n/10 NaOH to neutralise, and therefore 1000 c.c. require the addition of
226 c.c. n/10 NaOH, or 22.60 c.c. n/1 NaOH, or 2.26 c.c. n/10 NaOH.
Initial _titre_ of the bouillon = +22.6, and as such requires the
addition of (22.6 c.c. - 10 c.c.) = 12.6 c.c. of n/1 NaOH per litre to
leave its finished reaction +10.
But the three titrations, each on 25 c.c. of medium, have reduced the
original bulk of bouillon to (1000 - 75 c.c.) = 925 c.c. The amount of
n/1 NaOH required to render the reaction of this quantity of medium +10
may be deduced thus:
1000 c.c.:925 c.c.::12.6 c.c.:x.
Then x = 11.65 c.c. n/1 NaOH.
Whenever possible, however, the required reaction is produced by the
addition of dekanormal soda solution, on account of the minute increase
it causes in the bulk, and the consequent insignificant disturbance of
the percentage composition of the medium. By means of a pipette
graduated to 0.01 c.c. it is possible to deliver very small quantities;
but if the calculated amount runs into thousandth parts of a cubic
centimetre, these are replaced by corresponding quantities of normal or
even decinormal soda.
In the above example it is necessary to add 11.65 c.c. normal NaOH or
its equivalent, 1.165 c.c. dekanormal NaOH. The first being too bulky a
quantity, and the second inconveniently small for exact measurement, the
total weight of soda is obtained by substituting 1.16 c.c. dekanormal
soda solution, and either 0.05 c.c. of normal soda solution or 0.5 c.c.
of decinormal soda solution.
~Standardising Nutrient Agar and Gelatine.~--The method of standardising
agar and gelatine is precisely similar to that described under bouillon.
THE FILTRATION OF MEDIA.
~Fluid media~ are usually filtered through stout Swedish filter paper
(occasionally through a porcelain filter candle), and in order to
accelerate the rate of filtration the filter paper should be folded in
that form which is known as the "physiological filter," not in the
ordinary "quadrant" shape, as by this means a large surface is available
for filtration and a smaller area in contact with the glass funnel
supporting it.
To fold the filter proceed thus:
1. Take a circular piece of filter paper and fold it exactly through its
centre to form a semicircle (Fig. 100, a).
2. Fold the semicircle exactly in half to form a quadrant; make the
crease 2, distinct by running the thumbnail along it, then open the
filter out to a semicircle again.
3. Fold each end of the semicircle in to the centre and so form another
quadrant; smooth down the two new creases 3 and 3a, thus formed and
again open out to a semicircle.
4. The semicircle now appears as in figure 100, a, the dark lines
indicating the creases already formed.
5. Fold the point 1 over to the point 3, and 1a to 3a, to form the
creases 4 and 4a, indicated in the diagram by the light lines. Fold
point 1 over to 3a, and 1a to 3, to form the creases 5 and 5a.
[Illustration: FIG. 100.--Filter folding: a, Filter folded in half,
showing creases; b, appearance of filter on completion of folding;
c, filter opened out ready for use.]
6. Thus far the creases have all been made on the same side of the
paper. Now subdivide each of the eight sectors by a crease through its
centre on the opposite side of the paper, indicated by the faint broken
lines in the diagram. Fold up the filter gradually as each crease is
made, and when finished the filter has assumed the shape of a wedge, as
in figure 100, b.
When opened out the filter assumes the shape represented in figure 100,
c.
The folded filter is next placed inside a glass funnel supported on a
retort stand, and moistened with hot distilled water before the
filtration of the medium is commenced.
~Liquefiable solid media~ are filtered through a specially made filter
paper--"papier Chardin"--which is sold in boxes of twenty-five
ready-folded filters.
[Illustration: FIG. 101.--Hot-water filter funnel and ring burner.]
Gelatine, when properly made, filters through this paper as quickly as
bouillon does through the Swedish filter paper, and does _not_ require
the use of the hot-water funnel.
Agar, likewise, if properly made, filters readily, although not at so
rapid a rate as gelatine. If badly "egged," and also during the winter
months, it is necessary to surround the glass funnel, in which the
filtration of the agar is carried on, by a hot-water jacket. This is
done by placing the glass funnel inside a double-walled copper
funnel--the space between the walls being filled with water at about
90 deg. C.--and supporting the latter on a ring gas burner fixed to a
retort stand (Fig. 101). The gas is lighted and the water jacket
maintained at a high temperature until filtration is completed. If the
steam steriliser of the laboratory is sufficiently large, it is sometimes
more convenient to place the flask and filtering funnel bodily inside,
close the steriliser and allow filtration to proceed in an atmosphere of
live steam, than to use the gas ring and hot-water funnel.
STORING MEDIA IN BULK.
After filtration fill the medium into sterile litre flasks with
cotton-wool plugs and sterilise in the steamer for twenty minutes on
each of three consecutive days. After the third sterilisation, and when
the flasks and contents are cool, cut off the top of the cotton-wool
plug square with the mouth of the flask; push the plug a short distance
down into the neck of the flask and fill in with melted paraffin wax to
the level of the mouth. When the wax has set the flasks are stored in a
cool dark cupboard for future use.
[Illustration: FIG. 102.--Rubber cap closing store bottle. a, before,
and b, after sterilizing.]
This plan is not absolutely satisfactory, although very generally
employed on occasion, and it is preferable to fill the medium into
long-necked flint glass bottles (the quart size, holding nearly 1000
c.c., such as those in which Pasteurised milk is retailed) and to close
the neck of the bottle by a special rubber cap.[3] This cap is made of
soft rubber, the lower part, dome-shaped with thin walls, being slipped
over the neck of the bottle (Fig. 102, a). The upper part is solid,
but with a sharp clean-cut (made with a cataract or tenotomy knife)
running completely through its axis from the centre of the disc to the
top of the dome. During sterilisation the air in the neck of the bottle,
expanded by the heat, is driven out through the valvular aperture in the
solid portion of the stopper. On removing the bottle from the steam
chamber, the liquid contracts as it cools, and the pressure of the
external air drives the solid piece of rubber down into the neck of the
bottle, and forces together the lips of the slit (Fig. 102, b). Thus
sealed, the bottle will preserve its contents sterile for an indefinite
period without loss from evaporation.
TUBING NUTRIENT MEDIA.
After the final filtration, the nutrient medium is usually "tubed"--_i.
e._, filled into sterile tubes in definite measured quantities, usually
10 c.c. This process is sometimes carried out by means of a large
separator funnel fitted with a "three-way" tap which communicates with a
small graduated tube (capacity 20 c.c. and graduated in cubic
centimetres) attached to the side. The shape of this piece of apparatus,
known as Treskow's funnel, renders it particularly liable to damage. It
is better, therefore, to arrange a less expensive piece of apparatus
which will serve the purpose equally well (Fig. 103).
A Geissler's three-way stop-cock has the tube on one side of the tap
ground obliquely at its extremity, and the tube on the opposite side cut
off within 3 cm. of the tap. The short tube is connected by means of a
perforated rubber cork with a 10 cm. length of stout glass tubing (1.5
cm. bore). The third channel of the three-way tap is connected, by means
of rubber tubing, with the nozzle of an ordinary separator funnel.
Finally, the receiving cylinder above the three-way tap is graduated in
cubic centimetres up to 20, by pouring into it measured quantities of
water and marking the various levels on the outside with a writing
diamond.
Fluid media containing carbohydrates are filled into fermentation tubes
(_vide_ Fig. 21); or into ordinary media tubes which already have
smaller tubes, inverted, inside them (Fig. 104), to collect the products
of growth of gas-forming bacteria. When first filled, the small tubes
float on the surface of the medium after the first sterilisation nearly
all the air is replaced by the medium, and after the final sterilisation
the gas tubes will be submerged and completely filled with the medium.
[Illustration: FIG. 103.--Separatory funnel and three-way tap arranged
for tubing media.]
[Illustration: FIG. 104.--Gas tube (Durham).]
~Storing "Tubed" Media.~--Media after being tubed are best stored by
packing, in the vertical position, in oblong boxes having an internal
measurement of 37 cm. long by 12 cm. wide by 10 cm. deep. Each box (Fig.
105) has a movable partition formed by the vertical face of a weighted
triangular block of wood, sliding free on the bottom (Fig. 105, A); or
by a flat piece of wood sliding in a metal groove in the bottom of the
box, which can be fixed at any spot by tightening the thumbscrew of a
brass guide rod which transfixes the partition (Fig. 105, B). The front
of the box is provided with a handle and a celluloid label for the name
of the contained medium. These boxes are arranged upon shelves in a dark
cupboard--or preferably an iron safe--which should be rendered as nearly
air-tight as possible, and should have the words "media stores" painted
on its doors.
[Illustration: FIG. 105.--Medium box, showing alternative partitions A
and B.]
FOOTNOTES:
[3] This rubber cap has been made for me by the Holborn Surgical
Instrument Co., Thavies Inn, London, W. C.
XI. CULTURE MEDIA.
ORDINARY OR STOCK MEDIA.
~Nutrient Bouillon.~--
1. Measure out double strength meat extract, 500 c.c., into a litre
flask and add 300 c.c. distilled water.
2. Weigh out Witte's peptone, 10 grammes (= 1 per cent.), salt, 5
grammes (= 0.5 per cent.), and mix into a smooth paste with 200 c.c. of
distilled water previously heated to 60 deg. C. (Be careful to leave no
unbroken globular masses of peptone.)
3. Add the peptone emulsion to the meat extract in the flask and heat in
the steamer for forty-five minutes (to completely dissolve the peptone,
and to render the acidity of the meat extract stable).
4. Estimate the reaction of the medium; control the result; render the
reaction of the finished medium +10 (_vide_ page 155).
5. Heat for half an hour in the steamer at 100 deg. C. (to complete the
precipitation of the phosphates, etc.).
6. Filter through Swedish filter paper into a sterile flask.
7. Fill into sterile tubes (10 c.c. in each tube).
8. Sterilise in the steamer for twenty minutes on each of three
consecutive days--i. e., by the discontinuous method (_vide_ page 35).
NOTE.--As an alternative method when neither fresh nor
frozen meat is available nutrient bouillon may be prepared
from a commercial meat extract, as follows:
~Lemco Broth.~--
1. Measure out 250 c.c. distilled water into a litre flask.
2. Weigh out 10 grammes Liebig's Lemco Meat Extract on a
piece of clean filter paper and add to the water in the
flask. Shake the flask well to make an even emulsion of the
meat extract.
3. Weigh out Witte's peptone (10 grammes), salt (5 grammes).
Mix into smooth paste with 100 c.c. distilled water
previously heated to 60 deg. C.
4. Add the peptone salt emulsion to the meat extract
emulsion in the flask and add 650 c.c. distilled water. Heat
in the steamer for forty-five minutes.
5. Standardise the medium and complete as for nutrient
bouillon.
~Nutrient Gelatine.~--
1. Weigh a 2-litre flask on a trip balance (Fig. 106) and note the
weight, or counterpoise carefully.
[Illustration: FIG. 106.--Trip balance.]
An extremely useful counterpoise is a small sheet-brass cylinder about
38 mm. high and 38 mm. in diameter, with a funnel-shaped top and
provided with a side tube by which its contents, fine "dust" shot, may
be emptied out (Fig. 107).
[Illustration: FIG. 107.--Counterpoise; weight when empty, 35 grammes;
when full of dust shot, 200 grammes.]
2. Measure out double strength meat extract, 500 c.c., into the "tared"
flask.
3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and make
into a thick paste with 150 c.c. distilled water; then add the emulsion
to the meat extract in the flask; also add 100 grammes sheet gelatine
cut into small pieces; place the flask in the water-bath and raise to
the boil.
[Illustration: FIG. 108.--Arrangement of steam can and water-bath for
the preparation of media.]
4. Arrange a 5-litre tin can (with copper bottom, such as is used in the
preparation of distilled water) by the side of the water bath, fill the
can with boiling water and place a lighted Bunsen burner under it. Fit a
long safety tube to the neck of the can and also a delivery tube, bent
twice at right angles; adjust the tube to reach to the bottom of the
interior of the flask containing the gelatine, etc. (Fig. 108).
5. Keep the water in the steam can vigourously boiling, and so steam at
100 deg. C, bubbling through the medium mass, for ten minutes, by
which time complete solution of the gelatine is effected. A certain
amount of steam will condense as water in the medium flask during this
process--hence the necessity for the use of double strength meat
extract--but if the water bath is kept boiling this condensation will
not exceed 100 c.c.
6. Weigh the flask and its contents; then (1115[4] grammes + weight of
the flask) minus (weight of the flask and its contents) equals the
weight of water required to make up the bulk to 1 litre. The addition of
the requisite quantity of water is carried out as follows:
In one pan of the trip balance place the counterpoise of the tared flask
(or its equivalent in weights) together with the weights making up the
_calculated medium weight_. In the opposite pan place the flask
containing the medium mass. Now add boiling distilled water from a wash
bottle until the two pans are exactly balanced.
7. Titrate and estimate the reaction of the medium mass; control the
result. Calculate the amount of soda solution required to make the
reaction of the medium mass +10 (i. e., calculate for 1000 c.c., less
the quantity used for the titrations).
8. Add the necessary amount of soda solution and heat in the steamer at
100 deg. C. for twenty minutes, to precipitate the phosphates, etc.
9. Allow the medium mass to cool to 60 deg. C. Well whip the whites of
two eggs, add to the contents of the flask and replace in the steamer at
100 deg. C. for about half an hour (until the egg-albumen has coagulated
and formed large, firm masses floating on and in clear gelatine).
10. Filter through papier Chardin into a sterile flask.
11. Tube in quantities of 10 c.c.
12. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
three consecutive days--i. e., by the discontinuous method.
~Nutrient Agar-agar.~--
1. Weigh a 2-litre flask and note the weight--or counterpoise exactly.
2. Measure out double strength meat extract, 500 c.c., into the "tared"
flask.
3. Weigh out and mix 10 grammes of peptone, 5 grammes of salt, and 20
grammes of powdered agar, and make into a thick paste with 150 c.c.
distilled water, and add to the meat extract in the flask; place the
flask in a water-bath.
4. Arrange the steam can and water-bath as already directed (for the
preparation of gelatine) and figured.
5. Bubble live steam (at 100 deg. C.) through the medium mass, for
twenty-five minutes, by which time complete solution of the agar is
effected.
6. Now weigh the flask and its contents; then (1035[5] grammes + weight
of flask) minus (weight of flask and its contents) equals the weight of
water required to make up the bulk of the medium to 1 litre. Add the
requisite amount (see preparation of gelatine, page 166, step 6).
7. Titrate, and estimate the reaction of the medium mass; control the
result. Calculate the amount of soda solution required to make the
reaction of the medium mass + 10 (i. e., calculated for 1000 c.c.,
less the quantity used for the titrations).
8. Add the necessary amount of soda solution and replace in the steamer
for twenty minutes (to complete the precipitation of the phosphates,
etc.).
9. Allow the medium mass to cool to 60 deg. C. Well whip the whites of
two eggs, add to the contents of the flask, and replace in the steamer at
100 deg. C. for about _one hour_ (until the egg-albumen has coagulated
and formed large, firm masses floating on and in clear agar.)
10. Filter through papier Chardin, by the aid of a hot-water funnel, if
necessary (Fig. 101), into a sterile flask.
11. Tube in quantities of 10 c.c. or 15 c.c.
12. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
three consecutive days--i. e., by the discontinuous method.
~Blood-serum (Inspissated).~--
1. Sterilise cylindrical glass jar (Fig. 109) and its cover by dry heat,
or by washing first with ether and then with alcohol and drying.
2. Collect blood at the slaughter house from ox or sheep in the sterile
cylinder.
3. Allow the vessel to stand for fifteen minutes for the blood to
coagulate. (This must be done before leaving the slaughterhouse,
otherwise the serum will be stained with haemoglobin.)
4. Separate the clot from the sides of the vessel by means of a sterile
glass rod (the yield of serum is much smaller when this is not done),
and place the cylinder in the ice-chest for twenty-four hours.
5. Remove the serum with sterile pipettes, or syphon it off, and fill
into sterile tubes (5 c.c. in each) or flasks.
6. Heat tubes containing serum to 56 deg. C. in a water-bath for half an
hour on each of two successive days.
7. On the third day, heat the tubes, in a sloping position, in a serum
inspissator to about 72 deg. C. (A coagulum is formed at this temperature
which is fairly transparent; above 72 deg. C., a thick turbid coagulum is
formed.)
[Illustration: FIG. 109.--Blood-serum jar with wicker basket for
transport.]
The serum inspissator (Fig. 110) in its simplest form is a double-walled
rectangular copper box, closed in by a loose glass lid, and cased in
felt or asbestos--the space between the walls is filled with water. The
inspissator is supported on adjustable legs so that the serum may be
solidified at any desired "slant," and is heated from below by a Bunsen
burner controlled by a thermo-regulator. The more elaborate forms
resemble the hot-air oven (Fig. 26) in shape and are provided with
adjustable shelves so that any desired obliquity of the serum slope can
be obtained.
8. Place the tubes in the incubator at 37 deg. C. for forty-eight hours
in order to eliminate those that have been contaminated. Store the
remainder in a cool place for future use.
_Alternative Method._
_Steps 1-5 as above._
6. Sterilise the serum by the fractional method--that is, by exposure in
a water-bath to a temperature of 56 deg. C. for half an hour on each of
six consecutive days; store in the fluid condition.
7. Coagulate in the inspissator when needed.
[Illustration: FIG. 110.--Serum inspissator.]
~Serum Water.~--
This forms the basis of many useful media, and is prepared
as follows:
1. Collect blood in the slaughterhouse (see page 168) and
when firmly clotted collect all the expressed serum and
measure in a graduated cylinder.
2. For every 100 c.c. of serum add 300 c.c. distilled water
and mix in a flask.
3. Heat the mixture in the steamer at 100 deg. C. for thirty
minutes. (This destroys any diastatic ferment present in the
serum and partially sterilises the fluid.)
4. Filter if turbid.
5. If not needed at once complete the sterilisation of the
serum water by two subsequent steamings at 100 deg. C. for
twenty minutes at twenty-four hour intervals.
~Citrated Blood Agar. Guy's.~--
1. Kill a small rabbit with chloroform vapour, and nail it out on a
board (as for a necropsy); moisten the hair thoroughly with 2 per cent.
solution of lysol.
2. Sterilise several pairs of forceps, scissors, etc. by boiling.
3. Reflect the skin over the thorax with sterile instruments.
4. Open the thoracic cavity by the aid of a fresh set of sterile
instruments.
5. Open the pericardium with another set of sterile instruments.
6. Sear the surface of the left ventricle with a red-hot iron.
7. Take a sterile capillary pipette (Fig. 13, c); break off the sealed
extremity with a pair of sterile forceps.
8. Steady the heart in a pair of forceps and thrust the point of the
pipette through the wall of the ventricle and through the seared area,
apply suction to the plugged end of the pipette and fill it with blood.
9. Transfer the entire quantity of blood collected from the rabbit's
heart to a small Erlenmeyer flask containing a number of sterile glass
beads and 5 c.c. concentrated sod. citrate solution. (See page 378.)
10. Agitate thoroughly and set aside for a couple of hours.
11. Melt up several tubes of nutrient agar (see page 167) and cool to
42 deg. C.
12. With a sterile 10 c.c. graduated pipette transfer 1 c.c. citrated
blood from the Erlenmeyer flask to each tube of liquefied agar. Rotate
the tube between the hands in order to diffuse the citrated blood evenly
throughout the agar.
13. Place the tubes in a sloping position and allow the medium to set.
14. Place tubes of blood agar for forty-eight hours in the incubator at
37 deg. C. and at the end of that time eliminate any contaminated tubes.
15. Store such tubes as remain sterile for future use.
~Milk.~--
1. Pour 1 litre of fresh cow's or goat's milk into a large separating
funnel, and heat in the steamer at 100 deg. C. for one hour.
2. Remove from the steamer and estimate the reaction of the milk (normal
cows' milk averages +17). If of higher acidity than +20, or lower than
+10, reject this sample of milk and proceed with another supply of milk
from a different source.
Reject milk to which antiseptics have been added as preservatives.
3. Allow the milk to cool, when the fat or cream will rise to the
surface and form a thick layer.
4. Draw off the subnatant fat-free milk into sterile tubes (10 c.c. in
each).
5. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
five successive days.
6. Incubate at 37 deg. C. for forty-eight hours and eliminate any
contaminated tubes. Store the remainder for future use.
~Litmus Milk.~--
1. Prepare milk as described above, sections 1 to 3.
2. Draw off the subnatant fat-free milk into a flask.
3. Add sterile litmus solution, sufficient to colour the milk a deep
lavender.
4. Tube, sterilise, etc., as for milk.
~Nutrose Agar (Eyre).~--
(This is a modification of the well known Drigalski-Conradi medium
originally introduced for the isolation of B. typhosus).
1. Collect 250 c.c. perfectly fresh ox serum (_vide_ Blood Serum, page
168, steps 1 to 5) and add to it 450 c.c. sterile distilled water.
2. Weigh out agar powder, 20 grammes, and emulsify it with 250 c.c. of
the cold serum water.
3. Weigh out
Witte's peptone 10 grammes
Sodium chloride 5 grammes
Nutrose 10 grammes
and dissolve in 200 c.c. of serum water heated to 80 deg. C.
4. Mix the agar emulsion and the peptone-nutrose solution in a "tared"
flask of 2-litre capacity and add a further 100 c.c. serum water.
5. Complete the solution of the various ingredients by bubbling live
steam through the flask as in making nutrient agar.
6. Add further 250 c.c. serum water.
7. Weigh the flask and its contents: then (1045 grammes + weight of
flask) minus (weight of flask and its present contents) = weight of
fluid required to make up the bulk of the medium to 1 litre. Add the
requisite amount of sterile distilled water.
8. Titrate and estimate the reaction of the medium mass. Then
standardise to reaction of +2.5.
9. Clarify with egg, and filter as for nutrient agar. (In clarifying,
after the addition of the egg white the mixture should be in the steamer
for full two hours.)
10. After filtration is complete measure the filtrate, and to every 150
c.c. of the medium add:
Litmus solution (Kahlbaum) 20 c.c.
Krystal violet aqueous solution (1:1000) (B. Hoechst) 1.5 c.c.
Lactose 1.5 grammes
11. Tube in quantities of 15 c.c.