Bacteriological technology for physicians ; with seventy-two figures in the text

Author's Preface

THE first edition of this text-book was issued in 1885, as a reprint from the " Nordiskt Medicinskt Arkiv." The increased size of this new edition is partly due to the addition of an introduction to the microscopic investigation of bacteria; but the active development of bacteriology in the last few years has also necessitated a re-elaboration and extension of all of the chapters.

In the work, I have had two objects in view: the preparation of an outline adapted to bacteriological courses for physicians and veterinary surgeons, and a guide for those who are obliged to take up the subject at home without the assistance of an instructor, yet wish to carry out for themselves the fundamental experiments which are most important for pathology and hygiene. I have, therefore, not attempted to give an exhaustive presentation of the entire technology of the subject, but rather to describe the simplest and most easily managed apparatus and methods, so that the equipment of a home laboratory need not appear too expensive; while I have tried to describe these in a sufficiently elementary and detailed manner.

Those who wish a more comprehensive treatise, are referred to the last edition of Hueppe's " Methoden der Bakterienf orschung " (Wiesbaden, 1889).

C. J. S.

TRANSLATOR'S PREFACE.

So many works on bacteriology are now accessible, that anew one must possess decided merit to justify its appearance. The scope of the present little volume is such as to satisfy me that it should fill a place as yet vacant ; and the careful treatment of the subject gives rise to the hope that it may find a larger field of usefulness in the English language than could be possible while it was confined to the less-used Danish.

The physician who wishes to read a well-written account of the more important pathogenic bacteria, is further referred to Fraenkel's " Bakterienkunde." The fullest and most reliable attempt at a bacteriological flora, is to be found in Fluegge's " Ferment e und Mikroparasiten," which contains much additional information.

W. T.

BACTERIOLOGICAL TECHNOLOGY.

Chapter I. Sterilization

BACTERIOLOGICAL technology must in the first place put means in our hands for separating- the different kinds of bacteria from one another, and cultivating- each species in a state of complete purity. Pure cultivation is a necessary condition for a trustworthy study of the morphology and physiology of these microscopic organisms. Unless one has this constantly in mind during his work, he is certain to end in sad confusion; and the short history of bacteriology is only too full of worthless investigations and wrong- results, which are to be attributed to the untrustworthiness of the methods employed.

It is well known that the pure cultivation of a single kind of bacteria is surrounded by quite peculiar difficulties, due to the extreme smallness, the large numbers, and the extraordinary distribution, one might almost say omnipresence, of bacteria. Everything which is allowed to stand uncovered in the air for a short time is likely to receive spores capable of g-ermination, which fall on its surface with the dust. Every contact with hands or clothing- is attended with the same dang-er. We must learn first of all to meet and control this accidental and invisible source of contamination, for it is self-evident that all vessels, instruments, fluids, etc., which are to be used in cultures must be clean, not only in the sense in which chemists use the word, but also in the bacteriological sense, or sterile, that is, free from living germs.

Heat is the most generally used means of sterilization, but 1 it must be used in different ways, and of different degrees, according* to the nature of the material to be sterilized, a platinum needle, for example, is best sterilized by heating it to incandescence directly in the flame; but a knife blade, or a pair of scissors, can clearly not be heated to this point. These can only be " flamed/' that is, drawn slowly back and forth through the flame, until one is sure that all of their surface has been heated to at least 200° C., so that any germs have been burned.

But it is only exceptionally that glowing or flaming can be employed. If one has to deal with glass-ware, metallic articles, cotton, paper, etc., these are most frequently sterilized in air heated to about 150° C. In the preparation of most fluid and solid culture-media, one can most rapidly and surely reach the desired end by the help of a Papin digester, an " autoclave," in which the sterilization is effected by steam under pressure, at 110 to 120° C.; or one contents himself with using the boiling temperature, either by directly cooking the substance, or by heating it to 100° C., or about that point, by the aid of streaming steam. Occasionally, however, one may have to use a temperature lower than this (60 to 70° C.), to kill germs, for instance where serum is used. In some cases filtration and disinfecting solutions are used as means of sterilizing.

The entire collection of instruments needed for sterilizing, the apparatus and culture-media to be used in the investigations that we shall describe, are, in addition to a common Bunsen gas burner (or alcohol lamp), the following :

1. A sterilizing oven in which articles made of glass and metal, cotton, paper, etc., are heated to about 150° C., by means of hot air.

For this purpose one uses in the laboratory a double-walled sheet-iron box 30 cm. high, 35 cm. broad, and 23 cm. deep, with a tube in the top for inserting a thermometer, a door in the side, and with a loose middle piece in the bottom against which the large gas jet constantly plays, and which consequently will burn through after long use, when it is only necessary to put in a new piece of iron.

One can usually get along with less expense by making a serviceable sterilizing oven (Figs. 1, 2) of one of the common cracker boxes (about 24 cm. high, 22 cm. wide, and 24 cm.

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deep) to be obtained cheaply of any grocer. In the cover a round hole is to be cut in which a bored cork is fitted to carry a thermometer, registering- to 200° C. For ventilation, a small hole is punched in each of the four side-walls close to the top and bottom. In the box is set a four-sided piece of strong sheet-iron, with the sides bent down so as to hold it about 2 cm. from the bottom, so that the objects to be sterilized will not come into immediate contact with the latter. The box is inclosed in a substance which is a poor conductor of heat, felt being usually chosen. One piece covers the lid, and is perfor-

FlG. 1.

FIG. 2.

FIGS. 1 and 2.— Oven for sterilization at 150° C., formed of a cracker box surrounded with felt and provided with thermometer and support of iron netting.

ated for the passage of the cork and thermometer. Another piece is chosen long enough to be wrapped around the four sides of the box and wide enough to cover the upper three quarters of these, excepting- a small space where the air-holes occur. The lowermost quarter is best left uncovered to avoid scorching- or burning the felt. This strip of felt is fastened simply by tying around it tightly a few pieces of cord or wire. The box is set on one or two of the common iron tripods, such as are used in chemical laboratories, and is heated by one or more gas burners. Between these and the bottom of the box, a loose piece of sheet-tin can be placed to prevent burning through the bottom. If gas is not at hand, a'small oil stove with one or two burners can be used.

In comparison with the larger and more expensive ovens provided with ventilators and t her mo -regulator, this primitive apparatus offers some inconveniences, but these do not count for much in comparison with its perfect usefulness and extreme cheapness.

Care must be taken to guard the sterilized objects from fresh contamination when they are taken out of the sterilizer. Vessels which are guarded from the penetration of germs, by cotton plugs or otherwise, when it is not especially important to keep the outside free from germs (e.g., flasks and test-tubes) can simply be taken from the oven after they have been heated to 150° C. for half an hour or an hour, and set aside without any other precaution than covering or wrapping them up in paper to prevent them from becoming very dusty. But objects which must be perfectly sterilized, such as watch-glasses, slides, large glass plates, etc., must be carefully wrapped in a couple of thicknesses of thin, firm paper before they are put in the oven. Germs from the air cannot pass through this, and the objects can thus be kept satisfactorily sterile for a long time. It is well to wrap each object in several layers of thin paper, and then to wrap these in larger parcels.

Paper and cotton should be kept as far as possible from the bottom of the oven to prevent them from becoming charred by long heating. Paper and cotton (but not absorbent cotton) assume a yellowish-brown color after being kept at from 140° to 150° C. for a long time, and this discoloration consequently shows that the heat in the sterilizing oven has been sufficient even without the testimony of a thermometer.

2. The Steam Sterilizer of Koch. — Sterilization at the boiling temperature can evidently be effected by cooking in water, but since this method is not only inapplicable in many cases, but always more laborious and unreliable than sterilization by streaming steam as introduced by Koch, the latter is now the principal method used when it is desired to sterilize at 100° C.

It will be remembered that two advantages are gained by this: the heat penetrates rapidly into the objects to be sterilized, and steam of this temperature (99° to 100° C. according to altitude) sterilizes much more rapidly than atmospheric air

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of the same or a much higher temperature, probably for chemical reasons.

The partly diagrammatic illustration shows the Koch steam apparatus of a simple and cheap form. It consists of a tin

FIG. 3.

FIG. 4.

FIGS. 3 and 4.— Koch cylinder in its simplest form, with two tin shelves and pails. A, Hook for suspending long objects ; #, tin pail with sieve-bottom, containing plugged test-tubes ; C, tin cylinder for elongating the apparatus.

cylinder 50 cm. high and 17 cm. in diameter, and is provided with a small glass tube (a) fastened by rubber tubing in which the height of the water can be seen from the outside. The absolute size, as well as the proportion between the height and diameter, can naturally be varied within wide limits and the distance between the shelves is to be adapted to the height of the apparatus, etc., that are to be most frequently sterilized. The bottom, which is to be exposed to the action of the flame, is preferably made of somewhat thicker tin than the sides. The top of the apparatus is closed by a conical lid (b) the centre of which is pierced by a short tube (c) for the reception of a cork and thermometer. The lid and the side walls, to within 9 cm. of the bottom, are usually covered with felt, though this is not absolutely necessary. Within the cylinder, at a distance of 14 and 30 cm. from the bottom, are placed projecting1 ledges, on which can be placed two loosely fitting shelves of heavy tin perforated with large holes (del). These serve to support tin pails (ee) about 12 cm. high and 14 cm. in diameter, the bottom of which is best made of widemeshed galvanized iron netting (Fig. 4, B). These hold the objects to be sterilized, e.g., test-tubes plugged with cotton in the figure. When it is to be used, the apparatus is placed on a tripod (/) of stout strap-iron. The pails are taken out and the cylinder is filled with water to a depth of about 10 cm. The pails with their contents having been set in and the lid with its thermometer put in place, the water is heated to the boiling point. After some time steam will escape freely around the loosely-fitting lid, and the thermometer will also show about 100° C., after which the objects are left in this streaming steam for a longer or shorter time (as a rule, twenty minutes), the time being counted from the moment when the thermometer indicates 100° C. (or the steam begins to escape freely). Most objects which are to be sterilized by steam can be set on the tin shelves without difficulty. Longer objects can be hung in the apparatus after the pails and shelves have been removed. This is most easily done by using a piece of stout brass wire, bent and brought into the opening of the lid (Fig. 4, A).

The apparatus can be lengthened when this is necessary by using a tin cylinder, with a projecting collar near its base (Fig. 4, D C) to be inserted between the lid and the top of the cylinder. A stop-cock, near the bottom, for drawing off the water, increases the expense of the apparatus somewhat, but adds to its convenience. Moreover, one can easily form such a steam sterilizer from a common kitchen pot. It is only neces-

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sary to fit to it a tin cylinder open at the bottom, and furnished with a lid and felt covering1, but it is essential to provide this cylinder with a projecting- collar at a couple of centimetres from the bottom, by which it rests on the edge of the pot; and it is best to provide it with a couple of rings, by which the cylinder may be tied to the ears of the pot.

A word of explanation should be offered for the omission of the Papin digester in speaking- of necessary apparatus for sterilization. There are, indeed, bacillus spores which are able to survive a cooking for several hours in water or free steam, while they are killed after a few moments' heating- to 120° C. in the digester, consequently this is the only apparatus in which one can rapidly and surely effect sterilization by a single attempt. Still it is a very expensive piece of apparatus, which is the only reason that it has been omitted. Koch's steam cylinder, on the other hand, is very simple, cheap, and efficient when it is used with care. ' As a rule the spores of bacilli which always get into our culture media, from water, the air, etc., are not so resistant as those which have been mentioned. Generally, streaming1 steam at 100° C. will kill them, and experiments from a large number of bacteriological laboratories have long since" shown the adequac}^ of Koch's apparatus. However, in the preparation of his culture apparatus one must always have in mind that some very resistant form may have escaped destruction in the steam, and must make sure of absolute freedom from germs in his culture media by:

a. Cleanliness in the preparation. It is not sufficient to trust to the final sterilization of the materials, but at every step of the preparation one should work cleanly, to lessen the chances of resistant g-erms entering.

b. Discontinuous Heating. — This method was first applied by Tyndall in 1877 for the sterilization of hay infusion, which is known to contain some of the most resistant bacillus spores, the extraordinary vitality of which renders it impossible to sterilize this substance even by cooking- it for some minutes (c/. Chapter IV., Section 2). Tyndall reached his method of sterilizing- by the following train of thought : When a hay infusion has been boiled for some minutes all of the rod -shaped bacilli, unable to endure this treatment, are dead, while the spores of bacilli (more exactly, of some of them) are still alive.

Bacteriological Technology.

If now all of these surviving- spores are given time to germinate and the infusion is again cooked, it will be completely sterilized, because, when it is heated it contains no spores but only rods. However, as one cannot be sure that all spores have germinated at the same time, this process must usually be repeated several times. Hence it is recommended to submit the filled culture apparatus to steaming twice, with a day intervening. This is especially important for those culture media which, like gelatin, bear only a short cooking.

c. Waiting and Watching. — For greater certainty, the materials treated in this way are allowed to stand for some time before they are used, so that one may be sure that they contain nothing capable of germination. If they are placed in the thermostat at about 30° C., a delay of a couple of days may be looked on as sufficient.

3. Water Bath for Sterilizing at Temperatures below the Boiling Point. — Various fluids will not bear being sterilized at 100° C., because at so high a temperature they undergo chemical changes which it is wished to avoid for one reason or another. In such cases the short exposure to a high temperature can be replaced by long maintenance at a lower temperature, until no germ capable of growth remains in the fluids. This method was discovered by Pasteur, and by his advice applied on a large scale in the fabrication of wine (" Pasteurization ") to secure sterilization of the wines without at the same time injuring their quality. In what follows we shall make use of the preparation of blood serum according to Koch's directions. Rather complicated and expensive apparatus is often used for this, but one can get along very well with a simple cylindrical water-bath made of tin, such as Koch himself used in his first preparations. The tin receptacle (Fig. 5) is 22 cm. high, and 13 cm. in diameter. At the top it is provided with a collar 1.5 cm. broad. A piece of fine-meshed flexible galvanized iron netting (a) serves as a lid, and is fastened by bending its edges under the collar. The temperature of the water is read off on the thermometer (6) which projects through a hole in the lid. The bulb of the thermometer is held up several centimetres above the bottom of the cylinder, by another loose piece of wire cloth (c) resting upon its edges, which are bent down; when the water has reached a suitable temperature, usually 60° to 70° C., it is easy to hold it at about

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the same point for a long time, e.g., a couple of hours, "by the use of a small gas or alcohol flame. Though a range of a few degrees can usually be allowed, it is best to keep the temperature under observation. Further directions for the preparation of serum are given later.

4. Porcelain Filters. — Even a slight heating often causes in the culture fluid chemical changes which it may be important to avoid. In such cases the germs have been removed by filtration through burnt clay (Klebs), plaster (Pasteur and Miquel), porcelain (Chamberland), or sheet asbestos (Hesse),

FIG. 6.

Fio. 5.— Water-bath for Sterilization at Lower Temperatures.

FIG. G. — A, Chamberland Filter (X 1/6); B, bulb-pipette for collecting the contents of the filter; E, small porcelain filter (x 1 /6); F, reservoir, which can also be used as a culture-vessel; C and D are explained in the text.

which keep back all of the cells contained in the fluid. As filtration plays a prominent part in the separation of bacteria from their soluble products, we shall here dwell somewhat more fully on the use of

Ckamberland's Filter.— This consists of a hollow porcelain cylinder (Fig. 6 A\ which is closed at the bottom and at the upper end provided with a funnel-shaped glazed end-piece, over which a rubber tube can be pushed. The filter is immersed in the fluid to be sterilized and the upper (open) end is connected with some kind of aspirator. If the fluid to be fil-

Bacteriological Technology.

tered is in too great quantity to be contained in the cavity of the filter, another receptacle is inserted between the aspirator and filter. An ordinary wash-bottle (Fig-. ?) can be used for this purpose; and when the fluid to be sterilized is not too hard to filter, a common bottle aspirator (Fig-. 7, a) can be employed. The details of the process are then as follows: The open end of the filter is closed by a plug- of cotton batting-. It is then wrapped in paper and sterilized at 150° C. The open ends of both tubes of the wash-bottle are plugged with cotton, wrapped in paper and sterilized with the filter, as is the plugged wash-bottle. The rubber stopper of the latter (bored

FIG. 7.— Chamberland Filter set up for use, with Aspirator and Wash-bottle Reservoir.

with two holes for the tubes), and the two pieces of firm-walled rubber tubing- for connecting the flask with the filter and the aspirator, must be sterilized in some other manner. They are first laid for fifteen minutes in a -^ per cent sublimate solution, rinsed in sterilized distilled water, plug-g-ed with cotton that has been previously sterilized by dry heat, and after wrapping- in paper they are steamed for fifteen minutes. After this has been done, the filter is put together as rapidly as possible, and with the ulmost cleanliness, the fingers being first washed in sublimate. The glass tubes are fixed in the rubber stopper, and this is fitted to the wash-bottle after removing the plug from the latter with sterilized forceps. The long tube is then joined to the filter by one piece of rubber tubingafter removal of its cotton plug1. The only plug- left in place is that at * of Figure 7. The entire apparatus is now steamed for ten minutes, after which it can be used. For additional safety, it is usually customary after the last sterilization to bind the rubber tubing1 fast to the filter and glass tubes by cord, or better still by previously glowed coarse brass wire, which is tightly wound by the use of pliers.

If instead of the common wash-bottle, a so-called Pasteur flask, made entirely of glass, is used (cf. Chapter XL), the preparations for filtering1 are very much simplified. It is also evident that if an autoclave is at hand the preparatory sterilization, as well as the rapid and careful setting- up of the apparatus can be dispensed with. Without any especial precautions the glass and rubber parts can be joined and then sterilized in a few moments at 120° C.

Figure 7 shows the apparatus in use. The filter is immersed in a relatively narrow glass cylinder, which contains the fluid to be filtered, as this sinks in the glass it is tipped so as to use all of the filtering- surface and to prevent air from being sucked through, though this is also sterilized in the passage through the filter. The simple aspirator (a) is sufficient, when one has not to deal with fluids that are hard to filter. The bubbling- of air back through the aspirator, and the consequent hindrance to filtration, is avoided by reg-ulating- the size of the escape tube by a pineh-cock. In other cases, or when the filtering must be done rapidly, more powerful suction is employed, e.g., the small air pump recommended by Chamberland (Fig. 8, a), or a filter-pump worked by water. If the filtration is difficult and a powerful pump is used, care must be taken that the walls of the flask are strong enough to bear the atmospheric pressure. In such cases it is customary to use a thick- walled flask instead of a common wash-bottle, and under these circumstances exceptional use may be made of a lead tube within the rubber tubing. After repeated sterilizingby steam, even thick-walled rubber tubing- becomes too much softened for further use.

When the wash-bottle (b) is filled with the clear filtrate, it is first loosened from the aspirator, then the tube (c) is fastened by a clamp or glass plug, after which the flask can be separated from the filter and its contents kept sterile. The filtrate remaining- in the cavity of the filter can be saved, by pouring1 it into one or more sterile vessels, e.g., culture tubes, as soon as the filter is opened. This is best done by using a pipette (Fig. G, B), plugged at the top with cotton, while the tube below the bulb is long enough to reach the bottom of the filter and slender enough to pass through its opening. Before use, it is hermetically sealed, sterilized, and passed through the flame like an ordinary Pasteur pipette, of which, indeed, it is only an enlarged edition (c/. Fig. 22).

After being used, the Chamberland filter is cleaned according to circumstances, by simply brushing off the surface and washing- it in water, or by the additional use of chemicals, e.g., disinfectants. Occasionally it may also be desirable or necessary to rinse its pores out thoroughly with water, before it is dried and again sterilized. This is most quickly accomplished (Fig. 6, C), in the following way: the filter is filled with water and immersed in a large vessel of water, which is placed quite high, as on a shelf or cupboard. A rubber tube several feet long is attached to its open end. Somewhere in this a glass tube (Fig. 6, D), filled with water is inserted. Before putting together, the filter and tube arc both filled with water.

When one has to do with small quantities of fluid the small thick- walled filter (Fig. 6, E] can be used. This is 15 cm. long, with a cavity 2 mm. in diameter. These were first used in Pasteur's laboratory, inserted in a filtering apparatus of special construction, but they can be used in the same manner as the larger ones, by fastening a rubber tube to the upper end with copper wire. A strong suction apparatus is needed for this. The small bulb (Fig. 6, F) is intended to be used as a receiver of filtered fluid (Fig. 8) and also without special cnange, as a culture vessel, with tubular plug (cf. Chapter IL, p. 16).

It must be remembered that even filtration through porcelain does not always avoid the chemical modification of fluids, since the filter may hold back certain compounds, as well as the gerrns. If organic culture fluids must be absolutely unchanged, and at the same time free from g-erms, it is necessary to do without all sterilization, in which case they are to be collected or prepared with such care that all contaminations from their surroundings are avoided. Blood and urine.

for instance, are drawn directly from the heart, veins, or bladder with antiseptic precautions. The preparation of originally sterile infusions is further briefly discussed in Chapter XI. 5. Disinfecting Solutions. — As a help in sterilization, it is usual to have at hand a large quantity of corrosive sublimate solution (1 or 2 : 1,000), which is not only used in preparing some sorts of culture media, e.g.,. potatoes (see Chapter III.), but in many other cases, for cleansing the hands, instruments, morbid material, etc. If well water is used instead of distilled water in making this solution, care must be taken that no precipitate of insoluble mercurial salts is formed, or the fluid

FIG. 8.— Small Porcelain Filter joined to Bulb-reservoir and Air-pump (a).

will possess no disinfecting value. To prevent this, a small quantity of acetic acid may be added to the well water. Fiirbringer recommends 0.5 gram to each litre of 0.1 per cent solution. Carbolic acid solutions of various strengths are also to be kept ready for use.