the cloth with a few turns of copper wire, and place inside the muffle
(a small muffle 76x88x163 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 deg. C. destroys all bacteria, spores, etc:, in
about thirty minutes; a momentary exposure to a temperature of 175 deg. to
180 deg. 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 deg. or 250 deg. 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 deg. 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.
[Illustration: 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 deg. 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 deg. C.
3. When the temperature of the oven has reached 100 deg. C., close the
ventilating slide; reset the alarm to ring at 175 deg. C.
4. Run the temperature up to 175 deg. 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 deg. 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 deg. 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 deg.
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 deg. C. and retained at that temperature for
about thirty minutes.
~Moist Heat.~--_Water at 56 deg. 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
deg. 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 deg. C).
3. After thirty minutes' exposure to 56 deg. 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 deg. 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 deg. C. in a
water-bath for half an hour on each of six successive days is termed
_fractional sterilisation_.
_Water at 100 deg. 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).
[Illustration: 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.
2. Pour a sufficient quantity of water into the steriliser, shut down
the lid, and light the gas below.
[Illustration: FIG. 28.--Koch's steriliser.]
[Illustration: 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 100 deg. 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 deg. C.
for twenty minutes on each of three consecutive days is termed
_discontinuous_ or _intermittent sterilisation_.
Exposure to steam at 100 deg. 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 deg. 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 deg. 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 deg.
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.
[Illustration: FIG. 30.--Chamberland's Autoclave.]
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.
[Illustration: 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 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.
[Illustration: 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 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.
[Illustration: 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 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.
In this method the bacteria are retained in the pores of the filter
while the liquid passes through in a germ-free condition.
It is obvious that to be effective the pores of the filter must be
extremely minute, and therefore the rate of filtration will usually be
slow. Chamberland filter candles possess finer channels than Berkefeld
candles and consequently filter much more slowly. To overcome this
disadvantage, either aspiration or pressure, or a combination of these
two forces, may be employed to hasten the process.
Doultons white porcelain filters it may be noted are as efficient as the
Chamberland candles and filter rather more rapidly.
_Apparatus Required._--
1. Separatory funnel containing the unfiltered fluid.
2. Sterile filter candle (Fig. 34), the open end fitted with a rubber
stopper (Fig. 34, a) perforated to receive the delivery tube of the
separatory funnel, and its neck passed through a large rubber washer
(Fig. 34, b) which fits the mouth of the filter flask.
3. Sterile filter flask of suitable size, for the reception of the
filtered fluid, its mouth closed by a cotton-wool plug.
4. Water injector Sprengel (see Fig. 38, c) pump, or Geryk's pump (an
air pump on the hydraulic principle, sealed by means of low
vapor-tension oil, Fig. 35).
If this latter is employed, a Wulff's bottle, fitted as a wash-bottle
and containing sulphuric acid, must be interposed between the filter
flask and the pump, in order to prevent moist air reaching the oil in
the pump.
5. Air filter (_vide_ page 40) sterilised.
6. Pressure tubing.
7. Screw clamps (Fig. 36).
METHOD.--
1. Couple the exhaust pipe of the suction pump with the lateral tube of
the filter flask (first removing the cotton-wool plug from this latter),
by means of pressure tubing, interposing, if necessary, the wash-bottle
of sulphuric acid.
[Illustration: FIG. 34.--Porcelain filter candle.]
[Illustration: FIG. 35.--Geryk air pump.]
2. Remove the cotton-wool plug from the neck of the filter flask and
adjust the porcelain candle in its place.
[Illustration: FIG. 36.--Screw clamps.]
3. Attach the nozzle of the separatory funnel to the filter candle by
means of the perforated rubber stopper (Fig. 37).
[Illustration: FIG. 37.--Apparatus arranged for filtering--aspiration.]
4. Open the tap of the funnel, and exhaust the air from the filter flask
and wash-bottle; maintain the vacuum until the filtration is complete.
5. When the filtration is completed close the tap of the funnel; adjust
a screw clamp to the pressure tubing attached to the lateral branch of
the filter flask; screw it up tightly, and disconnect the acid
wash-bottle.
6. Attach the air filter to the open end of the pressure tubing; open
the screw clamp gradually, and allow filtered air to enter the flask, to
abolish the negative pressure.
7. Detach the rubber tubing from the lateral branch of the flask, flame
the end of the branch in the Bunsen, and plug its orifice with sterile
cotton-wool.
8. Remove the filter candle from the mouth of the flask, flame the
mouth, and plug the neck with sterile cotton-wool.
9. Disinfect the filter candle and separatory funnel by boiling.
If it is found necessary to employ pressure in addition to or in place
of suction, insert a perforated rubber stopper into the mouth of the
separatory funnel and secure in position with copper wire; next fit a
piece of glass tubing through the stopper, and connect the external
orifice with an air-pressure pump of some kind (an ordinary foot pump
such as is employed for inflating bicycle tyres is one of the most
generally useful, for this purpose) or with a cylinder of compressed air
or other gas.
In order to filter a large bulk of fluid very rapidly it is necessary to
use a higher pressure than glass would stand, and in these cases the
metal receptacle designed by Pakes (Fig. 38, a), to hold the filter
candle itself as well as the fluid to be filtered, should be employed.
(A vacuum must also be maintained in the filter flask, by means of an
exhaust pump, during the entire process.)
This piece of apparatus consists of a brass cylinder, capacity 2500
c.c., with two shoulders; and an opening in the neck at each end,
provided with screw threads.
A nut carrying a pressure gauge fits into the top screw; and into the
bottom is fitted a brass cylinder carrying the filter candle and
prolonged downwards into a delivery tube. Leakage is prevented by means
of rubber washers.
Into the top shoulder a tube is inserted, bent at right angles and
provided with a tap. All the brass-work is tinned inside (Fig. 38, a).
In use the reservoir is generally mounted on a tripod stand.
~To Sterilise.~--
1. Insert the filter candle into its cylinder and screw this loosely on.
[Illustration: FIG. 38.--Pakes' filtering reservoir--pressure and
aspiration.]
2. Wrap a layer of cotton-wool around the delivery tube and fasten in
position.