may appear both on the surface and in the depths of the medium) is
essential to the accurate study of the formation of colonies under
various conditions, but when the main object of the separation of the
bacteria is to obtain subcultivations from a number of individual
bacteria, "surface" plates must be prepared, since here colony formation
is restricted to the surface of the medium. The method adopted varies
slightly according to whether the medium employed is gelatine or agar,
or one of the derivatives or variants of the latter.
(a) ~Gelatine Surface Plates.~--
1. Liquefy three tubes of nutrient gelatine.
2. Pour each tube into a separate Petri dish and allow it to solidify.
Then turn each plate and its cover upside down.
[Illustration: FIG. 126.--Surface plate spreader.]
3. When quite cold raise the bottom of plate 1, revert it and deposit a
drop of the inoculum (whether a fluid culture or an emulsion from solid
culture) upon the surface of the gelatine with a platinum loop--close to
one side of the plate; replace the bottom half of the Petri dish in its
cover.
4. Take a piece of thin glass rod, stout platinum wire or best of all a
piece of aluminium wire (say 2 mm. diameter) about 28 cm. long. Bend the
terminal 4 cm. at right angles to the remainder, making an L-shaped rod
(Fig. 126). Sterilise the short arm and adjacent portion of the long
arm, in the Bunsen flame, and allow it to cool.
5. Now raise the bottom of the Petri dish in the left hand, leaving the
cover on the laboratory bench, and holding it vertically, smear the drop
of inoculum all over the surface of the gelatine with the short arm of
the spreader by a rotatory motion, (Fig. 127). Replace the dish in its
cover.
6. Raise the bottom of plate 2 and rub the infected spreader all over
the surface of the gelatine--then go on in like manner to the third
plate in the series.
7. Sterilise the spreader.
8. Label and incubate the plates.
[Illustration: FIG. 127.--Spreading surface plate.]
After incubation, plate No. 1 will probably yield an enormous number of
colonies; plate 2 will show fewer colonies, since only those bacteria
adhering to the rod after rubbing over plate 1 would be deposited on its
surface, and by the time the rod reached plate 3 but very few organisms
should remain upon it. So that the third plate as a rule will only show
a very few scattered colonies, eminently suitable for detailed study.
(b) ~Agar Surface Plates.~--
1. Liquefy three tubes of nutrient agar--nutrose agar or the like.
2. Pour each tube into a separate Petri dish and allow it to solidify.
3. When quite solid invert each dish, raise the bottom half and rest it
obliquely on its inverted cover (Fig. 128) and place it in this position
in an incubator at 60 deg. C. for forty-five minutes (or in an incubator
at 42 deg. C. for two hours). This evaporates the water of condensation
and gives the medium a firm, dry surface.
4. On removing the plates from the incubator close each dish and place
it--still upside down--on the laboratory bench.
[Illustration: FIG. 128.--Drying surface plate of agar.]
5. Inoculate the plates in series of three, as described for gelatine
surface plates 3-8.
Hanging-drop Cultivation.
~Apparatus Required.~--
Hanging-drop slides.
Cover-slips.
Section rack (Fig. 75).
Blotting paper.
Bell glass to cover slides.
Original culture.
Tubes of broth, or liquefied gelatine or agar.
Forceps.
Platinum loop.
Bunsen burner.
Grease pencil.
Sterile vaseline.
Lysol.
(a) ~Fluid Media.~--
1. Prepare first and second dilutions of the inoculum as directed for
plate cultivations (_vide_ pages 228-229, sections 4 to 6), substituting
tubes of nutrient broth for the liquefied gelatine.
2. Sterilise a hanging-drop slide by washing thoroughly in water and
drying, then plunging it into a beaker of absolute alcohol, draining off
the greater part of the spirit, grasping the slide in a pair of forceps,
and burning off the remainder of the alcohol in the flame.
3. Place the hanging-drop slide on a piece of blotting paper moistened
with 2 per cent. lysol solution and cover it with a small bell glass
that has been rinsed out with the same solution and _not dried_.
4. Raise the bell glass slightly and smear sterile vaseline around the
rim of the metal cell by means of a sterile spatula of stout platinum
wire.
5. Remove a clean cover-slip from the alcohol pot with sterile forceps
and burn off the alcohol; again raise the bell glass and place the
sterile cover-slip on the blotting paper by the side of the hanging-drop
slide.
6. Remove a drop of the broth from the second dilution tube with a large
platinum loop; raise the bell glass and deposit the drop on the centre
of the cover-slip. Sterilise the loop.
7. Raise the bell glass sufficiently to allow of the cover-slip being
grasped with forceps, inverted, and adjusted over the cell of the
hanging-drop slide. Remove the bell glass altogether and press the
cover-slip firmly on to the cell.
8. Either incubate and examine at definite intervals, or observe
continuously with the microscope, using a warm stage if necessary (Fig.
53).
(b) ~Solid Media.~--Observing precisely similar technique, a few drops of
liquefied gelatine or agar from the second dilution tube may be run over
the surface of the sterile cover-slip and a hanging-drop plate
cultivation thereby prepared.
This method is extremely useful in connection with the study of yeasts,
when the circular cell on the hanging-drop slide should be replaced by a
rectangular cell some 38 by 19 mm., and the gelatine spread over a
cover-slip of similar size. After sealing down the preparation, the
upper surface of the cover-slip may be ruled into squares by the aid of
the grease pencil or a writing diamond and numbered to facilitate the
subsequent identification of the colonies which are observed to develop
from solitary germs.
~Hanging-block Culture~ (Hill).--
_Apparatus required_: As for hanging-drop cultivation with the addition
of a scalpel.
Carry out the method as far as possible under cover of a bell glass, to
avoid aerial contamination.
1. Liquefy a tube of nutrient agar (or gelatine) and pour into a Petri
dish to the depth of about 4 mm. and allow to set.
2. With a sharp scalpel cut out a block some 8 mm. square, from the
entire thickness of the agar layer.
3. Raise the agar block on the blade of the scalpel and transfer it,
under side down, to the centre of a sterile slide.
4. Spread a drop of fluid cultivation (or an emulsion of growth from a
solid medium) over the upper surface of the agar block as if making a
cover-slip film.
5. Place the slide and block covered by the bell glass in the incubator
at 37 deg. C. for ten minutes to dry slightly.
6. Take a clean dry sterile cover-slip in a pair of forceps, and with
the help of a second pair of forceps lower it carefully on the
inoculated surface of the agar (avoiding air bubbles), so as to leave a
clear margin of cover-slip overlapping the agar block.
7. Invert the preparation and with the blade of the scalpel remove the
slide from the agar block.
8. With a platinum loop run a drop or two of melted agar around the
edges of the block. This solidifies at once and seals the block to the
cover-slip.
9. Prepare a sterile hanging-drop slide, and smear hard vaseline or
melted white wax on the rim of the metal cell.
10. Invert the cover-slip with the block attached on to the hanging-drop
slide, and seal the cover-slip firmly in place.
11. Observe as for hanging-drop cultivations.
ANAEROBIC CULTIVATIONS.
Numerous methods have been devised for the cultivation of anaerobic
bacteria, the majority requiring the employment of special apparatus.
The principle upon which any method is based and upon which it depends
for its success falls under one or another of the following headings:
(a) ~Exclusion of air~ from the cultivation.
(b) ~Exhaustion of air~ from the vessel containing the cultivation by
means of an air pump--i. e., cultivation _in vacuo_.
(c) ~Absorption of oxygen~ from the air in contact with the cultivation
by means of pyrogallic acid rendered alkaline with caustic soda--i. e.,
cultivation in an atmosphere of nitrogen.
(d) ~Displacement of air~ by an indifferent gas, such as hydrogen or coal
gas--i. e., cultivation in an atmosphere of hydrogen.
(e) A combination of two or more of the above methods.
A selection of the simplest and most generally useful methods is given
here.
Whenever possible, the nutrient media that are employed in any of the
processes should contain some easily oxidisable substance, such as
sodium formate (0.4 per cent.) or sodium sulphindigotate (0.1 per
cent.), which will absorb all the available oxygen held in solution by
the medium. The further addition of glucose, 2 per cent., favors the
growth of anaerobic bacteria (_vide_, pages 189-190).
Further, it is advisable to seal all joints between india-rubber
stoppers and tubulures or the mouths of the tubes with melted paraffin;
glass stoppers and taps should be lubricated with resin ointment or a
mixture of beeswax 1 part, olive oil 4 parts.
(A) ~Method I~ (Hesse's Method).--
1. Make a stab culture in gelatine or agar, choosing for the purpose a
straight tube containing a deep column of medium, and thrusting the
inoculating needle to the bottom of the tube.
2. Pour a layer of sterilised oil (olive oil, vaseline, or petroleum), 1
or 2 cm. deep, upon the surface of the medium.
3. Incubate.
~Method II.~--This method is only available when dealing with pure
cultivations.
1. Liquefy a tube of gelatine (or agar) by heat, pour it into a Petri
dish, and allow it to solidify.
2. Inoculate the surface of the medium in one spot only.
3. Remove a cover-slip from the pot of absolute alcohol with sterile
forceps; burn off the alcohol in the gas flame.
4. Lower the now sterile cover-slip carefully on to the inoculated
surface of the medium, carefully excluding air bubbles, and press it
down firmly with the points of the forceps. (A sterile disc of mica may
be substituted for the cover-slip.)
5. Incubate.
~Method III~ (Roux's Physical Method).--
1. Prepare tube cultures of fluid media (or solid media rendered fluid
by heat) in the usual way.
2. Aspirate some of the inoculated media into capillary pipettes.
3. Seal both ends of each pipette in the blowpipe flame.
4. Incubate.
~Method IV~ (Roux's Biological Method).--
1. Plant a deep stab, as in method I.
2. Pour a layer, 1 or 2 cm. deep, of broth cultivation of a vigourous
aerobe--e. g., B. aquatilis sulcatus or B. prodigiosus--upon the
surface of the medium; or an equal depth of liquefied gelatine, which is
then inoculated with the aerobic organism.
3. Incubate.
The growth of the aerobe will use up all the oxygen that reaches it and
will not allow any to pass through to the medium below, which will
consequently remain in an anaerobic condition.
(B) ~Method V.~--
1. Prepare tube or flask cultivations in the usual way.
2. Replace the cotton-wool plug by an india-rubber stopper perforated
with one hole and fitted with a length of glass tubing which has a
constriction about 3 cm. above the stopper and is then bent at right
angles (Fig. 129). The stopper and glass tubing are sterilised by being
boiled in a beaker of water for five minutes.
[Illustration: FIG. 129.--Vacuum culture.]
3. Connect the tube leading from the culture vessel with a water or air
pump, interposing a Wulff's bottle fitted as a wash-bottle and
containing sulphuric acid.
4. Exhaust the air from the culture vessel.
5. Before disconnecting the apparatus, seal the glass tube from the
culture vessel at the constriction, using the blowpipe flame.
6. Incubate.
(C) ~Method VI~ (Buchner's Method).
~Apparatus and Solutions Required.~--
Buchner's tube (a stout glass test-tube 23 cm. long and 4
cm. in diameter, fitted with india-rubber stopper, Fig.
130).
Pyrogallic acid in compressed tablets each containing 1
gram.
Dekanormal solution of caustic soda.
METHOD.--
1. Prepare the tube cultivation in the usual way.
2. Moisten the india-rubber stopper of the Buchner's tube with water and
see that it fits the mouth of the tube accurately.
3. Remove the stopper from the caustic soda bottle.
4. Drop one of the pyrogallic acid tablets[9] into the Buchner's tube
(roughly, use 1 gramme pyrogallic acid for every 100 c.c. air capacity
of the receiving vessel).
5. Add about 1 c.c. of the soda solution.
6. Place the inoculated tube inside the Buchner's tube. The pyrogallic
tablet acts as a buffer and prevents damage to either the inoculated
tube or the Buchner's tube even should it be slipped in hurriedly.
7. Fit the india-rubber stopper tightly into the mouth of the Buchner's
tube.
[Illustration: FIG. 130.--Buchner's tube.]
The pyrogallic acid tablet dissolves slowly in the soda solution and its
oxidation proceeds very slowly at first so that ample time is available
when this method is adopted.
8. Restopper the caustic soda bottle.
9. Place Buchner's tube in a wire support, and incubate.
~Method VII~ (Wright's Method).--
1. Prepare tube cultivation in the usual way.
2. Cut off that portion of the cotton-wool plug projecting above the
mouth of the tube with scissors, then push the plug into the tube for a
distance of 2 or 3 cm.
3. By means of a pipette drop about 1 c.c. of pyrogallic acid 10 per
cent. aqueous solution on to the plug. It will immediately be absorbed
by the cotton-wool.
4. With another pipette run in an equal quantity of the caustic soda
solution.
5. Quickly close the mouth of the tube with a tightly fitting
india-rubber stopper.
6. Incubate.
[Illustration: FIG. 131.--McLeod's anaerobic plate base with half petri
dish inverted _in situ_]
~Method VIII~ (McLeod's Method).--
~Apparatus and Solutions Required.~--
McLeod's plate base (a hollow glazed earthenware disc 9 cm.
in diameter and 2 cm. deep: the upper surface is pierced by
a central hole, 2 cm. in diameter, giving access to the
interior, the lower part of which is divided into two by a
low partition. A shallow groove encircles the upper surface
near to the edge).
Plasticine.
Pyrogallic acid (1 gramme) compressed tablets.
Sodic hydroxide (0.4 gramme) compressed tablets.
Wash bottle of distilled water.
Surface plates of one or other agar medium (in petri dishes
of 8 cm. diameter).
Surface plate spreader.
METHOD.--
1. Roll out a long cylinder of plasticine and fit it into the groove on
the upper surface of the earthenware base.
2. Place a tablet of pyrogallic acid in one division of the interior of
the plate base, and two tablets of sodic hydroxide in the other.
3. Prepare surface plate culture of the organism to be cultivated.
4. Run a few cubic centimetres of distilled water into that division of
the plate base containing the sodic hydroxide.
5. Invert the bottom half of the surface plate over the plate base and
press its edges firmly down into the plasticine filling the groove.
6. Label and incubate.
(D) ~Method IX.~--
~Apparatus Required.~--
Small Ruffer's or Woodhead's flask (Fig. 33).
Sterile india-rubber stopper.
India-rubber tubing.
Glass tubing.
Metal screw clips.
Cylinder of compressed hydrogen; or hydrogen gas apparatus
METHOD.--
1. Sterilise a glass vessel, shaped as in a Ruffer's or Woodhead's
flask, in the hot-air oven. (The tubulure and the side tubes are plugged
with cotton-wool.) After sterilisation, fix a short piece of rubber
tubing occluded by a metal clip to each side tube.
2. Inoculate a large quantity (e. g., 200 c.c.) of the medium. Where
solid media are employed they must first be liquefied by heat.
3. Remove the cotton-wool plug from the tubulure and pour the inoculated
medium into the glass vessel.
4. Close the tubulure by means of an india-rubber stopper previously
sterilised by boiling in a beaker of water.
[Illustration: FIG. 132.--Kipp's hydrogen apparatus, (a) connected up
to two washing bottles containing (b) lead acetate 10 per cent.
solution, to remove H_{2}S and (c) silver nitrate solution to remove
AsH_{3}. A third washing bottle containing pyrogallic acid 10 per cent.
solution, rendered alkaline, to remove any trace of oxygen, is sometimes
introduced.]
[Illustration: FIG. 133.--Improved gas apparatus; the metal is contained
in a perforated glass tube which is submerged in acid when the
triangular bottle is upright (a), but is above the level of the liquid
when the bottle is turned on its side (b).]
5. Connect up the india-rubber tubing on one of the side tubes with a
cylinder of compressed hydrogen (or the delivery tube of a Kipp's Fig.
132 or other hydrogen apparatus, Fig. 133), interposing a short piece of
glass tubing; and in like manner connect a long piece of rubber tubing
which should be led into a basin of water, to the opposite side tube.
6. Open both metal clips and pass hydrogen through the vessel until the
atmospheric air is replaced by hydrogen. This is determined by
collecting some of the gas which bubbles through the water in the basin
in a test-tube and testing it by means of a lighted taper.
7. Close the metal clip on the tube through which the gas is entering;
close the clip on the exit tube.
8. Disconnect the gas apparatus.
9. Incubate.
~Method X~ (Botkin's Method).--
~Apparatus Required.~--
Large glass dish 20 cm. diameter and 8 cm. deep. Flat leaden
cross slightly shorter than the internal diameter of the glass dish.
Bell glass about 15 cm. diameter and 20 to 25 cm. high.
Metal frame for plate cultivations.
_Or_, glass battery jar for tube cultivations.
Cylinder of compressed hydrogen.
Rubber tubing.
Two pieces of ~U~-shaped glass tubing (each arm 8 cm. in length).
Half a litre of glycerine (or metallic mercury).
METHOD.--
1. Place the leaden cross inside the glass dish, resting on the bottom.
2. Prepare the cultivations in the usual way.
3. Place the tube cultivations in a glass battery jar (or the plate
cultivations on a metal frame), resting on the centre of the leaden
cross.
4. Cover the cultivations with the bell jar.
5. Adjust the U-shaped pieces of glass tubing in a vertical position on
opposite sides of the bell jar, one arm of each inside the jar, the
other outside. These tubes are best held in position by embedding the
U-shaped bends in two lumps of plasterine stuck on the bottom of the
glass dish. Fix a short length of rubber tubing clamped with a metal
clip to each of the outside arms (Fig. 134).
6. Fill the glass dish with glycerine or metallic mercury to a depth of
about 5 cm.
[Illustration: FIG. 134.--Botkin's apparatus.]
7. Connect up one U-shaped tube with the hydrogen cylinder (or gas
apparatus) by means of rubber tubing. Replace the atmospheric air by
hydrogen, as in method IX.
8. Clamp the tubes and disconnect the gas apparatus.
9. Incubate.
~Method XI~ (Novy's Method).--
~Apparatus Required.~--
Jar for plate cultivations (Fig. 135).
_Or_, jar for tube cultivations (Fig. 136).
Lubricant for stopper of jar.
Rubber tubing.
Cylinder of compressed hydrogen.
METHOD.--
1. Prepare cultivations in the usual way.
2. Place these inside the jar.
3. Lubricate the stopper and insert it in the mouth of the jar, with the
handle in a line with the two side tubes.
4. Connect up the delivery tube a with the hydrogen gas supply by
means of rubber tubing.
[Illustration: FIG. 135.--Novy's jar for plate cultivations.]
[Illustration: FIG. 136.--Novy's jar for tube cultivations.]
5. Attach a piece of rubber tubing to the exit tube b and collect
samples of the issuing gas (over water) and test from time to time.
6. When the air is completely displaced by hydrogen, turn the handle of
the stopper at right angles to the line of entry and exit tubes; this
seals the orifice of both tubes.
7. Disconnect the gas apparatus and incubate.
(E) ~Method XII~ (Bulloch's Method).--
~Apparatus Required.~--
Bulloch's jar.
Pot of resin ointment.
Small glass dish 14 cm. diameter by 5 cm. deep.
Vessel for tube cultures or metal rack for plate cultures.
Pyrogallic acid tablets.
Cylinder of compressed hydrogen.
Geryk or other air pump.
Rubber pressure tubing.
10 c.c. pipette.
Glass tubing.
Dry granulated caustic soda or compressed tablets each, containing
0.4 grammes sodic hydroxide.
Small beaker of water.
METHOD.--
1. Prepare the cultivations in the usual way.
2. Place the glass dish in the centre of the glass slab, and stand the
cultivations inside this.
3. Place a sufficient number of pyrogallic acid tablets at one side of
the glass dish (i. e., 1 tablet for each 100 cubic centimeters air
capacity of the bell jar). Place a small heap of dry granulated soda (or
half a dozen tablets of sodic hydroxide) by the side of the pyro
tablets.
4. Smear the flange of the bell jar with resin ointment and apply the
jar firmly to the glass slab, covering the cultivations--so arranged
that the long tube passes with its lower end into the glass dish at a
point directly opposite to the pyrogallic acid tablets. Lubricate the
two stop-cocks with resin ointment (Fig. 137).
5. Connect up the short tube a with the gas-supply by means of rubber
pressure tubing and open both stop-cocks.
6. Connect a long, straight piece of glass tubing to the long tube b
by means of a piece of rubber tubing interposing a screw clamp: and
collect samples of the issuing gas from time to time and test.
7. When the air is displaced, shut off the stop-cock of the entry tube,
then that of the exit tube b. Screw down the clamp and remove the
glass tube from the rubber connection and connect up the short tube a
to the air pump by means of pressure tubing.
8. Open the stop-cock of tube a and with two or three strokes of the
air pump, aspirate a small quantity of gas, so creating a slight vacuum.
Then shut off the stop-cock and disconnect the air pump.
9. Fill the 10 c.c. bulb pipette with water; insert its point into the
rubber tubing on the long tube b as far as the screw clamp. Open the
screw clamp and run in water until stopped by the internal pressure.
Shut off stop-cock. (The water dissolves the soda and pyrogallic acid
converting the latter into alkaline pyro. and so bringing its latent
capacity for oxygen into action).
[Illustration: FIG. 137.--Bulloch's jar.]
10. Reverse the tubes from the tubulures so that they meet, out of
harm's way, over the top of the bell glass; again see that all joints
are tight and transfer the apparatus to the incubator.
This last method is the most satisfactory for anaerobic cultivations, as
by its means complete anaerobiosis can be obtained with the least
expenditure of time and trouble.
FOOTNOTES:
[8] See also method of opening and closing culture tubes, pages 74-76.
[9] If compressed tablets of pyrogallic acid cannot be obtained make up
a stock "acid pyro" solution
Pyrogallic acid, 10 grammes
Hydrochloric acid, 1.5 c.c.
Distilled water, 100 c.c.
and at step 4, run in 10 c.c. of the solution.
XV. METHODS OF ISOLATION.
The work in the preceding sections, arranged to demonstrate the chief
biological characters of bacteria in general, is intended to be carried
out by means of cultivations of various organisms previously isolated
and identified and supplied to the student in a state of purity. A
cultivation which comprises the progeny of a single cell is termed a
"pure culture"; one which contains representatives of two or more
species of bacteria is spoken of as an "impure," or "mixed"
"cultivation," and it now becomes necessary to indicate the chief
methods by which one or more organisms may be isolated in a state of
purity from a mixture; whether that mixture exists as an impure
laboratory cultivation, or is contained in pus and other morbid
exudations, infected tissues, or water or food-stuffs.
[Illustration: FIG. 138.--Haematocytometer cell, showing, a, section
through the centre of the cell, and b, a magnified image of the cell
rulings.]