A Manual of Clinical Diagnosis

by James Campbell Todd

INTRODUCTION

USE OF THE MICROSCOPE

There is probably no laboratory instrument whose usefulness depends so much upon proper manipulation as the microscope, and none is so frequently misused by beginners. Some suggestions as to its proper use are, therefore, given at this place. It is presumed that the reader is already familiar with its general construction (Fig. 1).

FIG. 1.—The microscope: 1, Eye-piece; 2, draw-tube; 3, main tube; 4, nose-piece with objectives attached; 5, objective in position; 6, stage; 7, substage; 8, adjustment of substage; 9, mirror; 10, coarse adjustment; 11, fine adjustment.

Illumination.—Good work cannot be done without proper illumination. It is difficult to lay too much stress upon this point.

The best light is that from a white cloud. A northern exposure is desirable, since direct sunlight is to be avoided. Good work can be done at night with a Welsbach light. Ordinary gas-light and the incandescent electric light are unsatisfactory, although the latter gives good results when subdued with a heavily frosted globe. The writer uses a frosted electric bulb in a dark-room lantern, and tones the light to the proper degree for low powers by means of frosted-glass plates which slide into the grooves which have held the ruby and orange glasses. One of these plates is made of blue glass, to overcome the yellow of the artificial light. It is not generally advised to do so, but it will be found convenient to use the Abbé condenser for all routine work. With daylight it is best to use the plane mirror: with artificial light, the concave mirror. To obtain best results, the light must be focused upon the object under examination by raising or lowering the condenser.

Illumination may be either central or oblique. Central illumination is to be used for all routine work. To obtain this, the mirror should be so adjusted that the light from the source selected is reflected directly up the tube of the microscope. This is easily done by removing the eye-piece and looking down the tube while adjusting the mirror. The eye-piece is then replaced, and the light reduced as much as desired by means of the diaphragm.

Oblique illumination is to be used only to bring out certain structures more clearly after viewing them by central light: as, for example, to show the edges of a hyaline cast by throwing one of its sides into shadow. Oblique illumination is obtained in the more simple instruments by swinging the mirror to one side, so that the light enters the microscope obliquely. The more complicated instruments obtain it by means of a rack and pinion, which moves the diaphragm laterally. Beginners frequently use oblique illumination without recognizing it. If the light be oblique, an object in the center of the field will appear to move from side to side when the fine adjustment is turned back and forth.

The amount of light is even more important than its direction. It is regulated by the diaphragm. It is always best to use the least light that will show the object well. Unstained objects require very subdued light. Beginners constantly use it too strong. Strong light will often render semitransparent structures, as hyaline casts, entirely invisible (Figs. 2 and 3). Stained objects, especially bacteria, require much greater light.

FIG. 2.—Hyaline casts, one containing renal cells; properly subdued illumination (from Greene's "Medical Diagnosis").	FIG. 3.—Same as Fig. 2; strong illumination. The casts are lost in the glare, and only the renal cells are seen (from Greene's "Medical Diagnosis").

If the reflection of the window-frame or other nearby object is seen in the field, the condenser should be lowered a little.

Focusing.—It is always best to "focus up," which saves annoyance and probable damage to slides and objectives. This is accomplished by bringing the objective nearer the slide than the proper focus, and then, with the eye at the eye-piece, turning the tube up until the object is clearly seen. The fine adjustment should be used only to get an exact focus with the higher power objectives after the instrument is in approximate focus. It should not be turned more than one revolution.

There will be less fatigue to the eyes if both are kept open while using the microscope, and if no effort is made to see objects which are out of distinct focus. Fine focusing should be done with the fine adjustment, not with the eye. An experienced microscopist keeps his fingers almost constantly upon one or other of the focusing adjustments. Greater skill in recognizing objects will be acquired if the same eye be always used. To be seen most clearly, an object should be brought to the center of the field.

Magnification.—The degree of magnification should always be expressed in diameters, not times, which is a misleading term. The former refers to increase of diameter; the latter, to increase of area. The comparatively low magnification of 100 diameters is the same as the apparently enormous magnification of 10,000 times.

Magnification may be increased—(a) by using a higher power objective, which is the best way; (b) by using a higher eye-piece; or (c) by increasing the length of tube.

Eye-pieces and Objectives.—The usual equipment consists of one- and two-inch eye-pieces, and two-thirds, one-sixth, and one-twelfth inch objectives. These are very satisfactory for clinical work. It is an advantage to add a one-half-inch eye-piece for occasional use with the two-thirds objective. The one-sixth should have an especially long working distance, otherwise it cannot be used satisfactorily with the Thoma-Zeiss blood-counting instrument, which has a very thick cover-glass. Such a "special one-sixth for blood work" is made by most of the microscope manufacturers.

Objectives are "corrected" for use under certain fixed conditions, and they will give the best results only when used under the conditions for which corrected. The most important corrections are: (a) For tube length; (b) for thickness of cover-glass; and (c) for the medium between objective and cover-glass.

(a) The tube length with which an objective is to be used is usually engraved upon it—in most cases it is 160 mm.

(b) The average No. 2 cover-glass is about the thickness for which most objectives are corrected. Low powers do not require any cover-glass. A cover should always be used with high powers, but its exact thickness is more important in theory than in practice.

(c) The correction for the medium between objective and cover-glass is very important. This medium may be either air or some fluid, and the objective is hence either a "dry" or an "immersion" objective. The immersion fluid generally used is cedar oil, which gives great optical advantages because its index of refraction is the same as that of crown glass. It is obvious that only objectives with very short working distance, as the one-twelfth, can be used with an immersion fluid.

To use an oil-immersion objective a drop of the cedar oil which is prepared for the purpose should be placed upon the cover, and the objective lowered into it and then brought to a focus in the usual way. Immediately after use the oil should invariably be wiped off with lens paper, or a soft linen handkerchief moistened with saliva.

Care of the Microscope.—The microscope is a delicate instrument and should be handled accordingly. It is so heavy that one is apt to forget that parts of it are fragile. It seems unnecessary to say that when there is unusual resistance to any manipulation, force should never be used to overcome it until its cause has first been sought; and yet it is no uncommon thing to see students, and even graduates, push a high power objective against a microscopic preparation with such force as to break not only the cover-glass, but even a heavy slide.

It is most convenient to carry a microscope with the fingers grasping the pillar and the arm which holds the tube; but since this throws a strain upon the fine adjustment, it is safer to carry it by the base. To bend the instrument at the joint, the force should be applied to the pillar and never to the tube or the stage.

Lens surfaces which have been exposed to dust only should be cleaned with a camel's-hair brush. Those which are exposed to finger-marks should be cleaned with lens paper, or a soft linen handkerchief wet with saliva. Particles of dirt which are seen in the field are upon the slide, the eye-piece, or the condenser. Their location can be determined by moving the slide, rotating the eye-piece, and lowering the condenser.

Oil and balsam which have dried upon the lenses and resist saliva may be removed with alcohol or xylol; but these solvents must be used sparingly and carefully, as there is danger of softening the cement. Care must be taken not to get any alcohol upon the brass parts, as it will remove the lacquer. Balsam and dried oil are best removed from the brass parts with xylol.

Measurement of Microscopic Objects.—Of the several methods, the most convenient is the use of a micrometer eye-piece. In its simplest form this is similar to an ordinary eye-piece, but has within it a glass disc upon which is ruled a graduated scale. When this eye-piece is placed in the tube of the microscope, the ruled lines appear in the microscopic field, and the size of an object is readily determined in terms of the divisions of this scale. The value of these divisions in inches or millimeters manifestly varies with different magnifications. Their value must, therefore, be determined separately for each objective. This is accomplished through use of a stage micrometer—a glass slide with carefully ruled scale divided into hundredths and thousandths of an inch, or into subdivisions of a millimeter. The stage micrometer is placed upon the stage of the microscope and brought into focus. From the number of divisions of the eye-piece scale corresponding to each division of the stage micrometer the value of the former in fractions of an inch or millimeter is easily calculated. The counting slide of the Thoma-Zeiss hemocytometer will answer in place of a stage micrometer, the lines which form the sides of the small squares being one-twentieth of a millimeter apart. Any eye-piece can be converted into a micrometer eye-piece by placing a micrometer disc—a small circular glass plate with ruled scale—ruled side down upon its diaphragm.

The principal microscopic objects which are measured clinically are animal parasites and their ova and abnormal blood-corpuscles. The metric system is used almost exclusively. For very small objects 0.001 mm. has been adopted as the unit of measurement, under the name micron. It is represented by the Greek letter µ. For larger objects, where exact measurement is not essential, the diameter of a red blood-corpuscle (7 to 8 µ) is sometimes taken as a unit.