The essentials of a microscope for bacteriological work may be briefly summed up as follows:

The instrument, of the monocular type, must be of good workmanship and well finished, rigid, firm, and free from vibration, not only when upright, but also when inclined to an angle or in the horizontal position. The various joints and movements must work smoothly and precisely, equally free from the defects of "loss of time" and "slipping." All screws, etc., should conform to the Royal Microscopical Society's standard. It must also be provided with good lenses and a sufficiently large stage. The details of its component parts, to which attention must be specially directed, are as follows:

1. The Base or Foot (Fig. 40, a).—Two elementary forms—the tripod (Fig. 41, a) and the vertical column set into a plate known as the "horse-shoe" (Fig. 41, b)—serve as the patterns for countless modifications in shape and size of this portion of the stand. The chief desiderata—stability and ease of manipulation—are attained in the first by means of the "spread" of the three feet, which are usually shod with cork; in the second, by the dead weight of the foot-plate. The tripod is mechanically the more correct form, and for practical use is much to be preferred. Its chief rival, the Jackson foot (Fig. 41, c), is based upon the same principle, and on the score of appearance has much to recommend it.

2. The body tube (Fig. 40, b) may be either that known as the "long" or "English" (length 250 mm.), or the "short" or "Continental" (length 160 mm.). Neither length appears to possess any material advantage over the other, but it is absolutely necessary to secure objectives which have been manufactured for the particular tube length chosen. In the high-class microscope of the present day the body tube is usually shorter than the Continental, but is provided with a draw tube which, when fully extended, gives a tube length greater than the English, thus permitting the use of either form of objective.

3. The coarse adjustment (Fig. 40, c) should be a rack-and-pinion movement, steadiness and smoothness of action being secured by means of accurately fitting dovetailed bearings and perfect correspondence between the teeth of the rack and the leaves of the pinion (Fig. 42). Also provision should be made for taking up the "slack" (as by the screws AA, Fig. 42).

4. The fine adjustment (Fig. 40, d) should on no account depend upon the direct action of springs, but should be of the lever pattern, preferably the Nelson (Fig. 43). In this form the unequal length of the arms of the lever secures very delicate movement, and, moreover, only a small portion of the weight of the body tube is transmitted to the thread of the vertical screw actuating the movement.

A spindle milled head (Fig. 44) will be found a very useful device to have fitted in place of the ordinary milled head controlling the fine adjustment. In this contrivance the axis of the milled head is prolonged upward in a short column, the diameter of which is one-sixth of that of the head. The spindle can be rapidly rotated between the fingers for medium power adjustments while the larger milled head can be slowly moved when focussing high powers.

5. The stage (Fig. 40, e) should be square in shape and large in area—at least 12 cm.—flat and rigid, in order to afford a safe support for the Petri dish used for plate cultivations; and should be supplied with spring clips (removable at will) to secure the 3 by 1 glass slides.

A mechanical stage must be classed as a necessity rather than a luxury so far as the bacteriologist is concerned, as when working with high powers, and especially when examining hanging-drop specimens, it is almost impossible to execute sufficiently delicate movements with the fingers. In selecting a mechanical stage, preference should be given to one which forms an integral part of the instrument (Fig. 45) rather than one which needs to be clamped on to an ordinary plain stage every time it is required, and its traversing movements should be controlled by stationary milled heads (Fig. 45, AA'). The shape of the aperture is a not unimportant point; it should be square to allow of free movement over the substage condenser. The mechanical stage should be tapped for three (removable) screw studs to be used in place of the sliding bar, so that if desired the Vernier finder (Fig. 45, BB'), such as is usually fitted to this class of stage, or a Maltwood finder, may be employed.

6. Diaphragm.—Separate single diaphragms must be avoided; a revolving plate pierced with different sized apertures and secured below the stage is preferable, but undoubtedly the best form is the "iris" diaphragm (Fig. 46) which enters into the construction of the substage condenser.

7. The substage condenser is a necessary part of the optical outfit. Its purpose is to collect the beam of parallel rays of light reflected by the plane mirror, by virtue of a short focus system of lenses, into a cone of large aperture (reducible at will by means of an iris diaphragm mounted as a part of the condenser), which can be accurately focussed on the plane of the object. This focussing must be performed anew for each object, on account of the variation in the thickness of the slides.

The form in most general use is that known as the AbbÉ (Fig. 47) and consists of a plano-convex lens mounted above a biconvex lens. This combination is carried in a screw-centering holder known as the substage below the stage of the microscope (Fig. 40 f), and must be accurately adjusted so that its optical axis coincides with that of the objective. Vertical movement of the entire substage apparatus effected by means of a rack and pinion is a decided advantage, and some means should be provided for temporarily removing the condenser from the optical axis of the microscope.

With the oil immersion objective, however, an achromatic condenser, giving an illuminating cone of about 0.9, should be used if the full value of the lens is to be obtained. It is generally assumed that a good objective requires an illuminating cone equivalent to two-thirds of its numerical aperture. The best AbbÉ condenser transmits a cone of about .45 whilst the aperture of the 1/12 inch immersion lenses of different makers varies from 1.0 to 1.4, hence, the efficiency of these lenses is much curtailed if the condenser is merely the AbbÉ. These improved condensers must be absolutely centered to the objective and capable of very accurate focussing otherwise much of their value is lost.

8. Mirrors.—Below the substage condenser is attached a gymbal carrying a reversible circular frame with a plane mirror on one side and a concave mirror on the other (Fig. 40, g). The plane mirror is that usually employed, but occasionally, as for example when using low powers and with the condenser racked down and thrown out of the optical axis, the concave mirror is used.

9. Oculars, or Eyepieces.—Those known as the Huyghenian oculars (Fig. 48) will be sufficient for all ordinary work without resorting to the more expensive "compensation" oculars. Two or three, magnifying the "real" image (formed by the objective) four, six, or eight times respectively, form a useful equipment.

As an accessory Ehrlich's Eyepiece is a very useful piece of apparatus when the enumeration of cells or bacteria has to be carried out. This is an ordinary eyepiece fitted with an adjustable square diaphragm operated by a lever projecting from the side of the mount. Three notches are made in one of the sides of the square and by moving the lever square aperture can be reduced to three-quarters, one-half or one-quarter of the original size.

10. Objectives.—Three objectives are necessary: one for low-power work—e. g., 1 inch, 2/3 inch, or 1/2 inch; one for high-power work—e. g., 1/12 inch oil immersion lens; and an intermediate "medium-power" lens—e. g., 1/6 inch or 1/8 inch (dry). These lenses must be carefully selected, especial attention being paid to the following points:

(a) Correction of Spherical Aberration.—Spherical aberration gives rise to an ill-defined image, due to the central and peripheral rays focussing at different points.

(b) Correction of Chromatic Aberration.—Chromatic aberration gives rise to a coloured fringe around the edges of objects due to the fact that the different-coloured rays of the spectrum possess varying refrangibilities and that a simple lens acts toward them as a prism.

(c) Flatness of Field.—The ideal visual field would be large and, above all, flat; in other words, objects at the periphery of the field would be as distinctly "in focus" as those in the centre. Unfortunately, however, this is an optical impossibility and the field is always spherical in shape. Some makers succeed in giving a larger central area that is in focus at one time than others, and although this may theoretically cause an infinitesimal sacrifice of other qualities, it should always be sought for. Successive zones and the entire peripheral ring should come into focus with the alteration of the fine adjustment. This simultaneous sharpness of the entire circle is an indication of the perfect centering of the whole of the lenses in the objective.

(d) Good Definition.—Actual magnification is, within limits, of course, of less value than clear definition and high resolving power, for it is upon these properties we depend for our knowledge of the detailed structure of the objects examined.

(e) Numerical Aperture (N. A.).—The numerical aperture may be defined, in general terms, as the ratio of the effective diameter of the back lens of the objective to its equivalent focal length. The determination of this point is a process requiring considerable technical skill and mathematical ability, and is completely beyond the powers of the average microscopist.[1]

Although with the increase in power it is correspondingly difficult to combine all these corrections in one objective, they are brought to a high pitch of excellence in the present-day "achromatic" objectives, and so remove the necessity for the use of the higher priced and less durable apochromatic lenses.

In selecting objectives the best "test" objects to employ are:

Accessories.—Eye Shade (Fig. 49).—This piece of apparatus consists of a pear-shaped piece of blackened metal or ebonite, hinged to a collar which rotates on the upper part of the body tube of the microscope. It can be used to shut out the image of surrounding objects from the unoccupied eye, and when carrying out prolonged observations will be found of real service.

Nosepiece.—Perhaps the most useful accessory is a nosepiece to carry two of the objectives (Fig. 50), or, better still, all three (Fig. 51). This nosepiece, preferably constructed of aluminium, must be of the covered-in type, consisting of a curved plate attached to the lower end of the body tube—a circular aperture being cut to correspond to the lumen of that tube. To the under surface of this plate is pivoted a similarly curved plate, fitted with three tubulures, each of which carries an objective. By rotating the lower plate each of the objectives can be brought successively in to the optical axis of the microscope.

For critical work and particularly for photo-micrography, however, the interchangeable nosepiece is by no means perfect as it is next to impossible to secure accurate centreing of each lens in the optical axis. For special purposes, therefore, it is necessary to employ a special nosepiece such as that made by Zeiss or Leitz into which each objective slides on its own carrier and upon which it is accurately centred.

Warm Stage (Fig. 52).—This is a flat metal case containing a system of tubes through the interior of which water of any required temperature can be circulated. It is made to clamp on to the stage of the microscope by the screws A A', and is perforated with a large hole coinciding with the optical axis of the microscope; a short tube B, projecting from one end of the warm stage permits water of the desired temperature to be conducted from a reservoir through a length of rubber tubing to the interior of the stage and a similar tube at the other end B' of the stage allows exit to the waste water. By raising the temperature of hanging-drop preparations, etc., placed upon it, above that of the surrounding atmosphere, the warm stage renders possible exact observations on spore germination, hanging-drop cultivations, etc.

A better form is the electrical hot stage designed by Lorrain Smith;[2] it requires the addition of a lamp resistance and sliding rheostat, also a delicate ammeter reading to .01 of an ampÈre. It consists of a wooden frame supporting a flat glass bulb with a long neck bent upward at an obtuse angle (Fig. 53). The bulb is filled with liquid paraffin, which rises in the open neck when expanded by heat. The neck also accommodates the thermometer. Two coils of manganin wire run in the paraffin at opposite sides of the bulb (outside the field of vision), coupled to brass terminals on the wooden frame by platinum wire fused into the glass. The resistance of the two coils in series is about 10 ohms. A current of 2-1/2 ampÈres is needed, and is conducted to the coils in the stage through the rheostat. With the help of the ammeter any desired temperature can be obtained and maintained, up to about 200° C. If immersion oil contact is made between the top lens of the condenser and the lower surface of the bulb, this stage works very well indeed with the 1/12-inch oil immersion lens.

Dark Ground or Paraboloid Condenser.—This is an immersion substage condenser of high aperture by means of which unstained objects such as bacteria can be shown as bright white particles upon a dense black background. The central rays of light are blocked out by means of an opaque stop while the peripheral rays are reflected from the paraboloidal sides of the condenser and refracted by the object viewed. To obtain the best results with this type of condenser a powerful illuminant—such as a small arc lamp or an incandescent gas lamp—is needed, together with picked slides of a certain thickness (specified for the particular make of condenser but generally 1 mm.) and specially thin cover-glasses (not more than 0.17 mm.) The objective must not have a higher NA than 1.0, consequently immersion lenses must be fitted with an internal stop to cut down the aperture.

Micrometer.—Some form of micrometer for the purpose of measuring bacteria and other objects is also essential. Details of those in general use will be found in the following pages.

Object Marker (Fig. 54).—This is an exceedingly useful piece of apparatus. Made in the form of an objective, the lenses are replaced by a diamond point, set slightly out of the centre, which can be rotated by means of a milled plate. Screwed on to the nosepiece in place of the objective, rotation of the diamond point will rule a small circle on the object slide to permanently record the position of an interesting portion of the specimen. The diamond is mounted on a spring which regulates the pressure, and the size of the circle can be adjusted by means of a lateral screw.

METHODS OF MICROMETRY.

The unit of length as applied to the measurement of microscopical objects is the one-thousandth part of a millimetre (0.001 mm.), denominated a micron (sometimes, and erroneously, referred to as a micro-millimetre), and indicated in writing by the Greek letter µ. Of the many methods in use for the measurement of bacteria, three only will be here described, viz.:

(a) By means of the Camera Lucida.

(b) By means of the ocular or Eyepiece Micrometer.

(c) By means of the Filar Micrometer (Ramsden's micrometer eyepiece).

For each of these methods a stage micrometer is necessary. This is a 3 by 1 inch glass slip having engraved on it a scale divided to hundredths of a millimetre (0.01 mm.), every tenth line being made longer than the intervening ones, to facilitate counting; and from these engraved lines the measurement in every case is evaluated. A cover-glass is cemented over the scale to protect it from injury.

(a) By means of the Camera Lucida.

1. Attach a camera lucida (of the Wollaston, Beale, or AbbÉ pattern) (Fig. 55) to the eyepiece of the microscope.

2. Adjust the micrometer on the stage of the microscope and accurately focus the divisions.

3. Project the scale of the stage micrometer on to a piece of paper and with pen or pencil sketch in the magnified image, each division of which corresponds to 10µ. Mark on the paper the optical combination (ocular objective and tube length) employed to produce this particular magnification.

4. Repeat this procedure for each of the possible combinations of oculars and objectives fitted to the microscope supplied, and carefully preserve the scales thus obtained.

To measure an object by this method simply project the image on to the scale corresponding to the particular optical combination in use at the moment. Read off the number of divisions it occupies and express them as micra.

In place of preserving a scale for each optical combination, the object to be measured and the micrometer scale may be projected and sketched, in turn, on the same piece of paper, taking particular care that the centre of the eyepiece is 25 cm. from the paper on which the divisions are drawn.

(b) By means of the Eyepiece Micrometer.

The eyepiece micrometer is a circular glass disc having engraved on it a scale divided to tenths of a millimetre (0.1 mm.) (Fig. 56), or the entire surface ruled in 0.1 mm. squares (the net micrometer) (Fig. 57). It can be fitted inside the mount of any ocular just above the aperture of the diaphragm and must be adjusted exactly in the focus of the eye lens.

Some makers mount the glass disc together with a circular cover-glass in such a way that when placed in position in any Huyghenian eyepiece of their own manufacture, the scale is exactly in focus for normal vision. Special eyepieces are also obtainable having a sledging adjustment to the eye lens for focussing the micrometer.

The value of one division of the micrometer scale must first be ascertained for each optical combination by the aid of the stage micrometer, thus:

1. Insert the eyepiece micrometer inside the ocular and adjust the stage micrometer on the stage of the microscope.

2. Focus the scale of the stage micrometer accurately; the lines will appear to be immediately below those of the eyepiece micrometer. Make the lines on the two micrometers parallel by rotating the ocular.

3. Make two of the lines on the ocular micrometer coincide with those bounding one division of the stage micrometer; this is effected by increasing or diminishing the tube length; and note the number of included divisions.

4. Calculate the value of each division of the eyepiece micrometer in terms of µ, by means of the following formula:

5. Note the optical combination employed in this experiment and record it with the calculated micrometer value.

Repeat this process for each of the other combinations. Carefully record the results.

To measure an object by this method read off the number of divisions of the eyepiece micrometer it occupies and express the result in micra by a reference to the standard value for the particular optical combination employed.

Zeiss prepares a compensating eyepiece micrometer for use with his apochromatic objectives, the divisions of which are so computed that (with a tube length of 160 mm.) the value of each is equivalent to as many micra as there are millimetres in the focal length of the objective employed.

Wright's Eikonometer is really a modification of the eyepiece micrometer for rapidly measuring microscopical objects by direct inspection, having previously determined the magnifying power of the particular optical combination employed. It is a small piece of apparatus resembling an eyepiece, with a sliding eye lens, which can be accurately focussed on a micrometer scale fixed within the instrument. When placed over the microscope ocular the divisions of this scale measure the actual size of the virtual image in millimetres.

In order to use this instrument for direct measurement, it is first necessary to determine the magnifying power of each combination of ocular, tube length and objective.

Place a stage micrometer divided into hundredths of a millimetre on the microscope stage and focus accurately.

Rest the eikonometer on the eyepiece. Observation through the eikonometer shows its micrometer scale superposed on the image of the stage micrometer.

Rotate the eikonometer until the lines on the two scales are parallel, and make the various adjustments to ensure that two lines on the eikonometer scale coincide with two lines on the stage micrometer.

For the sake of illustration it may be assumed that five of the divisions on the stage micrometer accurately fill one of the divisions of the eikonometer scale; this indicates a magnifying power of 500 as the constant for that particular optical combination, and a record should be made of the fact.

The magnification constants of the various other optical combinations should be similarly made and recorded.

To measure any object subsequently it should be first focussed carefully in the ordinary way.

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.

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 pass 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 µ.

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 quantity 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 glass bulb is warm, rubbing with a wad of cotton-wool will readily distribute the blue greasy material in an even film over the ground glass.