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

[Ill.u.s.tration: FIG. 28.--Koch's steriliser.]

[Ill.u.s.tration: FIG. 29.--Arnold's steriliser.]

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 100C.--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 a.s.surance 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 c.o.c.k, 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 c.o.c.k and light both rings of burners.

5. When steam is issuing in a steady, continuous stream from the vent tube, shut off the vent c.o.c.k 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.

[Ill.u.s.tration: FIG. 30.--Chamberland's Autoclave.]

8. Now open the vent c.o.c.k 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 pa.s.ses 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.

[Ill.u.s.tration: FIG. 31.--Electric signal timing clock.]

~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 gla.s.s-wool, the fibres of which entangle the micro-organisms and prevent their pa.s.sage.

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 pa.s.ses 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.

[Ill.u.s.tration: FIG. 32.--Cotton-wool air filter.]

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 gla.s.s 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.

[Ill.u.s.tration: FIG. 33.--Ruffer's flask.]

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 pa.s.s 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 pa.s.sage 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.