of three successive days--i. e., by the discontinuous method for three
days.
~Egg Medium (Dorset).~--
1. Prepare 1000 c.c. of a 0.85 per cent. solution of sodium chloride in
a stout 2-litre flask.
2. Sterilise in the autoclave at 120 deg. C. for twenty minutes. Cool
to 20 deg. C.
3. Take 12 fresh eggs; wash the shells first with water then with
undiluted formalin: allow the shells to dry.
4. Break the eggs into a sterile graduated cylinder and measure the
total volume of the mixed whites and yolks. Add one part sterile saline
solution to three parts mixed eggs.
5. Transfer this mixture to a large wide-mouthed stoppered bottle
previously sterilised. Add sterile glass beads and shake thoroughly in a
mechanical shaker for about thirty minutes, or whip with an egg-whisk.
6. Filter through coarse butter muslin into a sterile flask.
NOTE.--A few drops of alcoholic solution of basic fuchsin
(sufficient to give a definite pink colour), or a few drops
of waterproof Chinese ink added to the medium at this stage
facilitates the subsequent "fishing" of colonies.
7. Tube in quantities of 10 c.c.
8. Solidify in the sloping position in the inspissator at 75 deg. C.
for one hour.
9. Place the tubes for forty-eight hours in the incubator at 37 deg. C.,
and eliminate any contaminated tubes.
To prevent drying, 0.5 c.c. glycerine bouillon (see page 209) may be
added to each tube between steps 8 and 9.
10. Cap those tubes of media which remain sterile with india-rubber caps
and store for future use.
~Potato.~--
1. Choose fairly large potatoes, wash them well, and scrub the peel with
a stiff nail-brush.
2. Peel and take out the eyes.
3. Remove cylinders from the longest diameter of each potato by means of
an apple-corer or a large cork-borer (i. e., one of about 1.4 cm.
diameter).
The reaction of the fresh potato is strongly acid to phenolphthalein.
If, therefore, the potatoes are required to approximate +10, as for the
cultivation of some of the vibrios, the cylinders should be soaked in a
1 per cent. solution of sodium carbonate for thirty minutes.
4. Cut each cylinder obliquely from end to end, forming two wedge-shaped
portions.
5. Place a small piece of sterilised cotton-wool, moistened with sterile
water, at the bottom of a sterile test-tube; insert the potato wedge
into the tube so that its base rests upon the cotton-wool. Now plug the
tube with cotton-wool (Fig. 111).
6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
_five_ consecutive days.
[Illustration: FIG. 111.--Potato tube.]
NOTE.--The cork borer reserved for cutting the potato
cylinders should be silver electro-plated both inside and
out, and the knife used for dividing the cylinders should be
of silver or silver plated. When these precautions are
adopted the potato wedges will retain their white color and
will not show the discoloration so often observed when steel
instruments are employed.
~Beer Wort.~--Wort is chiefly used as a medium for the cultivation of
yeasts, moulds, etc., both in its fluid form and also when made solid by
the addition of gelatine or agar. The wort is prepared as follows:
1. Weigh out 250 grammes crushed malt and place in a 2-litre flask.
2. Add 1000 c.c. distilled water, heated to 70 deg. C., and close the
flask with a rubber stopper.
3. Place the flask in a water-bath regulated to 60 deg. C. and allow
the maceration to continue for one hour.
4. Strain through butter muslin into a clean flask and heat in the
steamer for thirty minutes.
5. Filter through Swedish filter paper.
6. Tube in quantities of 10 c.c. or store in flasks.
7. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
three consecutive days.
The natural reaction of the wort should _not_ be interfered with.
NOTE.--It is sometimes more convenient to obtain
"_unhopped_"[6] beer wort direct from the brewery. In this
case it is diluted with an equal quantity of distilled
water, steamed for an hour, filtered, filled into sterile
flasks or tubes, and sterilised by the discontinuous method.
~Wort Gelatine.~--
1. Measure out wort (prepared as above), 900 c.c., into a sterile flask.
2. Weigh out gelatine, 100 grammes (= 10 per cent.), and add it to the
wort in the flask.
3. Bubble live steam through the mixture for ten minutes, to dissolve
the gelatine.
4. Cool to 60 deg. C.; clarify with egg as for nutrient gelatine
(_vide_ page 164).
5. Filter through papier Chardin.
6. Tube, and sterilise as for nutrient gelatine.
~Wort Agar.~--
1. Measure out wort (as above), 700 c.c., into a sterile flask.
2. Weigh out powdered agar, 20 grammes; mix into a smooth paste with 200
c.c. of cold wort and add to the wort in the flask.
3. Bubble live steam through the mixture for twenty minutes, to dissolve
the agar.
4. Cool to 60 deg. C.; clarify with egg as for nutrient agar (_vide_ page
167).
5. Filter through papier Chardin, using the hot-water funnel.
6. Tube, and sterilise as for nutrient agar.
~Peptone Water (Dunham).~--
1. Weigh out Witte's peptone, 10 grammes, and salt, 5 grammes, and
emulsify with about 250 c.c. of distilled water previously heated to
60 deg. C.
2. Pour the emulsion into a litre flask and make up to 1000 c.c. by the
addition of distilled water.
3. Heat in the steamer at 100 deg. C. for thirty minutes.
4. Filter through Swedish filter paper.
5. Tube in quantities of 10 c.c. each.
6. Sterilise in the steamer at 100 deg. C. for twenty minutes on each of
three consecutive days.
~"Sugar" or "Carbohydrate" Media.~--
Formerly the ability of bacteria to induce hydrolytic changes in
carbohydrate substances was observed only in connection with a few
well-defined sugars, but of recent years it has been shown that when
using litmus as an indicator these so-called "fermentation reactions"
facilitate the differentiation of closely allied species, and the list
of substances employed in this connection has been considerably
extended. The media prepared with them are now no longer regarded as
special, but are comprised in the "stock media" of the laboratory. The
chief of these substances are the following, arranged in accordance with
their chemical constitution:
_Monosaccharides_ Dextrose (glucose), laevulose, galactose,
mannose, arabinose, xylose.
_Disaccharides_ Maltose, lactose, saccharose.
_Trisaccharides_ Raffinose (mellitose).
_Polysaccharides_ Dextrin, inulin, starch, glycogen, amidon.
_Glucosides_ Amygdalin, coniferin, salicin,
helicin, phlorrhizin.
_Polyatomic alcohols_ _Trihydric_, Glycerin.
_Tetrahydric_, Erythrite.
_Pentahydric_, Adonite.
_Hexahydric_, Dulcite, (dulcitol or
melampirite), isodulcite (rhamnose),
mannite (mannitol), sorbite (sorbitol),
inosite.
These substances should be obtained from Kahlbaum (of Berlin); in the
pure form, and when possible as large crystals, and the method of
preparing a medium containing either of them may be exemplified by
describing Dextrose Solution.
~Dextrose Solution.~--
1. Weigh out
Peptone 20 grammes
Glucose 10 grammes
and grind together in a mortar; then emulsify in 100 c.c. of distilled
water heated to 60 deg. C.
2. Place in a flask and add
Distilled water 850 c.c.
3. Steam in the steamer at 100 deg. C. for twenty minutes to dissolve
the peptone and glucose.
4. Add
Kubel-Tiemann litmus solution (Kahlbaum) 50 c.c.
(The substances enumerated above react acid to phenolphthalein, but
variously toward the neutral litmus solution. To such as react acid, add
very cautiously n/1 sodium hydrate solution to the medium in bulk until
the neutral tint has returned).
5. Fill into tubes in which have previously been placed the inverted
Durham's gas tubes.
6. Sterilise in the steamer at 100 deg. C. for _twenty minutes_ on
each of three successive days.
NOTE.--On no account should these media be sterilised in the
autoclave, as temperatures above 100 deg. C. themselves induce
hydrolytic changes in the substances in question. It is
equally important that the twenty minutes should not be
exceeded in sterilisation, as neglect of this precaution may
discolour the litmus or lead to the production of yellowish
tints when the tubes are subsequently inoculated with
acid-forming bacteria.
~Neutral Litmus Solution.~
The most satisfactory is the Kubel-Tiemann, prepared by Kahlbaum. It can
however be made in the laboratory as follows:
1. Weigh out
Commercial litmus 50 grammes,
and place in a well stoppered 500 c.c. bottle; measure out and add 300
c.c. alcohol 95 per cent.
2. Shake well at least once a day for seven days--the alcohol acquires a
green colour.
3. Decant off the green alcohol and fill a further 300 c.c. 95 per cent.
alcohol into the bottle and repeat the shaking.
4. Repeat this process until on adding fresh alcohol the fluid only
becomes tinged with violet.
5. Pour off the alcohol, leaving the litmus as dry as possible. Connect
up the bottle to an air pump and evaporate off the last traces of
alcohol.
6. Transfer the dry litmus to a litre flask, measure in 600 c.c.
distilled water and allow to remain in contact 24 hours with frequent
shakings.
7. Filter the solution into a clean flask and add one or two drops of
pure concentrated sulphuric acid until the litmus solution is distinctly
wine-red in colour.
8. Add excess of pure solid baryta and allow to stand until the reaction
is again alkaline.
9. Filter.
10. Bubble CO_{2} through the solution until reaction is definitely
acid.
11. Sterilise in the steamer at 100 deg. C. for thirty minutes on each of
three consecutive days. This sterilises the solution and also drives off
the carbon dioxide, leaving the solution neutral.
~Media for anaerobic cultures.~ In addition to the foregoing media, all of
which can be, and are employed in the cultivation of anaerobic bacteria,
certain special media containing readily oxidised substances are
commonly used for this purpose. The principal of these are as follows:
~Bile Salt Broth (MacConkey).~--
1. Weigh out Witte's peptone, 20 grammes (= 2 per cent.),
and emulsify with 200 c.c. distilled water previously warmed
to 60 deg. C.
2. Weigh out sodium taurocholate (commercial), 5 grammes (=
0.5 per cent.), and glucose, 5 grammes (= 0.5 per cent.),
and dissolve in the peptone emulsion.
3. Wash the peptone emulsion into a flask with 800 c.c.
distilled water, and heat in the steamer at 100 deg. C. for
twenty minutes.
4. Filter through Swedish filter paper into a sterile flask.
5. Add sterile litmus solution sufficient to colour the
medium to a deep purple, usually 13 per cent. required.
6. Fill, in quantities of 10 c.c., into tubes containing
small gas tubes (_vide_ Fig. 104, page 161). Sterilise in
the steamer at 100 deg. C. for twenty minutes on each of three
consecutive days.
~Glucose Formate Bouillon (Kitasato).~--
1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page
163, sections 1 to 6).
2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
formate, 4 grammes (= 0.4 per cent.), and dissolve in the
fluid.
3. Tube, and sterilise as for bouillon.
~Glucose Formate Gelatine (Kitasato).~--
1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to
7) and measure out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium
formate, 4 grammes (= 0.4 per cent.), and dissolve in the
hot gelatine.
3. Filter through papier Chardin.
4. Tube, and sterilise as for nutrient gelatine.
~Glucose Formate Agar (Kitasato).~--
1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8).
Measure out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
formate, 4 grammes (= 0.4 per cent.), and dissolve in the
agar.
3. Tube, and sterilise as for nutrient agar.
~Sulphindigotate Bouillon (Weyl).~--
1. Measure out nutrient bouillon (_vide_ page 163, sections
1 to 6 1000 c.c.).
2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in
the fluid.
3. Tube, and sterilise as for bouillon.
~Sulphindigotate Gelatine (Weyl).~--
1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to
7). Measure out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), and sodium
sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in
the hot gelatine.
3. Filter through papier Chardin.
4. Tube, and sterilise as for nutrient gelatine.
~Sulphindigotate Agar.~--
1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8).
Measure out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), sodium
sulphindigotate, 1 gramme (= 0.1 per cent.), and dissolve in
the hot agar.
3. Tube, and sterilise as for nutrient agar.
NOTE.--The Sulphindigotate media are of a blue colour, which
during the growth of anaerobic bacteria is oxidised and
decolourised to a light yellow.
FOOTNOTES:
[4] This figure is obtained by adding together 1 litre water, 1000
grammes; 10 per cent. gelatine, 100 grammes; 1 per cent. peptone, 10
grammes; 0.5 per cent. salt, 5 grammes; total, 1115 grammes.
Modifications of the above process, as to quantities and percentages,
will require corresponding alterations of the figures. The average
weight of a measured litre of 10 per cent. nutrient gelatine when
prepared in this way _after filtration_ is 1080 grammes.
[5] This figure is obtained by adding together 1 litre of water (meat
extract), 1000 grammes; 2 per cent. agar, 20 grammes; 1 per cent.
peptone, 10 grammes; 0.5 per cent. salt, 5 grammes--total 1035 grammes.
Modifications of the process as to quantities or percentages will
necessitate corresponding alterations in the calculated medium figure.
The average weight of a measured litre of 2 per cent. agar when prepared
in this way, _after filtration_, is 1010.5 grammes.
[6] "Hopped" wort exerts a toxic effect upon many bacteria, including
the lactic acid bacteria.
XII. SPECIAL MEDIA.
In this chapter are collected a number of media which have been
elaborated by various workers for special purposes, grouped together
under headings which indicate their chief utility. In many instances the
name of the originator of the medium is given, but without reference to
his original instructions, since these are in many cases inadequate to
the requirements of the isolated worker, who would probably fail to
reproduce the medium in a form giving the results attributed to it by
its author. Such modifications have therefore been introduced as make
for uniformity between the different batches of media.
A considerable number of coloured media, chiefly intended for work with
intestinal bacteria, have been included; but beyond the fact that the
author's modification of the Drigalski-Conradi medium has been included
amongst the routine media of the laboratory, no comment has been made
upon their relative values, since only by observation and practice can
the skill necessary to utilise their full value be acquired.
The instructions as to sterilisation are rarely given in full; the
routine method of exposure in the steam steriliser at 100 deg. C. (without
pressure) for twenty minutes on each of three successive days for all
fluid media, and thirty minutes on each of three successive days for all
liquefiable or solid media must be carried out; and only when these
general rules are to be departed from are further details given.
_Media for the Study of the Chemical Composition of Bacteria._
~Asparagin Medium (Uschinsky).~--
1. Weigh out and mix
Asparagin 3.4 grammes
Ammonium lactate 10.0 grammes
Sodium chloride 5.0 grammes
Magnesium sulphate 0.2 gramme
Calcium chloride 0.1 gramme
Acid potassium phosphate (KH_{2}PO_{4}) 1.0 gramme
2. Dissolve the mixture in distilled water 1000 c.c.
3. Add glycerine, 40 c.c.
4. Tube, and sterilise as for nutrient bouillon.
~Asparagin Medium (Frankel and Voges).~--
1. Weigh out and mix
Asparagin 4 grammes
Sodium phosphate, (Na_{2}HPO_{4}) 12OH 2 grammes
Ammonium lactate 6 grammes
Sodium chloride 5 grammes
and dissolve in
Distilled water 1000 c.c.
2. Tube, and sterilise as for nutrient bouillon.
NOTE.--Either of the above asparagin media, after the
addition of 10 per cent. gelatine or 1.5 per cent. agar, may
be advantageously employed in the solid condition.
~Proteid Free Broth (Uschinsky).~--
1. Weigh out and mix
Calcium chloride 0.1 gramme
Magnesium sulphate 0.2 gramme
Acid potassium phosphate (KH_{2}PO_{4}) 2.0 grammes
Potassium aspartate 3.0 grammes
Sodium chloride 5.0 grammes
Ammonium lactate 6.0 grammes
2. Dissolve the mixture in distilled water 1000 c.c.
3. Add glycerine 30 c.c.
4. Tube and sterilise as for nutrient broth.
_Media for the Study of Biochemical Reaction._
~Inosite-free Media--Bouillon (Durham).~--
1. Prepare meat extract, 1000 c.c. (_vide_ page 148), from bullock's
heart which has been "hung" for a couple of days.
2. Prepare nutrient bouillon (+10), 1000 c.c. (_vide_, page 161), from
the meat extract, and store in 1-litre flask.
3. Inoculate the bouillon from a pure cultivation of the B. lactis
aerogenes, and incubate at 37 deg. C. for forty-eight hours.
4. Heat in the steamer at 100 deg. C. for twenty minutes to destroy the
bacilli and some of their products.
5. Estimate the reaction of the medium and if necessary restore to +10.
6. Inoculate the bouillon from a pure cultivation of the B. coli
communis and incubate at 37 deg. C. for forty-eight hours.
7. Heat in the steamer at 100 deg. C. for twenty minutes.
Now fill two fermentation tubes with the bouillon, tint with litmus
solution, and sterilise; inoculate with B. lactis aerogenes. If no acid
or gas is formed, the bouillon is in a sugar-free condition; but if acid
or gas is present, again make the bouillon in the flask +10, reinoculate
with one or other of the above-mentioned bacteria, and incubate; then
test again. Repeat this till neither acid nor gas appears in the medium
when used for the cultivation of either of the bacilli referred to
above.
8. After the final heating, stand the flask in a cool place and allow
the growth to sediment. Filter the supernatant broth through Swedish
filter paper. If the filtrate is cloudy, filter through a porcelain
filter candle.
9. Tube, and sterilise as for bouillon.
Bouillon prepared in the above-described manner will prove to be
absolutely sugar-free; and from it may be prepared nutrient sugar-free
gelatine or agar, by dissolving in it the required percentage of
gelatine or agar respectively and completing the medium according to
directions given on pages 166 and 167. The most important application of
inosite-free bouillon is its use in the preparation of sugar bouillons,
whether glucose, maltose, lactose, or saccharose, of exact percentage
composition.
~Sugar (Dextrose) Bouillon.~--
1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
to 6) or sugar-free bouillon (_vide supra_).
2. Weigh out glucose (anhydrous), 20 grammes (= 2 per cent.), and
dissolve in the fluid.
3. Tube, and sterilise as for bouillon.
Ordinary commercial glucose serves the purpose equally well, but is not
recommended, as during the process of sterilisation it causes the medium
to gradually deepen in colour.
NOTE.--In certain cases a corresponding percentage of
lactose, maltose, or saccharose is substituted for glucose.
~Sugar Gelatine.~--
1. Prepare nutrient gelatine (_vide_ page 164, sections 1 to 7). Measure
out 1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the
hot gelatine.
3. Filter through papier Chardin.
4. Tube, and sterilise as for nutrient gelatine.
~Sugar Agar.~--
1. Prepare nutrient agar (_vide_ page 167, sections 1 to 8). Measure out
1000 c.c.
2. Weigh out glucose, 20 grammes (= 2 per cent.), and dissolve in the
clear agar.
3. Tube, and sterilise as for nutrient agar.
NOTE.--Other "sugar" media are prepared by substituting a
corresponding percentage of lactose, maltose (or any other
of the substances referred to under "Sugar Media," page 177)
for the glucose.
~Iron Bouillon.~--
1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 141, sections 1
to 6).
2. Weigh out ferric tartrate, 1 gramme (= 0.1 per cent.), and dissolve
it in the bouillon.
3. Tube, and sterilise as for bouillon.
NOTE.--The lactate of iron may be substituted for the
tartrate.
~Lead Bouillon.~--
1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
to 6).
2. Weigh out lead acetate, 1 gramme (= 0.1 per cent.), and dissolve it
in the bouillon.
3. Tube, and sterilise as for bouillon.
~Nitrate Bouillon.~--
1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
to 6).
2. Weigh out potassium nitrate, 5 grammes (= 0.5 per cent.), and
dissolve it in the bouillon.
3. Tube, and sterilise as for bouillon.
NOTE.--The nitrate of sodium or ammonium may be substituted
for that of potassium, or the salt may be added in the
proportion of from 0.1 to 1 per cent. to meet special
requirements.
~Iron Peptone Solution (Pakes).~--
1. Weigh out peptone, 30 grammes, and emulsify it with 200 c.c. tap
water, previously heated to about 60 deg. C.
2. Wash the emulsion into a litre flask with 800 c.c. tap water.
3. Weigh out salt, 5 grammes, and sodium phosphate, 3 grammes, and
dissolve in the mixture in the flask.
4. Heat the mixture in the steamer at 100 deg. C. for thirty minutes,
to complete the solution of the peptone, and filter into a clean flask.
5. Fill into tubes in quantities of 10 c.c. each.
6. Add to each tube 0.1 c.c. of a 2 per cent. neutral solution of ferric
tartrate. (A yellowish-white precipitate forms.)
7. Sterilise as for nutrient bouillon.
~Lead Peptone Solution.~--
Prepare as for iron peptone solution but in step 6 substitute 0.1 c.c.
of a 1 per cent. neutral aqueous solution of lead acetate.
~Nitrate Peptone Solution (Pakes).~--
1. Weigh out Witte's peptone, 10 grammes, and emulsify it with 200 c.c.
ammonia-free distilled water previously heated to 60 deg. C.
2. Wash the emulsion into a flask and make up to 1000 c.c., with
ammonia-free distilled water.
3. Heat in the steamer at 100 deg. C. for twenty minutes.
4. Weigh out sodium nitrate, 1 gramme, and dissolve in the contents of
the flask.
5. Filter through Swedish filter paper.
6. Tube, and sterilise as for nutrient bouillon.
~Litmus Bouillon.~--
1. Measure out nutrient bouillon, 1000 c.c. (_vide_ page 163, sections 1
to 6).
2. Add sufficient sterile litmus solution to tint the medium a dark
lavender colour. (Media rendered +10 will usually react very faintly
alkaline or occasionally neutral to litmus.)
3. Tube, and sterilise as for bouillon.
~Rosolic Acid Peptone Solution.~--
1. Weigh out rosolic acid (corallin), 0.5 gramme, and dissolve it in 80
per cent. alcohol, 100 c.c. Keep this as a stock solution.
2. Measure out peptone water (Dunham), 100 c.c., and rosolic acid
solution, 2 c.c., and mix.
3. Heat in the steamer at 100 deg. C. for thirty minutes.
4. Filter through Swedish filter paper.
5. Tube, and sterilise as for nutrient bouillon.
~Capaldi-Proskauer Medium, No. I.~--
1. Weigh out and mix
Sodium chloride 2.0 grammes
Magnesium sulphate 0.1 gramme
Calcium chloride 0.2 gramme
Monopotassium phosphate 2.0 grammes
2. Dissolve in water 1000 c.c. in a 2-litre flask
3. Weigh out and mix
Asparagin 2 grammes
Mannite 2 grammes
and add to contents of flask.
4. Measure out 25 c.c. of the solution and titrate it against decinormal
sodic hydrate, using litmus as the indicator. Control the result and
estimate the amount of sodic hydrate necessary to be added to render the
remainder of the solution neutral to litmus. Add this quantity of sodic
hydrate.
5. Filter.
6. Add litmus solution 47.5 c.c. (= 5 per cent.).
7. Tube, and sterilise as for nutrient bouillon.
~Capaldi-Proskauer Medium No. II.~--
1. Weigh out and mix
Peptone 20 grammes
Mannite 1 gramme
2. Dissolve in water 1000 c.c. in a 2-litre flask.
3. Neutralise to litmus as in No. I (_vide supra_, Step 4).
4. Filter.
5. Add litmus solution 47.5 c.c. (= 5 per cent.).
6. Tube, and sterilise as for nutrient bouillon.
~Urine Media. Bouillon.~--
1. Collect freshly passed urine in sterile flask.
2. Place the flask in the steamer at 100 deg. C. for thirty minutes.
3. Filter through two thicknesses of Swedish filter paper.