The Elements of Bacteriological Technique Part 10

The eikonometer should then be applied to the eyepiece and the size of the object read off on the eikonometer scale as millimetres, and the actual size calculated by dividing the observed size by the magnification constant for the particular optical combination employed in the observation.

(c) By means of the filar micrometer.

[Ill.u.s.tration: FIG. 58.--Ramsden's Filar micrometer.]

[Ill.u.s.tration: FIG. 59.--Ramsden's micrometer field, a, fixed wire; b, reference wire (fixed); c, travelling wire.]

The ~Filar~ or cobweb Micrometer (Ramsden's micrometer) eyepiece (Fig. 58) consists of an ocular having a fine "fixed" wire stretching horizontally across the field (Fig. 59), a vertical reference wire--fixed--adjusted at right angles to the first; and a fine wire, parallel to the reference wire, which can be moved across the field by the action of a micrometer screw; the drum head is divided into one hundred parts, which successively pa.s.s a fixed index as the head is turned. In the lower part of the field is a comb with the intervals between its teeth corresponding to one complete revolution of this screw-head.

As in the previous method, the value of each division of the micrometer scale (i. e., the comb) must first be determined for each optical combination. This is effected as follows:

1. Place the filar micrometer and the stage micrometer in their respective positions.

2. Rotate the screw of the filar micrometer until the movable wire coincides with the fixed one, and the index marks zero on the drum head.

(If when the drum head is at zero the two wires do not exactly coincide they must be adjusted by loosening the drum screw and resetting the drum.)

3. Focus the scale of each micrometer accurately, and make the lines on them parallel.

4. Rotate the head of the micrometer screw until the movable line has transversed one division of the stage micrometer. Note the number of complete revolutions (by means of the recording comb) and the fractions of a revolution (by means of scale on the head of the micrometer screw), which are required to measure the 0.01 mm.

5. Make several such estimations and average the results.

6. Note the optical combination employed in this experiment and record it carefully, together with the micrometer value in terms of .

7. Repeat this process for each of the different optical combinations and record the results.

To measure an object by this method, simply note the number of revolutions and fractions of a revolution of the screw-head required to traverse such object from edge to edge, and express the result as _micra_ by reference to the recorded values for that particular optical combination.

_Microscope Illuminant._--In tropical and subtropical regions diffuse daylight is the best illuminant. In temperate climes however daylight of the desirable quant.i.ty is not always available, and recourse must be had to oil lamps, gas lamps--preferably those with incandescent mantles--and electricity; and of these the last is undoubtedly the best.

A handy lamp holder which can be manufactured in the laboratory is shown in Fig. 60. It consists of a base board weighted with lead to which is attached the ordinary domestic lamp holder, and behind this is fastened a curved sheet-iron reflector. An obscured metal filament lamp of about 16 candle power gives the most suitable light, and if monochromatic light is needed, the blue grease pencil is streaked over the side of the lamp nearest the microscope; the current is switched on and when the gla.s.s bulb is warm, rubbing with a wad of cotton-wool will readily distribute the blue greasy material in an even film over the ground gla.s.s.

[Ill.u.s.tration: FIG. 60.--Electric microscope lamp.]

FOOTNOTES:

[1] Its importance will be realised, however, when it is stated in the words of the late Professor Abbe: "The numerical aperture of a lens determines all its essential qualities; the brightness of the image increases with a given magnification and other things being equal, as the square of the aperture; the resolving and defining powers are directly related to it, the focal depth of differentiation of depths varies inversely as the aperture, and so forth."

[2] Made by Mr. Otto Baumbach, 10, Lime Grove, Manchester.

V. MICROSCOPICAL EXAMINATION OF BACTERIA AND OTHER MICRO-FUNGI.

APPARATUS AND REAGENTS USED IN ORDINARY MICROSCOPICAL EXAMINATION.

The following comprises the essential apparatus and reagents for routine work with which each student should be provided.

1. India-rubber "change-mat" upon which cover-gla.s.ses may be rested during the process of staining.

2. Squares of blotting paper about 10 cm., for drying cover-slips and slides.

(The filter paper known as "German lined"--a highly absorbent, closely woven paper, having an even surface and no loose "fluff" to adhere to the specimens--is the most useful for this purpose.)

[Ill.u.s.tration: FIG. 61.--Disinfectant Jar.]

3. Gla.s.s jar filled with 2 per cent. lysol solution for the reception of infected cover-gla.s.ses and infected pipettes, etc.

4. A square glazed earthenware box with a loose lining containing 2 per cent. lysol solution for the reception of infected material and used slides. The bottom of the lining is perforated so that when full the lining and its contents can be lifted bodily out of the box, when the disinfectant solution drains away and the slides, etc., can easily be emptied out. The empty lining is then returned to the box with its disinfectant solution (Fig. 61).

5. Bunsen burner provided with "peep-flame" by-pa.s.s.

6. Porcelain trough holding five or six hanging-drop slides (Fig. 62).

[Ill.u.s.tration: FIG. 62.--Hanging-drop slides: a, Double cell seen from above; b, single cell seen from the side.]

The best form of hanging-drop slide is a modification of Boettcher's gla.s.s ring slide, and is prepared by cementing a circular cell of tin, 13 to 15 mm. diameter, and 1 to 2 mm. in height, to the centre of a 3 by 1 slip by means of Canada balsam. It is often extremely convenient to have two of these cells cemented close together on one slide (Fig. 62, a).

Another form of hanging-drop slide is made in which a circular or oval concavity or "cell" is ground out of the centre of a 3 by 1 slip. These are more expensive, less convenient to work with, and are more easily contaminated by drops of material under examination, and should be carefully avoided.

7. Three aluminium rods (Fig. 63), each about 25 cm. long and carrying a piece of 0.015 gauge platino-iridium wire 7.5 cm. in length. The end of one of the wires is bent round to form an oval loop, of about 1 mm. in its short diameter, and is termed a loop or an oese; the terminal 3 or 4 mm. of another wire is flattened out by hammering it on a smooth iron surface to form a "spatula"; the third is left untouched or is pointed by the aid of a file. These instruments are used for inoculating culture tubes and preparing specimens for microscopical examination.

[Ill.u.s.tration: FIG. 63.--Ends of platinum rods. a, loop; b, spatula; c, needle.]

The method of mounting these wires may be described as follows:

Take a piece of aluminium wire 25 cm. long and about 0.25 cm. in diameter, and drill a fine hole completely through the wire about a centimetre from one end. Sink a straight narrow channel along one side of the wire, in its long axis, from the hole to the nearest end, shallow at first, but gradually becoming deeper.

On the opposite side of the wire make a short cut, 2 mm. in length, leading from the hole in the same direction. [The use of a fine dental drill and small circular saw, worked by a dental motor facilitates the manufacture of these aluminium handled instruments.]

Now pa.s.s one end of the platinum wire through the hole, turn up about 2 mm. at right angles and press the short piece into the short cut. Turn the long end of the wire sharply, also at right angles, and sink it into the long channel so that it emerges from about the centre of the cut end of the aluminium wire (Fig. 63). A few sharp taps with a watch maker's hammer will now close in the sides of the two channels over the wire and hold it securely.

[Ill.u.s.tration: FIG. 64.--Platinum rod in aluminium handle--method of mounting.

The platinum wire may be fused into the end of a piece of gla.s.s rod, but such a handle is vastly inferior to aluminium and is not to be recommended.]

8. Two pairs of sharp-pointed spring forceps (10 cm. long), one of which must be kept perfectly clean and reserved for handling clean cover-slips, the other being for use during staining operations.

9. A box of clean 3 by 1 gla.s.s slips.

10. A gla.s.s capsule with tightly fitting (ground on) gla.s.s lid, containing clean cover-slips in absolute alcohol.

11. One of Faber's "grease pencils" (yellow, red, or blue) for writing on gla.s.s.

12. A wooden rack (Fig. 65) with twelve drop-bottles (Fig. 66) each 60 c.c. capacity, containing

Aniline water.