The Elements of Bacteriological Technique Part 2

They are prepared from soft-gla.s.s tubing of various-sized calibre (the most generally useful size being 8 mm. diameter) in the following manner: Hold a 10 cm. length of gla.s.s tube by each end, and whilst rotating it heat the central portion in the Bunsen flame or the blowpipe blast-flame until the gla.s.s 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.

[Ill.u.s.tration: FIG. 13.--Capillary pipettes. a, b, c.]

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).

[Ill.u.s.tration: FIG. 14.--Filling the capillary teat-pipette.]

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 quant.i.ties of blood (as suggested by Pakes) should also be prepared. These are made from _soft_ gla.s.s 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.

[Ill.u.s.tration: FIG. 15.--Blood pipettes and hair-lip pin in a test-tube.]

[Ill.u.s.tration: FIG. 16.--Blood-pipette in metal thermometer case.]

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 quant.i.ties of blood for agglutination reactions and the like, many prefer a short straight piece of narrow gla.s.s 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 quant.i.ties 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 quant.i.ties of fluid are available.

[Ill.u.s.tration: FIG. 17.--Capillary graduated pipettes.]

~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 quant.i.ties 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-pa.s.s 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 pa.s.sage of air but is too fine for metallic mercury to pa.s.s.

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.

[Ill.u.s.tration: FIG. 18.--Throttle pipette--small capacity.]

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).

[Ill.u.s.tration: FIG. 19.--Making throttle pipettes--large capacity]

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 gla.s.s tube, leaving about 5 mm. of hair-like extremity attached to the upper capillary portion (Fig. 19, D). Allow the gla.s.s 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 gla.s.s-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 gla.s.s 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 gla.s.s 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 gla.s.s mouthpiece. When it is necessary to observe sedimentation reactions in very small quant.i.ties of fluid, these tubes will be found much more convenient than the 5 by 0.5 cm. test-tubes previously mentioned.

[Ill.u.s.tration: FIG. 20.--Sedimentation pipette.]

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

[Ill.u.s.tration: FIG. 21.--Fermentation tubes.]

~Fermentation Tubes~ (Fig. 21).--These are used for the collection and a.n.a.lysis 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 gla.s.s 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 GLa.s.s APPARATUS.

All gla.s.sware 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.~--