Cultivations of micro-organisms are usually prepared in the laboratory in one of three ways:

These may be incubated either aerobically (i. e., in the presence of oxygen) or anaerobically (i. e., in the absence of oxygen, or in the presence of an indifferent gas, such as hydrogen, nitrogen, or carbon dioxide).

With regard to the temperature at which the cultivations are grown, it may be stated as a general rule that all media rendered solid by the addition of gelatine are incubated at 20°C., or at any rate at a temperature not exceeding 22° C. (that is, in the "cold" incubator); whilst fluid media and all other solid media are incubated at 37° C. (that is, in the "hot" incubator). Exceptions to this rule are numerous. For instance, in studying the growth of the psychrophylic bacteria, the yeasts and the moulds, the cold incubator is employed for all media.

Tube cultivations are usually packed in the incubator in small tin cylinders, such as those in which American cigarettes are sold, or in square tin boxes. Beakers or tumblers may be used for the same purpose, but being fragile are not so convenient. Metal test-tube racks, long enough to just fit into the interior of the incubator and each accommodating two rows of tubes, are also exceedingly useful.

AEROBIC.

The Preparation of Tube Cultivations.

The preparation of a tube cultivation consists in:

(a) Inoculating a tube of sterile nutrient medium with a portion of the material to be examined.

(b) Incubating the inoculated tube at a suitable temperature.

The details of the first of these processes must be varied somewhat according to whether the tubes of nutrient media are inoculated or "planted" from—

1. Pre-existing cultivations.

2. Morbid material previously collected (vide page 373).

3. Fluids, tissues, etc., or from the animal body direct.

The method of preparing tube cultivations from pre-existing cultivations is as follows:

1. Fluid Media (e. g., Nutrient Bouillon).—

1. Flame the cotton-wool plug of the tube containing the cultivation and also that of the tube of sterile bouillon.

2. Hold the two tubes, side by side, between the left thumb and the first and third fingers, allowing the sealed ends to rest on the dorsum of the hand, and separating the mouths of the tubes (which are pointed to the right) by the tip of the second finger. Keep the tubes as nearly horizontal as is possible without allowing the fluid in the bouillon tube to reach the cotton-wool plug (Fig. 117).

3. Sterilise the platinum loop and allow it to cool.[8]

4. Grasp the plug of the tube containing the cultivation between the little finger and palm of the hand and remove it from the tube.

5. Grasp the plug of the bouillon tube between the fourth finger and the ball of the thumb and remove it from the tube.

6. Pass the platinum loop into the tube containing the culture—do not allow the loop to touch the sides of the tube, or the handle to touch the medium—and remove a small portion of the growth; withdraw the loop from the tube, keeping the infected side of the loop downward.

7. Pass the loop into the bouillon tube almost down to the level of the fluid, reverse the loop so that the infected side faces upward, emulsify the portion of the growth in the moisture adhering to the side of the tube which is uppermost. Withdraw the loop.

8. Replug both tubes.

9. Sterilise the platinum loop.

10. Label the bouillon tube with (a) the name of the organism and (b) the date of inoculation.

11. Incubate.

2. Solid Media.—Solid media are stored in tubes in one of two ways:

1. Oblique tube or slanted tube (Fig. 118), in which the medium has been allowed to solidify whilst the tube was retained in an inclined position, so forming an extensive surface of medium extending from the bottom of the tube almost to its mouth.

This is employed for "streak" or "smear" cultivations (Strichcultur).

2. Straight tube (Fig. 119), in which the medium forms a cylindrical mass in the lower portion of the tube and presents an upper surface which is at right angles to the long axis of the tube.

This is employed for "stab" or "stick" cultivations (Stichcultur), or by inoculating the medium whilst fluid, and allowing to solidify in this position, for "shake" cultivations.

Streak Culture.—

1. Flame the plugs, sterilise the platinum loop (or spatula). Open the tubes and charge the loop as in previous inoculation.

2. Pass the infected loop to the bottom of the tube to be inoculated and draw it, as lightly as possible, along the centre of the surface of the medium, terminating the "streak" over the thin layer of medium near the mouth of the tube.

3. Replug the tubes, sterilise the platinum loop.

4. Label the newly inoculated tube and incubate.

Smear Culture.—Proceed generally as in streak culture, but rub the infected loop all over the surface of the medium, instead of restricting the inoculation to a narrow line.

Stab Culture.—

1. Flame the plugs, open the tubes, sterilise the platinum needle and charge it with the inoculum as in the previous cultivations.

2. Pass the platinum needle into the tube to be inoculated until it touches the centre of the surface of the medium. Now thrust it deeply into the substance of the medium, keeping the needle as nearly as possible in the axis of the cylinder of medium. Then withdraw the needle.

3. Replug the tubes. Sterilise the platinum needle.

4. Label the newly planted tube and incubate.

Shake Culture.—

1. Liquefy a tube of nutrient gelatine (or agar, or other similar medium), by heating in a water-bath (Fig. 121).

2. Inoculate the liquefied medium and label it, etc., precisely as if dealing with a tube of bouillon.

3. Place the newly planted tube in the upright position (e. g., in a test-tube rack) and allow it to solidify.

4. Label the tube; when solid, incubate.

The Preparation of Plate Cultures.

If a small number of bacteria are suspended in liquefied gelatine, agar, or other similar medium, and the infected medium spread out in an even layer over a flat surface and allowed to solidify, each individual micro-organism becomes fixed to a certain spot and its further development is restricted to the vicinity of this spot. After a variable interval the growth of this organism becomes visible to the naked eye as a "colony." This is the principle upon which the method of plate cultivation is based and its practice enables the bacteriologist to study the particular manner of development affected by each species of microbe when growing (a) unrestricted upon the surface of the medium, (b) in the depths of the medium. The method itself is as follows:

Method of "Pouring" Plates.—

1. Place the glass dish on the levelling tripod (Figs. 122, 123); if gelatine plates are to be poured fill the dish with ice water—gelatine solidifies so slowly that it is necessary to hasten the process; if agar is to be used fill with water at 50°C.—agar sets almost immediately at the room temperature and by slightly retarding the process lumpiness is avoided; cover the dish with the square sheet of glass.

2. Place the spirit level on the sheet of glass and by means of the levelling screws adjust the surface of the glass to the horizontal.

This leveling is an important matter since the development of a colony is to some extent proportionate to the supply of medium available for its nutrition. Thus in a "smear" on sloped tube culture, the colonies at the upper part of the medium are stunted and small but increase in size and luxuriance of growth the nearer they approach to the bottom of the tube, where there is the greatest depth of medium.

3. Place three sterile Petri dishes in a row on the surface of the glass plate and number them 1, 2, and 3, from left to right.

4. Number the previously liquefied tubes of nutrient media 1, 2, and 3. Flame the plugs and see that each plug can be readily removed from the mouth of its tube.

5. Add one loopful of the inoculum to tube No. 1, treating the liquefied medium as bouillon. After replugging, grasp the tube near its mouth by the thumb and first finger of the right hand, and with an even circular movement of the whole arm, diffuse the inoculum throughout the medium; avoid jerky movements, as these cause bubbles of air to form in the medium.

The knack of mixing evenly without producing air bubbles, is not always easily acquired, by this method. An alternative plan is to hold the inoculated tube vertically upright between the opposed palms and to rotate it between them by rapid backward and forward movements of the two hands (Fig. 124).

6. Sterilise the platinum loop, and add two loopfuls of diluted inoculum to tube No. 2, and mix as before.

7. In a similar manner transfer three loopfuls of liquefied medium from tube No. 2 to tube No. 3, and mix thoroughly.

8. Flame the plug of tube No. 1, remove it, then flame the lips of the tube; slightly raise the cover of Petri dish No. 1, introduce the mouth of the tube; then, elevating the bottom of the tube, pour the liquefied medium into the Petri dish, to form a thin layer. Remove the mouth of the tube and close the "plate." If the medium has failed to flow evenly over the bottom of the plate, raise the plate from the levelling platform and by tilting in different directions rectify the fault.

9. Pour plates No. 2 and No. 3, in a similar manner, from tubes Nos. 2 and 3.

10. Label the plates with the distinctive name or number of the inoculum, also the date; the number of the dilution having been previously indicated (step 3).

11. Place in the cool incubator for three or more days, as may be necessary.

In this way colonies may be obtained quite pure and separate from each other.

In plate No. 1, probably, the colonies will be so numerous and crowded, and therefore so small, as to render it useless. In plate No. 2 they will be more widely separated, but usually No. 3 is the plate reserved for careful examination, as in this the colonies are usually widely separated, few in number, and large in size.

Agar plates are poured in a similar manner, but the agar must be melted in boiling water and then allowed to cool to 45° C. or 42° C. in a carefully regulated water-bath before being inoculated, and the entire process must be carried out very rapidly, otherwise the agar will have solidified before the operation is completed.

Surface Plates.—

This method of pouring what may be termed "whole" plates (since colonies 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.

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.

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° C. for forty-five minutes (or in an incubator at 42° 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.

5. Inoculate the plates in series of three, as described for gelatine surface plates 3-8.

Hanging-drop Cultivation.

(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° 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.

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.—

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.

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.

Method VIII (McLeod's Method).—

Apparatus and Solutions Required.—

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.—

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.

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.—

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.

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.—

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.

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.—

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).

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.