The equipment of the bacteriological laboratory, so far as the glass apparatus is concerned, differs but little from that of a chemical laboratory, and the cleanliness of the apparatus is equally important. The glassware comprised in the following list, in addition to being clean, must be stored in a sterile or germ-free condition.

Test-tubes.—It is convenient to keep several sizes of test-tubes in stock, to meet special requirements, viz.:

1. 18 × 1.5 cm., to contain media for ordinary tube cultivations.

2. 18 × 1.3 cm., to contain media used for pouring plate cultivations, and also for holding sterile "swabs."

3. 18 × 2 cm., to contain wedges of potato, beetroot, or other vegetable media.

4. 13 × 1.5 cm., to contain inspissated blood-serum.

The tubes should be made from the best German potash glass, "blue-lined," stout and heavy, with the edge of the mouth of the tube slightly turned over, but not to such an extent as to form a definite rim. (Cost about $1.50, or 6 shillings per gross.) Such tubes are expensive it is true, but they are sufficiently stout to resist rough handling, do not usually break if accidentally allowed to drop (a point of some moment when dealing with cultures of pathogenic bacteria), can be cleaned, sterilised, and used over and over again, and by their length of life fully justify their initial expense.

A point be noted is that the manufacturers rarely turn out such tubes as these absolutely uniform in calibre, and a batch of 18 by 1.5 cm. tubes usually contains such extreme sizes as 18 by 2 cm. and 18 by 1.3 cm. Consequently, if a set of standard tubes is kept for comparison or callipers are used each new supply of so-called 18 by 1.5 cm. tubes may be easily sorted out into these three sizes, and so simplify ordering.

5. 5 × 0.7 cm., for use in the inverted position inside the tubes containing carbohydrate media, as gas-collecting tubes.

These tubes, "unrimmed," may be of common thin glass as less than two per cent. are fit for use a second time.

Bohemian Flasks (Fig. 1).—These are the ordinary flasks of the chemical laboratory. A good variety, ranging in capacity from 250 to 3000 c.c., should be kept on hand. A modified form, known as the "pear-shaped" (Fig. 2), is preferable for the smaller sizes—i. e., 250 and 500 c.c.

Erlenmeyer's Flasks (Fig. 3).—Erlenmeyer's flasks of 75, 100, and 250 c.c. capacity are extremely useful. For use as culture flasks care should be taken to select only such as have a narrow neck of about 2 cm. in length.

Kolle's Culture Flasks (Fig. 4).—These thin, flat flasks (to contain agar or gelatine, which is allowed to solidify in a layer on one side) are extremely useful on account of the large nutrient surface available for growth. A surface cultivation in one of these will yield as much growth as ten or twelve "oblique" tube cultures. The wide mouth, however, is a disadvantage, and for many purposes thin, flat culture bottles known as Roux's bottles (Fig. 5) are to be preferred.

An even more convenient pattern is that used in the author's laboratory (Fig. 6), as owing to the greater depth of medium which it is possible to obtain in these flasks an exceedingly luxuriant growth is possible; the narrow neck reduces the chance of accidental contamination to a minimum and the general shape permits the flasks to be stacked one upon the other.

Filter Flasks or Kitasato's Serum Flasks (Fig. 7).—Various sizes, from 250 to 2000 c.c. capacity. These must be of stout glass, to resist the pressure to which they are subjected, but at the same time must be thoroughly well annealed, in order to withstand the temperature necessary for sterilisation.

All flasks should be either of Jena glass or the almost equally well-known Resistance or R glass, the extra initial expense being justified by the comparative immunity of the glass from breakage.

Petri's Dishes or "Plates" (Fig. 8, a).—These have now completely replaced the rectangular sheets of glass introduced by Koch for the plate method of cultivation. Each "plate" consists of a pair of circular discs of glass with sharply upturned edges, thus forming shallow dishes, one of slightly greater diameter than the other, and so, when inverted, forming a cover or cap for the smaller. Plates having an outside diameter of 10 cm. and a height of 1.5 cm. are the most generally useful. A batch of eighteen such plates is sterilised and stored in a cylindrical copper box (30 cm. high by 12 cm. diameter) provided with a "pull-off" lid. Inside each box is a copper stirrup with a circular bottom, upon which the plates rest, and by means of which each can be raised in turn to the mouth of the box (Fig. 9) for removal.

Capsules (Fig. 8, b and c).—These are Petri's dishes of smaller diameter but greater depth than those termed plates. Two sizes will be found especially useful—viz., 4 cm. diameter by 2 cm. high, capacity about 14 c.c.; and 5 cm. diameter by 2 cm. high, capacity about 25 c.c. These are stored in copper cylinders of similar construction to those used for plates, but measuring 20 by 6 cm. and 20 by 7 cm., respectively.

Graduated Pipettes.—Several varieties of these are required, viz.:

1. Pipettes of 1 c.c. capacity graduated in 0.1 c.c.

2. Pipettes of 1 c.c. capacity graduated in 0.01 c.c. (Fig. 10, a).

3. Pipettes of 10 c.c. capacity graduated in 0.1 c.c. (Fig. 10, b).

These should be about 30 cm. in length (1 and 2 of fairly narrow bore), graduated to the extreme point, and having at least a 10 cm. length of clear space between the first graduation and the upper end; the open mouth should be plugged with cotton-wool. Each variety should be sterilised and stored in a separate cylindrical copper case some 36 by 6 cm., with "pull-off" lid, upon which is stamped, in plain figures, the capacity of the contained pipettes.

The laboratory should also be provided with a complete set of "Standard" graduated pipettes, each pipette in the set being stamped and authenticated by a certificate from one of the recognised Physical Measurement Laboratories, such as Charlottenburg. These instruments are expensive and should be reserved solely for standardising the pipettes in ordinary use, and for calibrating small pipettes manufactured in the laboratory. Such a set should comprise, at least, pipettes delivering 10 c.c., 5 c.c., 2.5 c.c., 2 c.c., 1 c.c., 0.5 c.c., 0.25 c.c., 0.2 c.c., 0.1 c.c., 0.05 c.c., and 0.01 c.c., respectively.

In the immediately following sections are described small pieces of glass apparatus which should be prepared in the laboratory from glass tubing of various sizes. In their preparation three articles are essential; first a three-square hard-steel file or preferably a glass-worker's knife of hard Thuringian steel for cutting glass tubes etc.; next a blowpipe flame, for although much can be done with the ordinary Bunsen burner, a blowpipe flame makes for rapid work; and lastly a bat's-wing burner.

1. The glass-cutting knife. This article is sold in two forms, a bench knife (Fig. 11) and a pocket knife. The former is provided with a blade some 8 cm. in length and having two cutting edges. The cutting edge when examined in a strong light is seen to be composed of small closely set teeth, similar to those in a saw. The knife should be kept sharp by frequent stroppings on a sandstone hone. The pocket form, about 6-cm. long over all, consists of a small spring blade with one cutting edge mounted in scales like an ordinary pocket knife.

2. For real convenience of work the blowpipe should be mounted on a special table connected up with cylindrical bellows operated by a pedal. That figured (Fig. 12) is made by mounting a teak top 60 cm. square upon the uprights of an enclosed double-action concertina bellows (Enfer's) and provided with a Fletcher's Universal gas blowpipe.

3. An ordinary bat's-wing gas-burner mounted at the far corner of the table top is invaluable in the preparation of tubular apparatus with sharp curves, and for coating newly-made glass apparatus with a layer of soot to prevent too rapid cooling, and its usually associated result—cracking.

6. Sedimentation tubes 5×0.5 cm., for sedimentation reactions, etc., and for containing small quantities of fluid to be centrifugalised in the hÆmatocrit. These are made by taking 14-cm. lengths of stout glass tubing of the requisite diameter and heating the centre in the Bunsen or blowpipe flame. When the central portion is quite soft draw the ends quickly apart and then round off the pointed ends of the two test-tubes thus formed. With the glass-cutting knife cut off whatever may be necessary from the open ends to make the tubes the required length.

A rectangular block of "plasticine" (modelling clay) into which the conical ends can be thrust makes a very convenient stand for these small tubes.

Capillary Pipettes or Pasteur's Pipettes (Fig. 13 a).—These little instruments are invaluable, and a goodly supply should be kept on hand. They are prepared from soft-glass tubing of various-sized calibre (the most generally useful size being 8 mm. diameter) in the following manner: Hold a 10 cm. length of glass tube by each end, and whilst rotating it heat the central portion in the Bunsen flame or the blowpipe blast-flame until the glass is red hot and soft. Now remove it from the flame and steadily pull the ends apart, so drawing the heated portion out into a roomy capillary tube; break the capillary portion at its centre, seal the broken ends in the flame, and round off the edges of the open end of each pipette. A loose plug of cotton-wool in the open mouth completes the capillary pipette. After a number have been prepared, they are sterilised and stored in batches, either in metal cases similar to those used for the graduated pipettes or in large-sized test-tubes—sealed ends downward and plugged ends toward the mouth of the case.

The filling and emptying of the capillary pipette is most satisfactorily accomplished by slipping a small rubber teat (similar to that on a baby's feeding bottle but not perforated) on the upper end, after cutting or snapping off the sealed point of the capillary portion. If pressure is now exerted upon the elastic bulb by a finger and thumb whilst the capillary end is below the surface of the fluid to be taken up, some of the contained air will be driven out, and subsequent relaxation of that pressure (resulting in the formation of a partial vacuum) will cause the fluid to ascend the capillary tube. Subsequent compression of the bulb will naturally result in the complete expulsion of the fluid from the pipette (Fig. 14).

A modification of this pipette, in which a constriction or short length of capillary tube is introduced just below the plugged mouth (Fig. 13, b), will also be found extremely useful in the collection and storage of morbid exudations.

A third form, where the capillary portion is about 4 or 5 cm. long and only forms a small fraction of the entire length of the pipette (Fig. 13, c), will also be found useful.

"Blood" Pipettes (Fig 15).—Special pipettes for the collection of fairly large quantities of blood (as suggested by Pakes) should also be prepared. These are made from soft glass tubing of 1 cm. bore, in a similar manner to the Pasteur pipettes, except that the point of the blowpipe flame must be used in order to obtain the sharp shoulder at either end of the central bulb. The terminal tubes must retain a diameter of at least 1 mm., in order to avoid capillary action during the collection of the fluid.

For sterilisation and storage each pipette is placed inside a test-tube, resting on a wad of cotton-wool, and the tube plugged in the ordinary manner. As these tubes are used almost exclusively for blood work, it is usual to place a lance-headed hare-lip pin or a No. 9 flat Hagedorn needle inside the tube so that the entire outfit may be sterilised at one time.

For the collection of small quantities of blood for agglutination reactions and the like, many prefer a short straight piece of narrow glass tubing drawn out at either extremity to almost capillary dimensions. Such pipettes, about 8 cm. in length over all, are most conveniently sterilized in ordinary metal thermometer cases (Fig. 16).

Graduated Capillary Pipettes (Fig. 17).—These should also be made in the laboratory—from manometer tubing—of simple, convenient shape, and graduated by the aid of "standard" pipettes (in hundredths) to contain such quantities as 10, 50, and 90 c. mm., and carefully marked with a writing diamond. These, previously sterilised in large test-tubes, will be found extremely useful in preparing accurate percentage solutions, when only minute quantities of fluid are available.

Automatic ("Throttle") Pipettes.—These ingenious pipettes, introduced by Wright, can easily be calibrated in the laboratory and are exceedingly useful for graduating small pipettes, for measuring small quantities of fluids, in preparing dilutions of serum for agglutination reactions, etc. They are usually made from the Capillary Pasteur pipettes (Fig. 13, a). The following description of the manufacture of a 5 c. mm. pipette will serve to show how the small automatic pipettes are calibrated.

1. Select a pipette the capillary portion of which is fairly roomy in bore and possesses regular even walls, and remove the cotton-wool plug from the open end.

2. Heat the capillary portion near the free extremity in the by-pass flame of the bunsen burner and draw it out into a very fine hair-like tube and break this across. This hair-like extremity will permit the passage of air but is too fine for metallic mercury to pass.

3. From a standard graduated pipette deliver 5 c. mm. clean mercury into the upper wide portion of the pipette.

4. Adjust a rubber teat to the pipette and by pressure on the bulb gradually drive the mercury in an unbroken column down the capillary tube until it is stopped by the filiform extremity.

5. Cut off the capillary tube exactly at the upper level of the column of mercury, invert it and allow the mercury to run out.

6. Snap off the remainder of the capillary tube from the broad upper portion of the pipette which is now destined to form the covering tube or air chamber, or what we may term the "barrel." This barrel now has the lower end in the form of a truncated cone, the upper end being cut square. Remove the teat.

7. Introduce the capillary tube into this barrel with the filiform extremity uppermost, and the square cut end projecting about 0.5 cm. beyond the tapering end of the barrel.

8. Drop a small pellet of sealing wax into the barrel by the side of the capillary tube and then warm the tube at the gas flame until the wax becomes softened and makes an air-tight joint between the capillary tube and the end of the barrel.

9. Fit a rubber teat to the open end of the barrel, and so complete a pipette which can be depended upon to always aspirate and deliver exactly 5 cm. of fluid.

Slight modification of this procedure is necessary in making tubes to measure larger volumes than say 75 c. mm. Thus to make a throttle pipette to measure 100 c. mm.:

1. Take a short length of quill tubing and draw out one end into a roomy capillary stem, and again draw out the extremity into a fine hair point, thus forming a small Pasteur pipette with a hair-like capillary extremity.

2. With a standard pipette fill 100 c. mm. into the neck of this pipette, and make a scratch with a writing diamond at the upper level (a) of the mercury meniscus (Fig. 19, A).

Now force the mercury down into the capillary stem as far as it will go, so as to leave the upper part of the tube in the region of the diamond scratch empty (Fig. 19, B).

3. Heat the tube in the region of the diamond scratch in the blowpipe flame, and removing the tube from the flame draw it out so that the diamond scratch now occupies a position somewhere near the centre of this new capillary portion (Fig. 19, C).

4. Heat the tube in this position in the peep flame of the Bunsen burner, and draw it out into a hair-like extremity. Snap off the glass tube, leaving about 5 mm. of hair-like extremity attached to the upper capillary portion (Fig. 19, D). Allow the glass to cool.

5. Lift up the bulb by the long capillary stem and allow the mercury to return to its original position—an operation which will be facilitated by snapping off the hair-like extremity from the long piece of capillary tubing.

6. Mark on the capillary stem with a grease pencil the position of the end of the column of mercury (Fig. 19, E.)

7. Warm the capillary tubing at this spot in the peep flame of the Bunsen burner, and draw it out very slightly so that when cut at this position a pointed extremity will be obtained.

8. With a glass-cutting knife cut the capillary tube through at the point "b," and allow the mercury to run out.

9. Now apply a thick layer of sealing wax to the neck of the bulb.

10. Take a piece of 5 mm. bore glass tubing and draw it out as if making an ordinary Pasteur pipette.

11. Break the capillary portion off so as to leave a covering tube similar to that already used for the smaller graduated pipettes. Into this covering tube drop the graduated bulb and draw the capillary stem down through the conical extremity until further progress is stopped by the layer of sealing wax.

12. Warm the pipette in the gas flame so as to melt the sealing wax and make an air-tight joint.

13. Fit an india-rubber teat over the open end of the covering tube, and the automatic pipette is ready for use (Fig. 19, F).

Sedimentation Pipettes (Fig. 20).—These are prepared from 10 cm. lengths of narrow glass tubing by sealing one extremity, blowing a small bulb at the centre, and plugging the open end with cotton-wool; after sterilisation the open end is provided with a short piece of rubber tubing and a glass mouthpiece. When it is necessary to observe sedimentation reactions in very small quantities of fluid, these tubes will be found much more convenient than the 5 by 0.5 cm. test-tubes previously mentioned.

Pasteur pipettes fitted with india-rubber teats will also be found useful for sedimentation tests when dealing with minute quantities of serum, etc.

Fermentation Tubes (Fig. 21).—These are used for the collection and analysis of the gases liberated from the media during the growth of some varieties of bacteria and may be either plain (a) or graduated (b). A simple form (Fig. 21, c) may be made from 14 cm. lengths of soft glass tubing of 1.5 cm. diameter. The Bunsen flame is applied to a spot some 5 cm. from one end of such a piece of tubing and the tube slightly drawn out to form a constriction, the constricted part is bent in the bat's-wing flame, to an acute angle, and the open extremity of the long arm sealed off in the blowpipe flame. The open end of the short arm is rounded off and then plugged with cotton-wool, and the tube is ready for sterilisation.

CLEANING OF GLASS APPARATUS.

All glassware used in the bacteriological laboratory must be thoroughly cleaned before use, and this rule applies as forcibly to new as to old apparatus, although the methods employed may vary slightly.

To Clean New Test-tubes.—

1. Place the tubes in a bucket or other convenient receptacle, fill with water and add a handful of "Sapon" or other soap powder. See that the tubes are full and submerged.

2. Fix the bucket over a large Bunsen flame and boil for thirty minutes—or boil in the autoclave for a similar period.

3. Cleanse the interior of the tubes with the aid of test-tube brushes, and rinse thoroughly in cold water.

4. Invert the tubes and allow them to drain completely.

5. Dry the tubes and polish the glass inside and out with a soft cloth, such as selvyt.

New flasks, plates, and capsules must be cleaned in a similar manner.

To Clean New Graduated Pipettes.—

1. Place the pipettes in a convenient receptacle, filled with water to which soap powder has been added.

2. Boil the water vigorously for twenty minutes over a Bunsen flame.

3. Rinse the pipettes in running water and drain.

4. Run distilled water through the pipettes and drain.

5. Run rectified spirits through the pipette and drain as completely as possible.

6. Place the pipettes in the hot-air oven (vide page 31), close the door, open the ventilating slide, and run the temperature slowly up to about 80° C. Turn off the gas and allow the oven to cool.

Or 6a. Attach each pipette in turn to the rubber tube of the foot bellows, or blowpipe air-blast, and blow air through the pipette until the interior is dry.

Glassware that has already been used is regarded as infected, and is treated in a slightly different manner.

Infected Test-tubes.—

1. Pack the tubes in the wire basket of the autoclave (having previously removed the cotton-wool plugs, caps, etc.), in the vertical position, and before replacing the basket see that there is a sufficiency of water in the bottom of the boiler. Now attach a piece of rubber tubing to the nearest water tap, and by means of this fill each tube with water.

2. Disinfect completely by exposing the tubes, etc., to a temperature of 120° C. for twenty minutes (vide page 37).

(If an autoclave is not available, the tubes must be placed in a digester, or even a large pan or pail with a tightly fitting cover, and boiled vigorously for some thirty to forty-five minutes to ensure disinfection.)

3. Whilst still hot, empty each tube in turn and roughly clean its interior with a stiff test-tube brush.

4. Place the tubes in a bucket or other convenient receptacle, fill with water and add a handful of Sapon or other soap powder. See that the tubes are full and submerged.

5. Fix the bucket over a large Bunsen flame and boil for thirty minutes.

6. Cleanse the interior of the tubes with the aid of test-tube brushes, and rinse thoroughly in cold water.

7. Drain off the water and immerse tubes in a large jar containing water acidulated with 2 to 5 per cent. hydrochloric acid. Allow them to remain there for about fifteen minutes.

8. Remove from the acid jar, drain, rinse thoroughly in running water, then with distilled water.

9. Invert the tubes and allow them to drain completely.

Dry the tubes and polish the glass inside and out with a soft cloth, such as selvyt.

Infected flasks, plates, and capsules must be treated in a similar manner.

Flasks which have been used only in the preparation of media must be cleaned immediately they are finished with. Fill each flask with water to which some soap powder and a few crystals of potassium permanganate have been added, and let boil over the naked flame. The interior of the flask can then usually be perfectly cleaned with the aid of a flask brush, but in some cases water acidulated with 5 per cent. nitric acid, or a large wad of wet cotton-wool previously rolled in silver sand, must be shaken around the interior of the flask, after which rinse thoroughly with clean water, dry, and polish.

Infected Pipettes.—

1. Plunge infected pipettes immediately after use into tall glass cylinders containing a 2 per cent. solution of lysol, and allow them to remain therein for some days.

2. Remove from the jar and drain. Boil in water to which a little soap has been added, for thirty minutes.

3. Rinse thoroughly in cold water.

4. Immerse in 5 per cent. nitric acid for an hour or two.

5. Rinse again in running water to remove all traces of acid.

6. Complete the cleaning as described under "new pipettes."

When dealing with graduated capillary pipettes employed for blood or serum work (whether new or infected), much time is consumed in the various steps from 5 onward, and the cleansing process can be materially hastened if the following device is adopted.

Fit up a large-sized Kitasato's filter flask to a Sprengel's suction pump or a Geryk air pump (see page 43). To the side tubulure of the filter flask attach a 20 cm. length of rubber pressure tubing having a calibre sufficiently large to admit the ends of the pipettes.

Next fill a small beaker with distilled water. Attach the first pipette to the free end of the rubber tubing, place the pipette point downward in the beaker of water and start the pump (Fig. 22).

When all the water has been aspirated through the pipette into the filter flask, fill the beaker with rectified spirit and when this is exhausted refill with ether. Detach the pipette and dry in the hot-air oven.

Slides and cover-slips (Fig. 23), when first purchased, have "greasy" surfaces, upon which water gathers in minute drops and effectually prevents the spreading of thin, even films.

Microscopical Slides.—The slides in general use are those known as "three by one" slips (measuring 3 inches by 1 inch, or 76 by 26 mm.), and should be of good white crown glass, with ground edges.

New slides should be allowed to remain in alcohol acidulated with 5 per cent. hydrochloric acid for some hours, rinsed in running water, roughly drained on a towel, dried, and finally polished with a selvyt cloth.

If only a few slides are required for immediate use a good plan is to rub the surface with jeweler's emery paper (Hubert's 00). A piece of hard wood 76×26×26 mm. with a piece of this emery paper gummed tightly around it is an exceedingly useful article on the microscope bench.

Cover-slips.—The most useful sizes are the 19 mm. squares for ordinary cover-glass film preparations, and 38 by 19 mm. rectangles for blood films and serial sections; both varieties must be of "No. 1" thickness, which varies between 0.15 and 0.22 mm., that they may be available for use with the high-power immersion lenses.

Cover-slips should be cleaned in the following manner:

1. Drop the cover-slips one by one into an enamelled iron pot or tall glass beaker, containing a 10 per cent. solution of chromic acid.

2. Heat over a Bunsen flame and allow the acid to boil gently for twenty minutes.

3. Turn the cover-slips out into a flat glass dish and wash in running water under the tap until all trace of yellow colour has disappeared. During the washing keep the cover-slips in motion by imparting a rotatory movement to the dish.

4. Wash in distilled water in a similar manner.

5. Wash in rectified spirit.

6. Transfer the cover-slips, by means of a pair of clean forceps, previously heated in the Bunsen flame to destroy any trace of grease, to a small beaker of absolute alcohol.

Drain off the alcohol and transfer the cover-slips, by means of the forceps, to a wide-mouthed glass pot, containing absolute alcohol, in which they are to be stored, and stopper tightly.

Used Slides and Cover-slips.—Used slides with the mounted cover-slip preparations, and cover-slips used for hanging-drop mounts, should, when discarded, be thrown into a pot containing a 2 per cent. solution of lysol.

After immersion therein for a week or so, even the cover-slips mounted with Canada balsam can be readily detached from their slides.

Slides.—

1. Wash the slides thoroughly in running water.

2. Boil the slides in water to which "sapon" has been added, for half an hour.

3. Rinse thoroughly in cold water.

4. Dry and polish with a dry cloth.

Cover-slips.—

1. Wash the cover-slips thoroughly in running water.

2. Boil the cover-slips in 10 per cent. solution of chromic acid, as for new cover-slips.

3. Wash thoroughly in running water.

4. Pick out those cover-slips which show much adherent dirty matter, and rub them between thumb and forefinger under the water tap. The dirt usually rubs off easily, as it has become friable from contact with the chromic acid.

5. Return all the cover-slips to the beaker, fill in fresh chromic acid solution, and treat as new cover-slips.

PLUGGING TEST-TUBES AND FLASKS.

Before sterilisation all test-tubes and flasks must be carefully plugged with cotton-wool, and for this purpose best absorbent cotton-wool (preferably that put up in cylindrical one-pound packets and interleaved with tissue paper—known as surgeons' wool) should be employed.

1. For a test-tube or a small flask, tear a strip of cotton-wool some 10 cm. long by 2 cm. wide from the roll.

2. Turn in the ends neatly and roll the strip of wool lightly between the thumb and fingers of both hands to form a long cylinder.

3. Double this at the centre and introduce the now rounded end into the open mouth of the tube or flask.

4. Now, whilst supporting the wool between the thumb and fingers of the right hand, rotate the test-tube between those of the left, and gradually screw the plug of wool into its mouth for a distance of about 2.5 cm., leaving about the same length of wool projecting.

The plug must be firm and fit the tube or flask fairly tightly, sufficiently tightly in fact to bear the weight of the glass plus the amount of medium the vessel is intended to contain, but not so tightly as to prevent it from being easily removed by a screwing motion when grasped between the fourth, or third and fourth, fingers, and the palm of the hand.

For a large flask a similar but larger strip of wool must be taken; the method of making and inserting the plug is identical.