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° 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° 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 76×26×26 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° 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:
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 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.
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° 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.
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° 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°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.
Note.—Neglect of this precautionary cooling of the oven to 60° 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° 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.
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° 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.