is fixed, which then passes through an insulating ring and projects inside the clock face, where it makes contact with the hour hand. The clock is mounted on a heavy base, with a key-board containing 20 numbered plugs. If one of the plugs is inserted in a hole in the plate it makes contact with the rod, and when the hour hand of the clock touches the other end the circuit is completed and the bell starts ringing. The period of this friction contact is approximately 20 seconds. The clock can therefore be used for electrically noting the periods of time from one minute by multiples of one minute up to one hour.



Filtration.—(a) Cotton-wool Filter.—Practically the only method in use in the laboratory for the sterilisation of air or of a gas is by filtration through dry cotton-wool or glass-wool, the fibres of which entangle the micro-organisms and prevent their passage.

Perhaps the best example of such a filter is the cotton-wool plug which closes the mouth of a culture tube. Not only does ordinary diffusion take place through it, but if a tube plugged in the usual manner with cotton-wool is removed from the hot incubator, the temperature of the contained air rapidly falls to that of the laboratory, and a partial vacuum is formed; air passes into the tube, through the cotton-wool plug, to restore the equilibrium, and, so long as the plug remains dry, in a germ-free condition. If, however, the plug becomes moist, either by absorption from the atmosphere, or from liquids coming into contact with it, micro-organisms (especially the mould fungi) commence to multiply, and the long thread forms rapidly penetrate the substance of the plug, and gain access to and contaminate the interior of the tube.



METHOD.—

If it is desired to sterilise gases before admission to a vessel containing a pure cultivation of a micro-organism, as, for instance, when forcing a current of oxygen over or through a broth cultivation of the diphtheria bacillus, this can be readily effected as follows:

1. Take a length of glass tubing of, say, 1.5 cm. diameter, in the centre of which a bulb has been blown, fill the bulb with dry cotton-wool (Fig. 32), wrap a layer of cotton-wool around each end of the tube, and secure in position with a turn of thin copper wire or string; then sterilise the piece of apparatus in the hot-air oven.

2. Prepare the cultivation in a Ruffer or Woodhead flask (Fig. 33) the inlet tube of which has its free extremity enveloped in a layer of cotton-wool, secured by thread or wire, whilst the exit tube is plugged in the usual manner.



3. Sterilise a short length of rubber tubing by boiling. Transfer it from the boiling water to a beaker of absolute alcohol.

4. When all is ready remove the rubber tube from the alcohol by means of a pair of forceps, drain it thoroughly, and pass through the flame of a Bunsen burner to burn off the last traces of alcohol.

5. Remove the cotton-wool wraps from the entry tube of the flask and from one end of the filter tube and rapidly couple them up by means of the sterile rubber tubing.

6. Connect the other end of the bulb tube with the delivery tube from the gas reservoir.

The gas in its passage through the dry sterile cotton-wool in the bulb of the filter tube will be freed from any contained micro-organisms and will enter the flask in a sterile condition.

(b) Porcelain Filter.—The sterilisation of liquids by filtration is effected by passing them through a cylindrical vessel, closed at one end like a test-tube, and made either of porous "biscuit" porcelain, hard-burnt and unglazed (Chamberland system), or of Kieselguhr, a fine diatomaceous earth (Berkefeld system), and termed a "bougie" or "candle" (Fig. 34).

NOTE.—In selecting candles for use in the laboratory avoid those with metal fittings, since during sterilisation cracks develop at the junction of the metal and the siliceous material owing to the unequal expansion.

In this method the bacteria are retained in the pores of the filter while the liquid passes through in a germ-free condition.

It is obvious that to be effective the pores of the filter must be extremely minute, and therefore the rate of filtration will usually be slow. Chamberland filter candles possess finer channels than Berkefeld candles and consequently filter much more slowly. To overcome this disadvantage, either aspiration or pressure, or a combination of these two forces, may be employed to hasten the process.

Doultons white porcelain filters it may be noted are as efficient as the Chamberland candles and filter rather more rapidly.

Apparatus Required.

1. Separatory funnel containing the unfiltered fluid.

2. Sterile filter candle (Fig. 34), the open end fitted with a rubber stopper (Fig. 34, a) perforated to receive the delivery tube of the separatory funnel, and its neck passed through a large rubber washer (Fig. 34, b) which fits the mouth of the filter flask.

3. Sterile filter flask of suitable size, for the reception of the filtered fluid, its mouth closed by a cotton-wool plug.

4. Water injector Sprengel (see Fig. 38, c) pump, or Geryk's pump (an air pump on the hydraulic principle, sealed by means of low vapor-tension oil, Fig. 35).

If this latter is employed, a Wulff's bottle, fitted as a wash-bottle and containing sulphuric acid, must be interposed between the filter flask and the pump, in order to prevent moist air reaching the oil in the pump.

5. Air filter (vide page 40) sterilised.

6. Pressure tubing.

7. Screw clamps (Fig. 36).

METHOD.—

1. Couple the exhaust pipe of the suction pump with the lateral tube of the filter flask (first removing the cotton-wool plug from this latter), by means of pressure tubing, interposing, if necessary, the wash-bottle of sulphuric acid.



2. Remove the cotton-wool plug from the neck of the filter flask and adjust the porcelain candle in its place.



3. Attach the nozzle of the separatory funnel to the filter candle by means of the perforated rubber stopper (Fig. 37).



4. Open the tap of the funnel, and exhaust the air from the filter flask and wash-bottle; maintain the vacuum until the filtration is complete.

5. When the filtration is completed close the tap of the funnel; adjust a screw clamp to the pressure tubing attached to the lateral branch of the filter flask; screw it up tightly, and disconnect the acid wash-bottle.

6. Attach the air filter to the open end of the pressure tubing; open the screw clamp gradually, and allow filtered air to enter the flask, to abolish the negative pressure.

7. Detach the rubber tubing from the lateral branch of the flask, flame the end of the branch in the Bunsen, and plug its orifice with sterile cotton-wool.

8. Remove the filter candle from the mouth of the flask, flame the mouth, and plug the neck with sterile cotton-wool.

9. Disinfect the filter candle and separatory funnel by boiling.

If it is found necessary to employ pressure in addition to or in place of suction, insert a perforated rubber stopper into the mouth of the separatory funnel and secure in position with copper wire; next fit a piece of glass tubing through the stopper, and connect the external orifice with an air-pressure pump of some kind (an ordinary foot pump such as is employed for inflating bicycle tyres is one of the most generally useful, for this purpose) or with a cylinder of compressed air or other gas.

In order to filter a large bulk of fluid very rapidly it is necessary to use a higher pressure than glass would stand, and in these cases the metal receptacle designed by Pakes (Fig. 38, a), to hold the filter candle itself as well as the fluid to be filtered, should be employed. (A vacuum must also be maintained in the filter flask, by means of an exhaust pump, during the entire process.)

This piece of apparatus consists of a brass cylinder, capacity 2500 c.c., with two shoulders; and an opening in the neck at each end, provided with screw threads.

A nut carrying a pressure gauge fits into the top screw; and into the bottom is fitted a brass cylinder carrying the filter candle and prolonged downwards into a delivery tube. Leakage is prevented by means of rubber washers.

Into the top shoulder a tube is inserted, bent at right angles and provided with a tap. All the brass-work is tinned inside (Fig. 38, a). In use the reservoir is generally mounted on a tripod stand.

To Sterilise.

1. Insert the filter candle into its cylinder and screw this loosely on.



2. Wrap a layer of cotton-wool around the delivery tube and fasten in position.

3. Remove the nut carrying the pressure gauge and plug the neck with cotton-wool.

4. Heat the whole apparatus in the autoclave at 120 deg. C. for twenty minutes.

METHOD.—

1. Remove the apparatus from the autoclave, and allow it to cool.

2. Screw home the box carrying the bougie.

3. Set the apparatus up in position, with its delivery tube (from which the cotton-wool wrapping has been removed) passing through a perforated rubber stopper in the neck of a filter flask.



4. Fill the fluid to be filtered into the cylinder and screw on the nut carrying the pressure gauge. (This nut should be immersed in boiling water for a few minutes previous to screwing on, in order to sterilise it.)

5. Connect the horizontal arm of the entry tube with a cylinder of compressed oxygen (or carbon dioxide, Fig. 38, b), by means of pressure tubing.

6. Connect the lateral arm of the filter flask with the exhaust pump (Fig. 38, c) and start the latter working.

7. Open the tap of the gas cylinder; then open the tap on the entry tube of the filter cylinder and raise the pressure in its interior until the desired point is recorded on the manometer. Maintain this pressure, usually one or one and a half atmospheres, until filtration is completed, by regulating the tap on the entry tube.

Some forms of filter candle are made with the open end contracted into a delivery nozzle, which is glazed. In this case the apparatus is fitted up in a slightly different manner; the fluid to be filtered is contained in an open cylinder into which the candle is plunged, while its delivery nozzle is connected with the filter flask by means of a piece of flexible pressure tubing (previously sterilised by boiling), as in figure 39.



IV. THE MICROSCOPE.

The essentials of a microscope for bacteriological work may be briefly summed up as follows:



The instrument, of the monocular type, must be of good workmanship and well finished, rigid, firm, and free from vibration, not only when upright, but also when inclined to an angle or in the horizontal position. The various joints and movements must work smoothly and precisely, equally free from the defects of "loss of time" and "slipping." All screws, etc., should conform to the Royal Microscopical Society's standard. It must also be provided with good lenses and a sufficiently large stage. The details of its component parts, to which attention must be specially directed, are as follows:



1. The Base or Foot (Fig. 40, a).—Two elementary forms—the tripod (Fig. 41, a) and the vertical column set into a plate known as the "horse-shoe" (Fig. 41, b)—serve as the patterns for countless modifications in shape and size of this portion of the stand. The chief desiderata—stability and ease of manipulation—are attained in the first by means of the "spread" of the three feet, which are usually shod with cork; in the second, by the dead weight of the foot-plate. The tripod is mechanically the more correct form, and for practical use is much to be preferred. Its chief rival, the Jackson foot (Fig. 41, c), is based upon the same principle, and on the score of appearance has much to recommend it.

2. The body tube (Fig. 40, b) may be either that known as the "long" or "English" (length 250 mm.), or the "short" or "Continental" (length 160 mm.). Neither length appears to possess any material advantage over the other, but it is absolutely necessary to secure objectives which have been manufactured for the particular tube length chosen. In the high-class microscope of the present day the body tube is usually shorter than the Continental, but is provided with a draw tube which, when fully extended, gives a tube length greater than the English, thus permitting the use of either form of objective.



For practical purposes the tube length = distance from the end of the nosepiece to the eyeglass of the ocular. This is the measurement referred to in speaking of "long" or "short" tube.

3. The coarse adjustment (Fig. 40, c) should be a rack-and-pinion movement, steadiness and smoothness of action being secured by means of accurately fitting dovetailed bearings and perfect correspondence between the teeth of the rack and the leaves of the pinion (Fig. 42). Also provision should be made for taking up the "slack" (as by the screws AA, Fig. 42).

4. The fine adjustment (Fig. 40, d) should on no account depend upon the direct action of springs, but should be of the lever pattern, preferably the Nelson (Fig. 43). In this form the unequal length of the arms of the lever secures very delicate movement, and, moreover, only a small portion of the weight of the body tube is transmitted to the thread of the vertical screw actuating the movement.



A spindle milled head (Fig. 44) will be found a very useful device to have fitted in place of the ordinary milled head controlling the fine adjustment. In this contrivance the axis of the milled head is prolonged upward in a short column, the diameter of which is one-sixth of that of the head. The spindle can be rapidly rotated between the fingers for medium power adjustments while the larger milled head can be slowly moved when focussing high powers.

5. The stage (Fig. 40, e) should be square in shape and large in area—at least 12 cm.—flat and rigid, in order to afford a safe support for the Petri dish used for plate cultivations; and should be supplied with spring clips (removable at will) to secure the 3 by 1 glass slides.

A mechanical stage must be classed as a necessity rather than a luxury so far as the bacteriologist is concerned, as when working with high powers, and especially when examining hanging-drop specimens, it is almost impossible to execute sufficiently delicate movements with the fingers. In selecting a mechanical stage, preference should be given to one which forms an integral part of the instrument (Fig. 45) rather than one which needs to be clamped on to an ordinary plain stage every time it is required, and its traversing movements should be controlled by stationary milled heads (Fig. 45, AA'). The shape of the aperture is a not unimportant point; it should be square to allow of free movement over the substage condenser. The mechanical stage should be tapped for three (removable) screw studs to be used in place of the sliding bar, so that if desired the Vernier finder (Fig. 45, BB'), such as is usually fitted to this class of stage, or a Maltwood finder, may be employed.



6. Diaphragm.—Separate single diaphragms must be avoided; a revolving plate pierced with different sized apertures and secured below the stage is preferable, but undoubtedly the best form is the "iris" diaphragm (Fig. 46) which enters into the construction of the substage condenser.

7. The substage condenser is a necessary part of the optical outfit. Its purpose is to collect the beam of parallel rays of light reflected by the plane mirror, by virtue of a short focus system of lenses, into a cone of large aperture (reducible at will by means of an iris diaphragm mounted as a part of the condenser), which can be accurately focussed on the plane of the object. This focussing must be performed anew for each object, on account of the variation in the thickness of the slides.

The form in most general use is that known as the Abbe (Fig. 47) and consists of a plano-convex lens mounted above a biconvex lens. This combination is carried in a screw-centering holder known as the substage below the stage of the microscope (Fig. 40 f), and must be accurately adjusted so that its optical axis coincides with that of the objective. Vertical movement of the entire substage apparatus effected by means of a rack and pinion is a decided advantage, and some means should be provided for temporarily removing the condenser from the optical axis of the microscope.



With the oil immersion objective, however, an achromatic condenser, giving an illuminating cone of about 0.9, should be used if the full value of the lens is to be obtained. It is generally assumed that a good objective requires an illuminating cone equivalent to two-thirds of its numerical aperture. The best Abbe condenser transmits a cone of about .45 whilst the aperture of the 1/12 inch immersion lenses of different makers varies from 1.0 to 1.4, hence, the efficiency of these lenses is much curtailed if the condenser is merely the Abbe. These improved condensers must be absolutely centered to the objective and capable of very accurate focussing otherwise much of their value is lost.

8. Mirrors.—Below the substage condenser is attached a gymbal carrying a reversible circular frame with a plane mirror on one side and a concave mirror on the other (Fig. 40, g). The plane mirror is that usually employed, but occasionally, as for example when using low powers and with the condenser racked down and thrown out of the optical axis, the concave mirror is used.

9. Oculars, or Eyepieces.—Those known as the Huyghenian oculars (Fig. 48) will be sufficient for all ordinary work without resorting to the more expensive "compensation" oculars. Two or three, magnifying the "real" image (formed by the objective) four, six, or eight times respectively, form a useful equipment.

As an accessory Ehrlich's Eyepiece is a very useful piece of apparatus when the enumeration of cells or bacteria has to be carried out. This is an ordinary eyepiece fitted with an adjustable square diaphragm operated by a lever projecting from the side of the mount. Three notches are made in one of the sides of the square and by moving the lever square aperture can be reduced to three-quarters, one-half or one-quarter of the original size.

10. Objectives.—Three objectives are necessary: one for low-power work—e. g., 1 inch, 2/3 inch, or 1/2 inch; one for high-power work—e. g., 1/12 inch oil immersion lens; and an intermediate "medium-power" lens—e. g., 1/6 inch or 1/8 inch (dry). These lenses must be carefully selected, especial attention being paid to the following points:

(a) Correction of Spherical Aberration.—Spherical aberration gives rise to an ill-defined image, due to the central and peripheral rays focussing at different points.

(b) Correction of Chromatic Aberration.—Chromatic aberration gives rise to a coloured fringe around the edges of objects due to the fact that the different-coloured rays of the spectrum possess varying refrangibilities and that a simple lens acts toward them as a prism.

(c) Flatness of Field.—The ideal visual field would be large and, above all, flat; in other words, objects at the periphery of the field would be as distinctly "in focus" as those in the centre. Unfortunately, however, this is an optical impossibility and the field is always spherical in shape. Some makers succeed in giving a larger central area that is in focus at one time than others, and although this may theoretically cause an infinitesimal sacrifice of other qualities, it should always be sought for. Successive zones and the entire peripheral ring should come into focus with the alteration of the fine adjustment. This simultaneous sharpness of the entire circle is an indication of the perfect centering of the whole of the lenses in the objective.



(d) Good Definition.—Actual magnification is, within limits, of course, of less value than clear definition and high resolving power, for it is upon these properties we depend for our knowledge of the detailed structure of the objects examined.

(e) Numerical Aperture (N. A.).—The numerical aperture may be defined, in general terms, as the ratio of the effective diameter of the back lens of the objective to its equivalent focal length. The determination of this point is a process requiring considerable technical skill and mathematical ability, and is completely beyond the powers of the average microscopist.[1]

Although with the increase in power it is correspondingly difficult to combine all these corrections in one objective, they are brought to a high pitch of excellence in the present-day "achromatic" objectives, and so remove the necessity for the use of the higher priced and less durable apochromatic lenses.

In selecting objectives the best "test" objects to employ are:

1. A thin (one cell layer), even } { 1", 2/3", 1/2": "blood film," stained with Jenner's } for { 1/6", 1/8" or Romanowsky's stain. } { 1/12" oil

2. A thin cover-slip preparation } of a young cultivation of } { 1/8" dry B. diphtheriae (showing } for { segmentation) stained with } { 1/12" oil methylene-blue. }

Accessories.Eye Shade (Fig. 49).—This piece of apparatus consists of a pear-shaped piece of blackened metal or ebonite, hinged to a collar which rotates on the upper part of the body tube of the microscope. It can be used to shut out the image of surrounding objects from the unoccupied eye, and when carrying out prolonged observations will be found of real service.

Nosepiece.—Perhaps the most useful accessory is a nosepiece to carry two of the objectives (Fig. 50), or, better still, all three (Fig. 51). This nosepiece, preferably constructed of aluminium, must be of the covered-in type, consisting of a curved plate attached to the lower end of the body tube—a circular aperture being cut to correspond to the lumen of that tube. To the under surface of this plate is pivoted a similarly curved plate, fitted with three tubulures, each of which carries an objective. By rotating the lower plate each of the objectives can be brought successively in to the optical axis of the microscope.



For critical work and particularly for photo-micrography, however, the interchangeable nosepiece is by no means perfect as it is next to impossible to secure accurate centreing of each lens in the optical axis. For special purposes, therefore, it is necessary to employ a special nosepiece such as that made by Zeiss or Leitz into which each objective slides on its own carrier and upon which it is accurately centred.



Warm Stage (Fig. 52).—This is a flat metal case containing a system of tubes through the interior of which water of any required temperature can be circulated. It is made to clamp on to the stage of the microscope by the screws A A', and is perforated with a large hole coinciding with the optical axis of the microscope; a short tube B, projecting from one end of the warm stage permits water of the desired temperature to be conducted from a reservoir through a length of rubber tubing to the interior of the stage and a similar tube at the other end B' of the stage allows exit to the waste water. By raising the temperature of hanging-drop preparations, etc., placed upon it, above that of the surrounding atmosphere, the warm stage renders possible exact observations on spore germination, hanging-drop cultivations, etc.



A better form is the electrical hot stage designed by Lorrain Smith;[2] it requires the addition of a lamp resistance and sliding rheostat, also a delicate ammeter reading to .01 of an ampere. It consists of a wooden frame supporting a flat glass bulb with a long neck bent upward at an obtuse angle (Fig. 53). The bulb is filled with liquid paraffin, which rises in the open neck when expanded by heat. The neck also accommodates the thermometer. Two coils of manganin wire run in the paraffin at opposite sides of the bulb (outside the field of vision), coupled to brass terminals on the wooden frame by platinum wire fused into the glass. The resistance of the two coils in series is about 10 ohms. A current of 2-1/2 amperes is needed, and is conducted to the coils in the stage through the rheostat. With the help of the ammeter any desired temperature can be obtained and maintained, up to about 200 deg. C. If immersion oil contact is made between the top lens of the condenser and the lower surface of the bulb, this stage works very well indeed with the 1/12-inch oil immersion lens.



Dark Ground or Paraboloid Condenser.—This is an immersion substage condenser of high aperture by means of which unstained objects such as bacteria can be shown as bright white particles upon a dense black background. The central rays of light are blocked out by means of an opaque stop while the peripheral rays are reflected from the paraboloidal sides of the condenser and refracted by the object viewed. To obtain the best results with this type of condenser a powerful illuminant—such as a small arc lamp or an incandescent gas lamp—is needed, together with picked slides of a certain thickness (specified for the particular make of condenser but generally 1 mm.) and specially thin cover-glasses (not more than 0.17 mm.) The objective must not have a higher NA than 1.0, consequently immersion lenses must be fitted with an internal stop to cut down the aperture.

Micrometer.—Some form of micrometer for the purpose of measuring bacteria and other objects is also essential. Details of those in general use will be found in the following pages.



Object Marker (Fig. 54).—This is an exceedingly useful piece of apparatus. Made in the form of an objective, the lenses are replaced by a diamond point, set slightly out of the centre, which can be rotated by means of a milled plate. Screwed on to the nosepiece in place of the objective, rotation of the diamond point will rule a small circle on the object slide to permanently record the position of an interesting portion of the specimen. The diamond is mounted on a spring which regulates the pressure, and the size of the circle can be adjusted by means of a lateral screw.

METHODS OF MICROMETRY.

The unit of length as applied to the measurement of microscopical objects is the one-thousandth part of a millimetre (0.001 mm.), denominated a micron (sometimes, and erroneously, referred to as a micro-millimetre), and indicated in writing by the Greek letter mu. Of the many methods in use for the measurement of bacteria, three only will be here described, viz.:

(a) By means of the Camera Lucida.

(b) By means of the ocular or Eyepiece Micrometer.

(c) By means of the Filar Micrometer (Ramsden's micrometer eyepiece).

For each of these methods a stage micrometer is necessary. This is a 3 by 1 inch glass slip having engraved on it a scale divided to hundredths of a millimetre (0.01 mm.), every tenth line being made longer than the intervening ones, to facilitate counting; and from these engraved lines the measurement in every case is evaluated. A cover-glass is cemented over the scale to protect it from injury.



(a) By means of the Camera Lucida.

1. Attach a camera lucida (of the Wollaston, Beale, or Abbe pattern) (Fig. 55) to the eyepiece of the microscope.

2. Adjust the micrometer on the stage of the microscope and accurately focus the divisions.

3. Project the scale of the stage micrometer on to a piece of paper and with pen or pencil sketch in the magnified image, each division of which corresponds to 10 mu. Mark on the paper the optical combination (ocular objective and tube length) employed to produce this particular magnification.

4. Repeat this procedure for each of the possible combinations of oculars and objectives fitted to the microscope supplied, and carefully preserve the scales thus obtained.

To measure an object by this method simply project the image on to the scale corresponding to the particular optical combination in use at the moment. Read off the number of divisions it occupies and express them as micra.

In place of preserving a scale for each optical combination, the object to be measured and the micrometer scale may be projected and sketched, in turn, on the same piece of paper, taking particular care that the centre of the eyepiece is 25 cm. from the paper on which the divisions are drawn.



(b) By means of the Eyepiece Micrometer.

The eyepiece micrometer is a circular glass disc having engraved on it a scale divided to tenths of a millimetre (0.1 mm.) (Fig. 56), or the entire surface ruled in 0.1 mm. squares (the net micrometer) (Fig. 57). It can be fitted inside the mount of any ocular just above the aperture of the diaphragm and must be adjusted exactly in the focus of the eye lens.

Some makers mount the glass disc together with a circular cover-glass in such a way that when placed in position in any Huyghenian eyepiece of their own manufacture, the scale is exactly in focus for normal vision. Special eyepieces are also obtainable having a sledging adjustment to the eye lens for focussing the micrometer.

The value of one division of the micrometer scale must first be ascertained for each optical combination by the aid of the stage micrometer, thus:

1. Insert the eyepiece micrometer inside the ocular and adjust the stage micrometer on the stage of the microscope.

2. Focus the scale of the stage micrometer accurately; the lines will appear to be immediately below those of the eyepiece micrometer. Make the lines on the two micrometers parallel by rotating the ocular.

3. Make two of the lines on the ocular micrometer coincide with those bounding one division of the stage micrometer; this is effected by increasing or diminishing the tube length; and note the number of included divisions.

4. Calculate the value of each division of the eyepiece micrometer in terms of mu, by means of the following formula:

x = 10 y.

Where x = the number of included divisions of the eyepiece micrometer.

y = the number of included divisions of the stage micrometer.

5. Note the optical combination employed in this experiment and record it with the calculated micrometer value.

Repeat this process for each of the other combinations. Carefully record the results.

To measure an object by this method read off the number of divisions of the eyepiece micrometer it occupies and express the result in micra by a reference to the standard value for the particular optical combination employed.

Zeiss prepares a compensating eyepiece micrometer for use with his apochromatic objectives, the divisions of which are so computed that (with a tube length of 160 mm.) the value of each is equivalent to as many micra as there are millimetres in the focal length of the objective employed.

Wright's Eikonometer is really a modification of the eyepiece micrometer for rapidly measuring microscopical objects by direct inspection, having previously determined the magnifying power of the particular optical combination employed. It is a small piece of apparatus resembling an eyepiece, with a sliding eye lens, which can be accurately focussed on a micrometer scale fixed within the instrument. When placed over the microscope ocular the divisions of this scale measure the actual size of the virtual image in millimetres.

In order to use this instrument for direct measurement, it is first necessary to determine the magnifying power of each combination of ocular, tube length and objective.

Place a stage micrometer divided into hundredths of a millimetre on the microscope stage and focus accurately.

Rest the eikonometer on the eyepiece. Observation through the eikonometer shows its micrometer scale superposed on the image of the stage micrometer.

Rotate the eikonometer until the lines on the two scales are parallel, and make the various adjustments to ensure that two lines on the eikonometer scale coincide with two lines on the stage micrometer.

For the sake of illustration it may be assumed that five of the divisions on the stage micrometer accurately fill one of the divisions of the eikonometer scale; this indicates a magnifying power of 500 as the constant for that particular optical combination, and a record should be made of the fact.

The magnification constants of the various other optical combinations should be similarly made and recorded.

To measure any object subsequently it should be first focussed carefully in the ordinary way.

The eikonometer should then be applied to the eyepiece and the size of the object read off on the eikonometer scale as millimetres, and the actual size calculated by dividing the observed size by the magnification constant for the particular optical combination employed in the observation.

(c) By means of the filar micrometer.



The Filar or cobweb Micrometer (Ramsden's micrometer) eyepiece (Fig. 58) consists of an ocular having a fine "fixed" wire stretching horizontally across the field (Fig. 59), a vertical reference wire—fixed—adjusted at right angles to the first; and a fine wire, parallel to the reference wire, which can be moved across the field by the action of a micrometer screw; the drum head is divided into one hundred parts, which successively pass a fixed index as the head is turned. In the lower part of the field is a comb with the intervals between its teeth corresponding to one complete revolution of this screw-head.

As in the previous method, the value of each division of the micrometer scale (i. e., the comb) must first be determined for each optical combination. This is effected as follows:

1. Place the filar micrometer and the stage micrometer in their respective positions.

2. Rotate the screw of the filar micrometer until the movable wire coincides with the fixed one, and the index marks zero on the drum head. (If when the drum head is at zero the two wires do not exactly coincide they must be adjusted by loosening the drum screw and resetting the drum.)

3. Focus the scale of each micrometer accurately, and make the lines on them parallel.

4. Rotate the head of the micrometer screw until the movable line has transversed one division of the stage micrometer. Note the number of complete revolutions (by means of the recording comb) and the fractions of a revolution (by means of scale on the head of the micrometer screw), which are required to measure the 0.01 mm.

5. Make several such estimations and average the results.

6. Note the optical combination employed in this experiment and record it carefully, together with the micrometer value in terms of mu.

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