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° C. (for the sterilisation of certain albuminous fluids).

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

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

4. Superheated steam at 115° C. or 120° 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:

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.

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 76×88×163 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.

Hot Air.—Hot air at 150° C. destroys all bacteria, spores, etc:, in about thirty minutes; a momentary exposure to a temperature of 175° to 180° 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° or 250°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°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.

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°C.

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

4. Run the temperature up to 175°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°C.—but not before—the door may be opened and the sterile articles removed and stored away.

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° 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° C. and retained at that temperature for about thirty minutes.

Moist Heat.—Water at 56° 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° 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° C).

3. After thirty minutes' exposure to 56° 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° 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° C. in a water-bath for half an hour on each of six successive days is termed fractional sterilisation.

Water at 100°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°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.

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° C. for twenty minutes on each of three consecutive days is termed discontinuous or intermittent sterilisation.

Exposure to steam at 100° 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° 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° 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° 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.