CHAPTER IV

HOW TO USE THE MICROSCOPE

In beginning work with the compound microscope, place the base of the microscope opposite your right shoulder, if you are right-handed; or opposite your left shoulder, if you are left-handed. Incline the body so that the ocular is on a level with your eye, if necessary; but if not, work with the body of the microscope in an erect position. In viewing the specimen, keep both eyes open. Use one eye for observation and the other for sketching. In this way it will not be necessary to remove the observation eye from the ocular unless it be to complete the details of a sketch.

=Learn to use both eyes.= Most workers, however, accustom themselves to using one eye; when they are sketching, they use both eyes, although it is not necessary to do so.

=Open the iris diaphragm=, and incline the mirror so that white light is reflected on the Abbé condenser. Place the slide on the centre of the stage, and if the slide contains a section of a plant, move the slide so as to place this specimen over the centre of the Abbé condenser. Then lower the body by means of the coarse adjustment until the low-power object, which should always be in position when work is begun, is within one-fourth of an inch of the stage. Then raise the body by means of the coarse adjustment until the object, or objects, in case a powder is being examined, is seen. Open and close the iris diaphragm, finally adjusting the opening so that the best possible illumination is obtained for bringing out clearly the structure of the object or objects viewed. Then regulate the focus by moving the body up or down by turning the fine adjustment. When studying cross-sections or large particles of powders, it is sometimes desirable to make low-power sketches of the specimen. In most cases, however, only sufficient time should be spent in studying the specimen to give an idea of the size, structure, and general arrangement or plan or structure if a section of a plant, or, if a powder, to note its striking characters. All the finer details of structure are best brought out with the high-power objective in position.

In =placing the high-power objective in position=, it is first necessary to raise the body by the coarse adjustment; then open the iris diaphragm, and lower the body until the objective is within about one-eighth of an inch of the slide. Now raise the tube by the fine adjustment until the object is in focus, then gradually close the iris diaphragm until a clear definition of the object is obtained. Now proceed to make an accurate sketch of the object or objects being studied.

In =using the water or oil-immersion objectives= it is first necessary to place a drop of distilled water or oil, as the case may be, immediately over the specimen, then lower the body by the coarse adjustment until the lens of the objective touches the water or the oil. Raise the tube, regulate the light by the iris diaphragm, and proceed as if the high-power objectives were in position.

The water or oil should be removed from the objectives and from the slide when not in use.

After the higher-powered objective has been used, the body should be raised, and the low-power objective placed in position. If the draw-tube has been drawn out during the examination of the object, replace it, but be sure to hold one hand on the nose-piece so as to prevent scratching the objective and Abbé condenser by their coming in forceful contact. Lastly, clean the mirror with a soft piece of linen. In returning the microscope to its case, or to the shelf, grasp the limb, or the pillar, firmly and carry as nearly vertical as possible in order not to dislodge the eye-piece.

ILLUMINATION

The illumination for microscopic work may be from natural or artificial sources.

[Illustration: FIG. 30.--Micro Lamp]

It has been generally supposed that the best possible illumination for microscopic work is diffused sunlight obtained from a northern direction. No matter from what direction diffused sunlight is obtained, it will be found suitable for microscopic work. In no case should direct sunlight be used, because it will be found blinding in its effects upon the eyes. Natural illumination--diffused sunlight--varies so greatly during the different months of the year, and even during different periods of the day, that individual workers are resorting more and more to artificial illumination. The

## particular advantage of such illumination is due to the fact that

its quality and intensity are uniform at all times. There are many ways of securing such artificial illumination, no one of which has any particular advantage over the other. Some workers use an ordinary gas or electric light with a color screen placed in the sub-stage below the iris diaphragm. In other cases a globe filled with a weak solution of copper sulphate is placed in such a way between the source of light and the microscope that the light is focused on the mirror. Modern mechanical ingenuity has devised, however, a number of more convenient micro lamps (Fig. 30). These lamps are a combination of light and screen. In some forms a number of different screens come with each lamp, so that it is possible to obtain white-, blue-, or dark-ground illumination. The type of the screen used will be varied according to the nature of the object studied.

CARE OF THE MICROSCOPE

If possible, the microscope should be stored in a room of the same temperature as that in which it is to be used. In any case, avoid storing in a room that is cooler than the place of use, because when it is brought into a warmer room, moisture will condense on the ocular objectives and mirrors.

Before beginning work remove all moisture, dust, etc., from the inner and outer lenses of the ocular, the objectives, the Abbé condenser, and the mirror by means of a piece of soft, old linen. When the work is finished the optical parts should be thoroughly cleaned.

If reagents have been used, be sure that none has got on the objectives or the Abbé condenser. If any reagent has got on these parts, wash it off with water, and then dry them thoroughly with soft linen.

The inner lenses of the eye-pieces and the under lens of the Abbé condenser should occasionally be cleaned. The mechanical parts of the stand should be cleaned if dust accumulates, and the movable surfaces should be oiled occasionally. Never attempt to make new combinations of the ocular or objective lenses, or transfer the objectives or ocular from one microscope to another, because the lenses of any given microscope form a perfect lens system, and this would not be the case if they were transferred. Keep clean cloths in a dust-proof box. Under no circumstances touch any of the optical parts with your fingers.

PREPARATION OF SPECIMENS FOR CUTTING

Most drug plants are supplied to pharmacists in a dried condition. It is necessary, therefore, to boil the drug in water, the time varying from a few minutes, in the case of thin leaves and herbs, up to a half hour if the drug is a thick root or woody stem. If a green (undried) drug is under examination, this first step is not necessary.

If the specimen to be cut is a leaf, a flower-petal, or other thin, flexible part of a plant, it may be placed between pieces of elder pith or slices of carrot or potato before cutting.

SHORT PARAFFIN PROCESS

In most cases, however, more perfect sections will be obtained if the specimens are embedded in paraffin, by the quick paraffin process, which is easily carried out.

After boiling the specimen in water, remove the excess of moisture from the outer surface with filter paper or wait until the water has evaporated. Next make a mould of stiff cardboard and pour melted paraffin (melting at 50 or 60 degrees) into the mould to a height of about one-half inch, when the paraffin has solidified. This may be hastened by floating it on cool or iced water instead of allowing it to cool at room temperature.

The specimens to be cut are now placed on the paraffin, with glue, if necessary, to hold them in position, and melted paraffin poured over the specimens until they are covered to a depth of about one-fourth of an inch. Cool on iced water, trim off the outer paraffin to the desired depth, and the Specimen will be in a condition suitable for cutting.

Good workable sections may be cut from specimens embedded by this quick paraffin method. After a little practice the entire process can be carried out in less than an hour. This method of preparing specimens for cutting will meet every need of the pharmacognosist.

LONG PARAFFIN PROCESS

In order to bring out the structure of the =protoplast= (living part of the cell), it will be necessary to begin with the living part of the plant and to use the long paraffin method or the collodion method.

Small fragments of a leaf, stem, or root-tip are placed in chromic-acid solution, acetic alcohol, picric acid, chromacetic acid, alcohol, etc., depending upon the nature of the specimen under observation. The object of placing the living specimen in such solutions is to kill the protoplast suddenly so that the parts of the cell will bear the same relationship to each other that they did in the living plant, and to fix the parts so killed.

After the fixing process is complete, the specimen is freed of the fixing agent by washing in water. From the water-bath the specimens are transferred successively to 10, 20, 40, 60, 70, 80, 90, and finally 100 per cent alcohol. In this 100 per cent alcohol-bath the last traces of moisture are removed. The length of time required to leave the specimens in the different percentages of alcohols varies from a few minutes to twenty-four hours, depending upon the size and the nature of the specimen.

[Illustration: FIG. 31.--Paraffin-embedding Oven]

After dehydration the specimen is placed in a clearing agent--chloroform or xylol--both of which are suitable when embedding in paraffin. The clearing agents replace the alcohol in the cells, and at the same time render the tissues transparent. From the clearing agent the specimen is placed in a weak solution of paraffin, dissolved xylol, or chloroform. The strength of the paraffin solution is gradually increased until it consists of pure paraffin. The temperature of the paraffin-embedding oven (Fig. 31) should not be much higher than the melting-point of the paraffin.

The specimen is now ready to be embedded. First make a mould of cardboard or a lead-embedding frame (Fig. 32), melt the paraffin, and then place the specimen in a manner that will facilitate cutting. Remove the excess of paraffin and cut when desired.

[Illustration: FIG. 32.--Paraffin Blocks]

In using the collodion method for embedding fibrous specimens, as wood, bark, roots, etc., the specimen is first fixed with picric acid, washed with water, cleared in ether-alcohol, embedded successively in two, five, and twelve per cent ether-alcohol collodion solution, and finally embedded in a pure collodion bath.

CUTTING SECTIONS

Specimens prepared as described above may be cut with a hand microtome or a machine microtome.

HAND MICROTOME

In cutting sections by a =hand microtome=, it is necessary to place the specimen, embedded in paraffin or held between pieces of elder pith, carrot, or potato, over the second joints of the fingers, then press the first joints firmly upon the specimen with the thumb pressed against it. If they are correctly held, the specimens will be just above the level of the finger and the end of the thumb, and the joint will be below the level of the finger.

[Illustration: FIG. 33.--Hand Microtome]

Hold the section cutter (Fig. 33) firmly in the hand with the flat surface next to the specimen. While cutting the section, press your arm firmly against your chest, and bend the wrist nearly at right angles to the arm. Push the cutting edge of the microtome toward the body and through the specimen in such a way as to secure as thin a section as possible. Do not expect to obtain nice, thin sections during the first or second trials, but continued practice will enable one to become quite efficient in cutting sections in this manner.

When the examination of drugs is a daily occurrence, the above method will be found highly satisfactory.

MACHINE MICROTOMES

When a number of sections are to be prepared from a given specimen, it is desirable to cut the sections on a machine microtome,

## particularly when the sections are to be prepared for the use of

students, in which case they should be as uniform as possible.

Great care should be exercised in cutting sections with a machine microtome--first, in the selection of the type of the microtome; and secondly, in the style of knife used in cutting.

For soft tissues embedded in paraffin or collodion, the =rotary microtome= with vertical knife will give best results. The thickness of the specimen is regulated by mechanical means, so that in cutting the sections it is only necessary to turn a crank and remove the specimens from the knife-edge, unless there is a ribbon-carrier attachment. If the sections are being cut from a specimen embedded by the quick paraffin method, it is best to drop the section in a metal cup partly filled with warm water. This will cause the paraffin to straighten out, and the specimen will uncoil. After sufficient specimens have been cut, the cup should be placed in a boiling-water bath until the paraffin surrounding the sections melts and floats on the water. Before removing the specimen from the water-bath, it is advisable to shake the glass vigorously in order to cause as many specimens as possible to settle to the bottom of the cup. The cup is then placed in iced water or set aside until the paraffin has solidified. The cake-like mass is then removed from the cup, and the sections adhering to its under surface are removed by lifting them carefully off with the flat side of the knife and transferring them, together with the sections at the bottom of the cup, to a wide-mouth bottle, and covered with alcohol, glycerine, and water mixture; or if it is desired to stain the specimens, they should be placed in a weak alcoholic solution.

Specimens having a hard, woody texture should be cut on a =sliding microtome= by means of a special wood knife, which is especially tempered to cut woody substances. Woody roots, wood, or thick bark may be cut readily on this microtome when they have been embedded by the quick paraffin process. The knife in the sliding microtome is placed in a horizontal position, slanting so that the knife-edge is drawn gradually across the specimen. After cutting, the sections are treated as described above.

The thickness of the sections is regulated by mechanical means. After a section has been cut, the block containing the specimen is raised by turning a thumb-screw. In this microtome the knife, as in the rotary type, is fixed, and the block containing the specimen is movable.

If the specimen has been infiltrated with, and embedded in, paraffin or collodion, the treatment of the sections after cutting should be different.

In the case of paraffin, the sections are fastened directly to the slide, and the paraffin is dissolved by either chloroform or xylol. The specimen is then placed in 100, 95, and 45 per cent alcohol, and then washed in water. These sections are now stained with water-stains, brought back through alcohol, cleared, and mounted in Canada balsam.

If alcoholic stains are used, it will not be necessary to dehydrate before staining, and the dehydration after staining will also be eliminated.

Sections infiltrated with collodion are either stained directly without removing the collodion or after removal.

FORMS OF MICROTOMES

The =hand cylinder microtome= (Fig. 34) consists of a cylindrical body. The clamp for holding the specimen is near the top below the cutting surface. At the lower end is attached a micrometer screw with a divided milled head. When moved forward one division, the specimen is raised 0.01 mm. This micrometer screw has an upward movement of 10 mm. The cutting surface consists of a cylindrical glass ring.

[Illustration: FIG. 34.--Hand Cylinder Microtome]

[Illustration: FIG. 35.--Hand Table Microtome]

The =hand table microtome= (Fig. 35) is provided with a clamp, by which it may be attached to the edge of a table or desk. The cutting surface consists of two separated but parallel glass benches. The object is held by a clamp and is raised by a micrometer screw, which, when moved through one division by turning the divided head, raises the specimen 0.01 mm.

The =sliding microtome= has a track of 250 mm. The object is held by a clamp and its height regulated by hand. The disk regulating the micrometer screw is divided into one hundred parts. When this is turned through one division, the object is raised 0.005 mm. or 5 microns, at the same time a clock-spring in contact with teeth registers by a clicking sound. If the disk is turned through two divisions, there will be two clicks, etc. In this way is regulated the thickness of the sections cut. When the micrometer screw has been turned through the one hundred divisions, it must be unscrewed, the specimen raised, and the steps of the process repeated. The knife is movable and is drawn across the specimen in making sections.

[Illustration: FIG. 36.--Base Sledge Microtome]

The =base sledge microtome= (Fig. 36) has a heavy iron base which supports a sliding-way on which the object-carrier moves. The object-carrier is mounted on a solid mass of metal, and is provided with a clamp for holding the object. The object is raised by turning a knob which, when turned once, raises the specimen one to twenty microns, according to how the feeding mechanism is set.

Sections thicker than twenty microns may be obtained by turning the knob two or more times. The knife is fixed and is supported by two pillars, the base of which may be moved forward or backward in such a manner that the knife can be arranged with an oblique or right-angled cutting surface.

[Illustration: FIG. 37.--Minot Rotary Microtome]

The =Minot rotary microtome= (Fig. 37) has a fixed knife, held in position by two pillars, and a movable object-carrier. The object is firmly secured by a clamp, and it is raised by a micrometer screw. The screw is attached to a wheel having five hundred teeth on its periphery. A pawl is adjusted to the teeth in such a way that, when moved by turning a wheel to which it is attached, specimens varying from one to twenty-five microns in thickness may be cut, according to the way the adjusting disk is set. When the mechanism has been regulated and the object adjusted for cutting, it is only necessary to turn a crank in cutting sections.

CARE OF MICROTOMES

When not in use, microtomes should be protected from dust, and all parts liable to friction should be oiled.

Microtome knives should be honed as often as is necessary to insure a proper cutting edge. After cutting objects, the knives should be removed, cleaned, and oiled.

It should be kept clearly in mind that special knives are required for cutting collodion, paraffin, and frozen and woody sections. The cutting edges of the different knives vary considerably, as is shown in the preceding cuts.

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