The Elements of Bacteriological Technique Part 5

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 cla.s.s 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 cla.s.s (in order of general utility) belong:

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

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

NOTE.--Tubes, flasks, filter flasks, pipettes, gla.s.s tubing, etc., may be rapidly sterilised, in case of emergency, by was.h.i.+ng, 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-gla.s.ses, 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 a.s.sumed that the apparatus so treated is sterile.

[Ill.u.s.tration: FIG. 25.--m.u.f.fle furnace.]

_m.u.f.fle 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 m.u.f.fle (a small m.u.f.fle 7688163 mm. will hold perhaps four small filter candles).

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

3. Extinguish the gas, and when the m.u.f.fle 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 m.u.f.fle 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 gla.s.s and metallic substances, and the small bulk of cotton-wool comprised in the test-tube plugs, etc. Large ma.s.ses 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 250C., 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., 175C.) 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.

[Ill.u.s.tration: FIG. 26.--Hot-air oven.]

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 100C.

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

4. Run the temperature up to 175C.

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 60C.--_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 alb.u.minous "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 alb.u.minous 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 a.s.sumed 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 100C._ 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 gla.s.s tubing, and other small apparatus, and is effected in what is usually spoken of as the "water steriliser" (Fig. 27).

[Ill.u.s.tration: FIG. 27.--Water sterilizer.]

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.