HISTOLOGY OF
MEDICINAL PLANTS

BY

WILLIAM MANSFIELD, A.M., Phar.D.

Professor of Histology and Pharmacognosy, College of
Pharmacy of the City of New York
Columbia University

TOTAL ISSUE, FOUR THOUSAND

NEW YORK
JOHN WILEY & SONS, Inc.
London: CHAPMAN & HALL, Limited

Copyright, 1916, by
WILLIAM MANSFIELD

PREFACE

The object of the book is to provide a practical scientific course in vegetable histology for the use of teachers and students in schools and colleges.

The medicinal plants are studied in great detail because they constitute one of the most important groups of economic plants. The cells found in these plants are typical of the cells occurring in the vegetable kingdom; therefore the book should prove a valuable text-book for all students of histology.

The book contains much that is new. In Part II, which is devoted largely to the study of cells and cell contents, is a new scientific, yet practical, classification of cells and cell contents. The author believes that his classification of bast fibres and hairs will clear up much of the confusion that students have experienced when studying these structures.

The book is replete with illustrations, all of which are from original drawings made by the author. As most of these illustrations are diagnostic of the plants in which they occur, they will prove especially valuable as reference plates.

The material of the book is the outgrowth of the experience of the author in teaching histology at the College of Pharmacy of the City of New York, Columbia University, and of years of practical experience gained by examining powdered drugs in the laboratory of a large importing and exporting wholesale drug house.

The author is indebted to Ernest Leitz and Bausch & Lomb Optical Company for the use of cuts of microscopic apparatus used in Part I of the book.

The author also desires to express his appreciation to Professor Walter S. Cameron, who has rendered him much valuable aid.

William Mansfield.

Columbia University,
September, 1916.

CONTENTS

TABLE OF ILLUSTRATIONS

Part I
SIMPLE AND COMPOUND MICROSCOPES
AND MICROSCOPIC TECHNIC

CHAPTER I
THE SIMPLE MICROSCOPES

The construction and use of the simple microscope (magnifiers) undoubtedly date back to very early times. There is sufficient evidence to prove that spheres of glass were used as burning spheres and as magnifiers by people antedating the Greeks and Romans.

The simple microscopes of to-day have a very wide range of application and a corresponding variation in structure and in appearance.

Simple microscopes are used daily in classifying and studying crude drugs, testing linen and other cloth, repairing watches, in reading, and identifying insects. The more complex simple microscopes are used in the dissection and classification of flowers.

The watchmaker’s loupe, the linen tester, the reading glass, the engraver’s lens, and the simplest folding magnifiers consist of a double convex lens. Such a lens produces an erect, enlarged image of the object viewed when the lens is placed so that the object is within its focal distance. The focal distance of a lens varies according to the curvature of the lens. The greater the curvature, the shorter the focal distance and the greater the magnification.

The more complicated simple microscope consists of two or more lenses. The double and triple magnifiers consist of two and three lenses respectively.

When an object is viewed through three lenses, the magnification is greater than when viewed through one or two lenses, but a smaller part of the object is magnified.

FORMS OF SIMPLE MICROSCOPES

TRIPOD MAGNIFIER

The tripod magnifier (Fig. 1) is a simple lens mounted on a mechanical stand. The tripod is placed over the object and the focus is obtained by means of a screw which raises or lowers the lens, according to the degree it is magnified.

WATCHMAKER’S LOUPE

The watchmaker’s loupe (Fig. 2) is a one-lens magnifier mounted on an ebony or metallic tapering rim, which can be placed over the eye and held in position by frowning or contracting the eyelid.

Fig. 1.—Tripod Magnifier Fig. 2.—Watchmaker’s Loupe

FOLDING MAGNIFIER

The folding magnifier (Fig. 3) of one or more lenses is mounted in such a way that, when not in use, the lenses fold up like the blade of a knife, and when so folded are effectively protected from abrasion by the upper and lower surfaces of the folder.

Fig. 3.—Folding Magnifier Fig. 4.—Reading Glass

READING GLASSES

Reading glasses (Fig. 4) are large simple magnifiers, often six inches in diameter. The lens is encircled with a metal band and provided with a handle.

Fig. 5.—Steinheil Aplanatic Lens

STEINHEIL APLANATIC LENSES

Steinheil aplanatic lenses (Fig. 5) consist of three or four lenses cemented together. The combination is such that the field is large, flat, and achromatic. These lenses are suitable for field, dissecting, and pocket use. When such lenses are placed in simple holders, they make good dissecting microscopes.

Fig. 6.—Dissecting microscope

DISSECTING MICROSCOPE

The dissecting microscope (Fig. 6) consists of a Steinheil lens and an elaborate stand, a firm base, a pillar, a rack and pinion, a glass stage, beneath which there is a groove for holding a metal plate with one black and one white surface. The nature of the object under observation determines whether a plate is used. When the plate is used and when the object is studied by reflected light it is sometimes desirable to use the black and sometimes the white surface. The mirror, which has a concave and a plain surface, is used to reflect the light on the glass stage when the object is studied by transmitted light. The dissecting microscope magnifies objects up to twenty diameters, or twenty times their real size.

CHAPTER II
COMPOUND MICROSCOPES

The compound microscope has undergone wonderful changes since 1667, the days of Robert Hooke. When we consider the crude construction and the limitations of Robert Hooke’s microscope, we marvel at the structural perfection and the unlimited possibilities of the modern instrument. The advancement made in most sciences has followed the gradual perfection of this instrument.

The illustration of Robert Hooke’s microscope (Fig. 7) will convey to the mind more eloquently than words the crudeness of the early microscopes, especially when it is compared with the present-day microscopes.

STRUCTURE OF THE COMPOUND MICROSCOPE

The parts of the compound microscope (Fig. 8) may be grouped into—first, the mechanical, and, secondly, into the optical parts.

THE MECHANICAL PARTS

1. The foot is the basal part, the part which supports all the other mechanical and optical parts. The foot should be heavy enough to balance the other parts when they are inclined. Most modern instruments have a three-parted or tripod-shaped base.

2. The pillar is the vertical part of the microscope attached to the base. The pillar is joined to the limb by a hinged joint. The hinges make it possible to incline the microscope at any angle, thus lowering its height. In this way, short, medium, and tall persons can use the microscope with facility. The part of the pillar above the hinge is called the limb. The limb may be either straight or curved. The curved form is preferable, since it offers a more suitable surface to grasp in transferring from box or shelf to the desk, and vice versa.

Fig. 7.—Compound Microscope of Robert Hooke

3. The stage is either stationary or movable, round or square, and is attached to the limb just above the hinge. The upper surface is made of a composition which is not easily attacked by moisture and reagents. The centre of the stage is perforated by a circular opening.

4. The sub-stage is attached below the stage and is for the purpose of holding the iris diaphragm and Abbé condenser. The raising and lowering of the sub-stage are accomplished by a rack and pinion.

5. The iris diaphragm, which is held in the sub-stage below the Abbé condenser, consists of a series of metal plates, so arranged that the light entering the microscope may be cut off completely or its amount regulated by moving a control pin.

6. The fine adjustment is located either at the side or at the top of the limb. It consists of a fine rack and pinion, and is used in focusing an object when the low-power objective is in position, or in finding and focusing the object when the high-power objective is in position.

7. The coarse adjustment is a rack and pinion used in raising and lowering the body-tube and in finding the approximate focus when either the high- or low-power objective is in position.

8. The body-tube is the path traveled by the rays of light entering the objectives and leaving by the eye-piece. To the lower part of the tube is attached the nose-piece, and resting in its upper part is the draw-tube, which holds the eye-piece. On the outer surface of the draw-tube there is a scale which indicates the distance it is drawn from the body-tube.

9. The nose-piece may be simple, double, or triple, and it is protected from dust by a circular piece of metal. Double and triple nose-pieces may be revolved, and like the simple nose-piece they hold the objectives in position.

THE OPTICAL PARTS

1. The mirror is a sub-stage attachment one surface of which is plain and the other concave. The plain surface is used with an Abbé condenser when the source of light is distant, while the concave surface is used with instruments without an Abbé condenser when the source of light is near at hand.

Fig. 8.—Compound Microscope

2. The Abbé condenser (Fig. 9) is a combination of two or more lenses, arranged so as to concentrate the light on the specimen placed on the stage. The condenser is located in the opening of the stage, and its uppermost surface is circular and flat.

Fig. 9—Abbé Condenser

3. Objectives (Figs. 10, 11, and 12). There are low, medium, and high-power objectives. The low-power objectives have fewer and larger lenses, and they magnify least, but they show more of the object than do the high-power objectives. There are three chief types of objectives: First, dry objectives; second, wet objectives, of which there are the water-immersion objectives; and third, the oil-immersion objectives. The dry objectives are used for most histological and pharmacognostical work. For studying smaller objects the water objective is sometimes desirable, but in bacteriological work the oil-immersion objective is almost exclusively used. The globule of water or oil, as the case may be, increases the amount of light entering the objective, because the oil and water bend many rays into the objective which would otherwise escape.

Fig. 10. Fig. 11. Fig. 12.
Objectives.

4. Eye-pieces (Figs. 13, 14, and 15) are of variable length, but structurally they are somewhat similar. The eye-piece consists of a metal tube with a blackened inner tube. In the centre of this tube there is a small diaphragm for holding the ocular micrometer. In the lower end of the tube a lens is fastened by means of a screw. This, the field lens, is the larger lens of the ocular. The upper, smaller lens is fastened in the tube by a screw, but there is a projecting collar which rests, when in position, on the draw-tube.

Fig. 13. Fig. 14 Fig. 15
Eye-Pieces.

The longer the tube the lower the magnification. For instance, a two-inch ocular magnifies less than an inch and a half, a one-inch less than a three-fourths of an inch, etc.

The greater the curvature of the lenses of the ocular the higher will be the magnification and the shorter the tube-length.

FORMS OF COMPOUND MICROSCOPES

The following descriptions refer to three different models of compound microscopes: one which is used chiefly as a pharmacognostic microscope, one as a research microscope stand, while the third type represents a research microscope stand of highest order, which is used at the same time for taking microphotographs.

Fig. 16.—Pharmacognostic Microscope

PHARMACOGNOSTIC MICROSCOPE

The pharmacognostic microscope (Fig. 16) is an instrument which embodies only those parts which are most essential for the examination of powdered drugs, bacteria, and urinary sediments. This microscope is provided with a stage of the dimensions 105 × 105 mm. This factor and the distance of 80 mm. from the optical centre to the handle arm render it available for the examination of even very large objects and preparations, or preparations suspended in glass dishes. The stand is furnished with a side micrometer, a fine adjustment having knobs on both sides, thereby permitting the manipulation of the micrometer screw either by left or right hand. The illuminating apparatus consists of the Abbé condenser of numerical aperture of 1.20, to which is attached an iris diaphragm for the proper adjustment of the light. A worm screw, mounted in connection with the condenser, serves for the raising and lowering of the condenser, so that the cone of illuminating pencils can be arranged in accordance to the objective employed and to the preparation under observation. The objectives necessary are those of the achromatic type, possessing a focal length of 16.2 mm. and 3 mm. Oculars which render the best results in regard to magnification in connection with the two objectives mentioned are the Huyghenian eye-pieces II and IV so that magnifications are obtained varying from 62 to 625. It is advisable, however, to have the microscope equipped with a triple revolving nose-piece for the objectives, so that provision is made for the addition of an oil-immersion objective at any time later should the microscope become available for bacteriological investigations.

THE RESEARCH MICROSCOPE

The research microscope used in research work (Fig. 17) must be equipped more elaborately than the microscope especially designed for the use of the pharmacognosist. While the simple form of microscope is supplied with the small type of Abbé condenser, the research microscope is furnished with a large illuminating apparatus of which the iris diaphragm is mounted on a rack and pinion, allowing displacement obliquely to the optical centre, also to increase resolving power in the objectives when observing those objects which cannot be revealed to the best advantage with central illumination. Another iris is furnished above the condenser; this iris becomes available the instant an object is to be observed without the aid of the condenser, in which case the upper iris diaphragm allows proper adjustment of the light. The mirror, one side plane, the other concave, is mounted on a movable bar, along which it can be slid—another convenience for the adjustment of the light. The microscope stage of this stand is of the round, rotating and centring pattern, which permits a limited motion to the object slide: The rotation of the microscope stage furnishes another convenience in the examination of objects in polarized light, allowing the preparation to be rotated in order to distinguish the polarization properties of the objects under observation.

Fig. 17.—Research Microscope Fig. 18.—Special Research Microscope

SPECIAL RESEARCH MICROSCOPE

A special research microscope of the highest order (Fig. 18) is supplied with an extra large body tube, which renders it of special advantage for micro-photography. Otherwise in its mechanical equipment it resembles very closely the medium-sized research microscope stand, with the exception that the stand is larger in its design, therefore offering universal application. In regard to the illuminating apparatus, it is advisable to mention that the one in the large research microscope stand is furnished with a three-lens condenser of a numerical aperture of 1.40, while the medium-sized research stand is provided with a two-lens condenser of a numerical aperture of 1.20. The stage of the microscope is provided with a cross motion—the backward and forward motion of the preparation is secured by rack and pinion, while the side motion is controlled by a micrometric worm screw. In cases where large preparations are to be photographed, the draw-tube with ocular and the slider in which the draw-tubes glide are removed to allow the full aperture of wide-angle objectives to be made use of.

Fig. 19.—Greenough Binocular Microscope

BINOCULAR MICROSCOPE

The Greenough binocular microscope, as shown in Fig. 19, consists of a microscope stage with two tubes mounted side by side and moving on the same rack and pinion for the focusing adjustment. Either tube can be used without the other. The oculars are capable of more or less separation to suit the eyes of different observers. In each of the drub-like mountings, near the point where the oculars are introduced, porro-prisms have been placed, which erect the image. This microscope gives most perfect stereoscopic images, which are erect instead of inverted, as in the monocular compound microscopes. The Greenough binocular microscope is especially adapted for dissection and for studying objects of considerable thickness.

POLARIZATION MICROSCOPE

The polarization microscope (Fig. 20) is used chiefly for the examination of crystals and mineral sections as well as for the observation of organic bodies in polarized light. It can, however, also be used for the examination of regular biological preparations.

Fig. 20.—Polarization Microscope

If compared with the regular biological microscope, the polarization microscope is found characteristic of the following points: it is supplied with a polarization arrangement. The latter consists of a polarizer and analyzer. The polarizer is situated in a rotating mount beneath the condensing system. The microscope, of which the diagram is shown, possesses a triple “Ahrens” prism of calcite. The entering light is divided into two polarized parts, situated perpendicularly to each other. The so-called “ordinary” rays are reflected to one side by total reflection, which takes place on the inner cemented surface of the triple prism, allowing the so-called “extraordinary” rays to pass through the condenser. If the prism is adjusted to its focal point, it is so situated that the vibration plane of the extra-ordinary rays are in the same position as shown in the diagram of the illustration.

The analyzer is mounted within the microscope-tube above the objective. Situated on a sliding plate, it can be shifted into the optical axis whenever necessary. The analyzer consists of a polarization prism after Glan-Thompson. The polarization plane of the active extraordinary rays is situated perpendicularly to the plane as shown in the diagram. The polarization prisms are ordinarily crossed. In this position the field of the microscope is darkened as long as no substance of a double refractive index has been introduced between the analyzer and polarizer. In rotating the polarizer up to the mark 90, the polarization prisms are mounted parallel and the field of the microscope is lighted again. Immediately above the analyzer and attached to the mounting of the analyzer a lens of a comparatively long focal length has been placed in order to overcome the difference in focus created by the introduction of the analyzer into the optical rays.

The condensing system is mounted on a slider, and, furthermore, can be raised and lowered along the optical centre by means of a rack-and-pinion adjustment. If lowered sufficiently, the condensing system can be thrown to the side to be removed from the optical rays. The condenser consists of three lenses. The two upper lenses are separately mounted to an arm, which permits them to be tilted to one side in order to be removed from the optical rays. The complete condenser is used only in connection with high-power objectives. As far as low-power objectives are concerned, the lower condensing lens alone is made use of, and the latter is found mounted to the polarizer sleeve. Below the polarizer and above the lower condensing lens an iris diaphragm is found.

The microscope table is graduated on its periphery, and, furthermore, carries a vernier for more exact reading.

The polarization microscope is not furnished with an objective nose-piece. Every objective, however, is supplied with an individual centring head, which permits the objective to be attached to an objective clutch-changer, situated at the lower end of the microscope-tube. The centring head permits the objectives to be perfectly centred and to remain centred even if another objective is introduced into the objective clutch-changer.

At an angle of 45 degrees to the polarization plane of polarizer and analyzer, a slot has been provided, which serves for the introduction of compensators.

Between analyzer and ocular, another slot is found which permits the Amici-Bertrand lens to be introduced into the optical axis. The slider for the Bertrand lens is supplied with two centring screws whereby this lens can be perfectly and easily centred. The Bertrand lens serves the purpose of observing the back focal plane of the microscope objective. In order to allow the Bertrand lens to be focused, the tube can be raised and lowered for this purpose. An iris diaphragm is mounted above the Bertrand lens.

If the Bertrand lens is shifted out of the optical axis, one can observe the preparation placed upon the microscope stage and, depending on its thickness or its double refraction, the interference color of the specimen. This interference figure is called the orthoscopic image and, accordingly, one speaks of the microscope as being used as an “orthoscope.”

After the Bertrand lens has been introduced into the optical axis, the interference figure is visible in the back focal plane of the objective. Each point of this interference figure corresponds to a certain direction of the rays of the preparation itself. This arrangement permits observation of the change of the reflection of light taking place in the preparation, this in accordance with the change of the direction of the rays. This interference figure is called the conoscopic image, and, accordingly, the microscope is used as a “conoscope.”

Many types of polarization microscopes have been constructed; those of a more elaborate form are used for research investigations; others of smaller design for routine investigations.

CHAPTER III
MICROSCOPIC MEASUREMENTS

In making critical examinations of powdered drugs, it is frequently necessary to measure the elements under observation, particularly in the case of starches and crystals.

OCULAR MICROMETER

Microscopic measurements are made by the ocular micrometer (Fig. 21). This consists of a circular piece of transparent glass on the centre of which is etched a one- or two-millimeter scale divided into one hundred or two hundred divisions respectively. The value of each line is determined by standardizing with a stage micrometer.

Fig. 21.—Ocular Micrometer Fig. 22.—Stage Micrometer

STAGE MICROMETER

The stage micrometer (Fig. 22) consists of a glass slide upon which is etched a millimeter scale divided into one hundred equal parts or lines: each line has a value of one hundredth of a millimeter.

STANDARDIZATION OF OCULAR MICROMETER
WITH LOW-POWER OBJECTIVE

Having placed the ocular micrometer in the eye-piece and the stage micrometer on the centre of the stage, focus until the lines of the stage micrometer are clearly seen. Then adjust the scales until the lines of the stage micrometer are parallel with and directly under the lines of the ocular micrometer.

Ascertain the number of lines of the stage micrometer covered by the one hundred lines of the ocular micrometer. Then calculate the value of each line of the ocular. This is done in the following manner:

If the one hundred lines of the ocular cover seventy-five lines of the stage micrometer, then the one hundred lines of the ocular micrometer are equivalent to seventy-five one-hundredths, or three-fourths, of a millimeter. One line of the ocular micrometer will therefore be equivalent to one-hundredth of seventy-five one-hundredths, or .0075 part of a millimeter, and as a micron is the unit for measuring microscopic objects, this being equivalent to one one-thousandth of a millimeter, the value of each line of the ocular will therefore be 7.5 microns.

Fig. 23.—Micrometer Eye-Piece

With the high-power objective in place, ascertain the value of each line of the ocular. If one hundred lines of the ocular cover only twelve lines of the stage micrometer, then the one hundred lines of the ocular are equivalent to twelve one-hundredths of a millimeter, the value of one line being equivalent to one one-hundredth of twelve one-hundredths, or twelve ten-thousandths of a millimeter, or .0012, or 1.2 µ.

It will therefore be seen that objects as small as a thousandth of a millimeter can be accurately measured by the ocular micrometer.

In making microscopic measurements it is only necessary to find how many lines of the ocular scale are covered by the object. The number of lines multiplied by the equivalent of each line will be the size of the object in microns, or micromillimeters.

Fig. 24.—Micrometer Eye-Piece

MICROMETER EYE-PIECES

Micrometer eye-pieces (Figs. 23 and 24) may be used in making measurements. These eye-pieces with micrometer combinations are preferred by some workers, but the ocular micrometer will meet the needs of the average worker.

MECHANICAL STAGES

Moving objects by hand is tiresome and unsatisfactory, first, because of the possibility of losing sight of the object under observation, and secondly, because the field cannot be covered so systematically as when a mechanical stage is used for moving slides.

The mechanical stage (Fig. 25) is fastened to the stage by a screw. The slide is held by two clamps. There is a rack and pinion for moving the slide to left or right, and another rack and pinion for moving the slide forward and backward.

Fig. 25. Mechanical Stage Fig. 26.—Camera Lucida

CAMERA LUCIDA

The camera lucida is an optical mechanical device for aiding the worker in making drawings of microscopic objects. The instrument is particularly necessary in research work where it is desirable to reproduce an object in all its details. In fact, all reproductions illustrating original work should be made by means of the camera lucida or by microphotography.

Fig. 27.—Camera Lucida

A great many different types of camera lucidas or [drawing apparatus] are obtainable, varying from simple-inexpensive to complex-expensive forms. Figs. 26, 27, and 28 show simple and complex forms.

Fig. 28.—Drawing Apparatus

MICROPHOTOGRAPHIC APPARATUS

The microphotographic apparatus (Fig. 29), as the name implies, is an apparatus constructed in such a manner that it may be attached to a microscope when we desire to photograph microscopic objects. It consists of a metal base and a polished metal pillar for holding the bellows, slide holder, ground-glass observation plate, and eye-piece. In making photographs, the small end of the bellows is attached to the ocular of the microscope, the focus adjusted, and the object or objects photographed. More uniform results are obtained in making such photographs if an artificial light of an unvarying candle-power is used.

Fig. 29.—Microphotographic Apparatus

There are obtainable more elaborate microphotographic apparatus than the one figured and described, but for most workers this one will prove highly satisfactory. It is possible, by inclining the tube of the microscope, to make good microphotographs with an ordinary plate camera. This is accomplished by removing the lens of the camera and attaching the bellows to the ocular, focusing, and photographing.

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].

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.

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.

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.

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.

Fig. 34.—Hand Cylinder Microtome

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.

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.

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.

CHAPTER V
REAGENTS

Little attention is given in the present work to micro-chemical reactions for the reason that their value has been much overrated in the past. A few reagents will be found useful, however, and these few are given, as well as their special use. They are as follows:

LIST OF REAGENTS

Distilled Water is used in the alcohol, glycerine, and water mixture as a general mounting medium. It is used when warm as a test for inulin and it is used in preparing various reagents.

Glycerine is used in preparing the alcohol, glycerine, and water mixture, in testing for aleurone grains, and as a temporary mounting medium.

Alcohol is used in preparing the alcohol, glycerine, and water mixture, in testing for volatile oils.

Acetic Acid. Both dilute and strong solutions are used in testing for aleurone grains, cystoliths, and crystals of calcium oxalate.

Hydrochloric Acid is used in connection with phloroglucin as a test for lignin and as a test for calcium oxalate.

Ferric Chloride Solution is used as a test for tannin.

Sulphuric Acid is used as a test for calcium oxalate.

Tincture Alkana is used when freshly prepared by macerating the granulated root with alcohol and filtering, as a test for resin.

Sodium Hydroxide. A five per cent solution is used as a test for suberin and as a clearing agent.

Copper Ammonia is used as a test for cellulose.

Ammonical Solution of Potash is used as a test for fixed oils. The solution is a mixture of equal parts of a saturated solution of potassium hydroxide and stronger ammonia.

Oil of Cloves is used as a clearing fluid for sections preparatory to mounting in Canada balsam.

Canada Balsam is used as a permanent mounting medium for dehydrated specimens, and as a cement for ringing slides.

Paraffin is used for general embedding and infiltrating.

Lugol’s Solution is used as a test for starch and for aleurone grains and proteid matters.

Osmic Acid. A two per cent solution is used as a test for fixed oils.

Alcohol, Glycerine, and Water Mixture is used as a temporary mounting medium and as a qualitative test for fixed oils.

Chlorzinc Iodide is used as a test for suberin, lignin, cellulose, and starch.

Analine Chloride is used as a test for lignified cell walls of bast fibres and of stone cells.

Phloroglucin. A one per cent alcoholic solution is used in connection with hydrochloric acid as a test for lignin.

Hæmatoxylin-Delifields is used as a test for cellulose.

Fig. 38.—Reagent Set

REAGENT SET

Each worker should be provided with a set of reagent bottles (Fig. 38). Such a set may be selected according to the taste of the individual, but experience has shown that a 30 c.c. bottle with a ground-in pipette and a rubber bulb is preferable to other types. In such forms the pipettes are readily cleaned, and the rubber bulbs can be replaced when they become old and brittle. The entire set should be protected from dust by keeping it in a case, the cover of which should be closed when the set is not in use.

MEASURING CYLINDER

In order accurately to measure micro-chemical reagents, it is necessary to have a standard 50 c.c. cylinder (Fig. 39) graduated to c.c.’s. Such a cylinder should form a part of the reagent set.

Fig. 39.—Measuring CylinderFig. 40.—Staining Dish

STAINING DISHES

There is a great variety of staining dishes (Fig. 40), but for general histological work a glass staining dish with groves for holding six or more slides and a glass cover is most desirable.

CHAPTER VI
HOW TO MOUNT SPECIMENS

The method of procedure in mounting specimens for study varies according to the nature of the specimen, its preliminary treatment, and the character of the mount to be made. As to duration, mounts are either temporary or permanent.

TEMPORARY MOUNTS

In preparing a temporary mount, place the specimen in the centre of a clean slide and add two or more drops of the temporary mounting medium, which may be water, or a mixture of equal parts of alcohol, glycerine, and water, or some micro-chemical reagent, as weak Lugol’s solution, solution of chloral hydrate, etc. Cover this with a cover glass and press down gently. Remove the excess of the mounting medium with a piece of blotting paper. Now place the slide on the stage and proceed to examine it. Such mounts can of course be used only for short periods of study; and when the period of observation is finished, the specimen should be removed and the slide washed, or the slide washing may be deferred until a number of such slides have accumulated. At any rate, when the mounting medium dries, the specimen is no longer suitable for observation.

PERMANENT MOUNTS

Permanent mounts are prepared in much the same way as temporary, but of course the mounting medium is different. The kind of permanent mounting medium used depends upon the previous treatment of the specimen. If the specimen has been preserved in alcohol or glycerine and water, it is usually mounted in glycerine jelly. If the specimen in question is a powder, it is placed in the centre of the slide and a drop or two of glycerine, alcohol, and water mixture added, unless the powder was already in suspension in such a mixture. Cut a small cube of glycerine jelly and place it in the centre of the powder mixture. Lift up the slide by means of pliers, or grasp the two edges between the thumb and finger and hold over a small flame of an alcohol lamp, or place on a steam-bath until the glycerine jelly has melted. Next sterilize a dissecting needle, cool, and mix the powder with the glycerine jelly, being careful not to lift the point of the needle from the slide during the operation. If the mixing has been carefully done, few or no air-bubbles will be present; but if they are present, heat the needle, and while it is white hot touch the bubbles with its point, and they will disappear. Now take a pair of forceps and, after securing a clean cover glass near the edge, pass them three times through the flame of the alcohol lamp. While holding it in a slanting position, touch one side of the powder mixture and slowly lower the cover glass until it comes in complete contact with the mixture. Now press gently with the end of the needle-handle, and set it aside to cool. When it is cool, place a neatly trimmed label on one end of the slide, on which write the name of the specimen, the number of the series of which it is to form a part, etc. Any excess of glycerine jelly, which may have been pressed out from the edges of the cover glass, should not be removed at once, but should be allowed to remain on the slide for at least one month in order to allow for shrinkage due to evaporation. At the end of a month remove the glycerine jelly by first passing the blade of a knife, held in a vertical position, the back of the knife being next to the slide, around the edge of the cover glass. After turning the knife-blade so that the flat side is in contact with slide, remove the jelly outside of the cover glass. Any remaining fragments should be removed with a piece of old linen or cotton cloth. Finally, ring the edge of the cover glass with microscopical cement, of which there are many types to be had. If the cleaning has been done thoroughly, there is no better ringing cement than Canada balsam.

In mounting cross-sections, the method of procedure is similar to the above, with the exception that the glycerine jelly is placed at the side of the specimen and not in the centre. While melting the jelly, incline the slide in order to allow the melted glycerine jelly to flow gradually over the specimen, thus replacing the air contained in the cells and intercellular spaces. Finish the mounting as directed above, but under no conditions should you stir the glycerine jelly with the section.

If specimens, after having been embedded in paraffin or collodion, are cut, cleared, stained, and dehydrated, they are usually mounted in Canada balsam. A small drop of this substance, which may be obtained in collapsible tubes, is placed at one side of the specimen. While inclining the slide, gently heat until the Canada balsam covers the specimen. Secure a cover glass by the aid of pliers, pass it through the flame three times, and lower it slowly while holding it in an inclined position. Press gently on the cover glass with the needle-handle, and keep in a horizontal position for twenty-four hours, then place directly in a slide box or cabinet, since no sealing is required.

Glycerine is sometimes used to make permanent mounts, but it is unsatisfactory, because the cover glass is easily removed and the specimen spoiled or lost, unless ringed—a procedure which is not easily accomplished. If the specimen is to be mounted in glycerine, it must first be placed in a mixture of alcohol, glycerine, and water, and then transferred to glycerine. Lactic acid is another permanent liquid-mounting medium, which is unsatisfactory in the same way as glycerine, but like glycerine, there are certain special cases where it is desirable to use it. When this is used, the slides should be kept in a horizontal position, unless ringed.

COVER GLASSES

Great care should be used In the selection of cover glasses, however, not only as regards their shape but as to their thickness. The standard tube length of the different manufacturers makes an allowance of a definite thickness for cover glasses. It is necessary, therefore, to use cover glasses made by the manufacturer of the microscope in use.

Cover glasses are either square or round. Of each there are four different thicknesses and two different sizes. The standard thicknesses are: The small size is designated three-fourths and the large size seven-eighths.

Fig. 41.—Round Cover Glass Fig. 42.—Square Cover Glass Fig. 43.—Rectangular Cover Glass

Cover glasses are circular (Fig. 41), square (Fig. 42), or rectangular (Fig. 43) pieces of transparent glass used in covering the specimens mounted on glass slides. A few years ago much difficulty was experienced in obtaining uniformly thick and transparent cover glasses, but no such difficulty is experienced to-day. The type of cover glass used depends largely upon the character of the specimen to be mounted. The square and rectangular glasses are selected when a series of specimens are to be mounted, but in mounting powdered drugs and histological specimens the round cover glasses are preferable because they are more sightly and more readily cleaned and rinsed.

GLASS SLIDES

Fig. 44.—Glass Slide

Glass slides (Fig. 44) are rectangular pieces of transparent glass used as a mounting surface for microscopic objects. The slides are usually three inches long by one inch wide, and they should be composed of white glass, and they should have ground and beveled edges. Slides should be of uniform thickness, and they should not become cloudy upon standing.

SLIDE AND COVER-GLASS FORCEPS

Slides and cover glasses should be grasped by their edges. To the beginner this is not easy. In order to facilitate holding slides and cover glasses during the mounting process, one may use a slide and a cover-glass forceps. The slide forceps consists of wire bent and twisted in such a way that it holds a slide firmly when attached to its two edges.

Fig. 45.—Histological Forceps

Fig. 46.—Forceps

Fig. 47.—Sliding-pin Forceps

There are various forms of cover-glass holders, but only two types as far as the method of securing the cover glass is concerned. First, there are the bacteriological and the histological forceps (Fig. 45), which are self-closing. The two blades of such forceps must be forced apart by pressure in securing the cover glass. The second type of forceps is that in which the two blades are normally separated (Fig. 46), it being necessary to press the blades to either side of the cover glass in order to secure and hold it. There is a modification of this type of forceps which enables one to lock the blades by means of a sliding pin (Fig. 47), after the cover glass has been secured. It is well to accustom oneself to one type, for by so doing one may become dexterous in its use.

NEEDLES

Fig. 48.—Dissecting Needle

Two dissecting needles (Fig. 48) should form a part of the histologist’s mounting set. The handles may be of any material, but the needle should be of tempered steel and about two inches long.

SCISSORS

Fig. 49.—Scissors

Almost any sort of scissors (Fig. 49) will do for histology work, but a small scissors with fine pointed blades, are preferred. Scissors are useful in trimming labels and in cutting strips of leaves and sections of fibrous roots that are to be embedded and cut.

SCALPELS

Fig. 50.—Scalpels

Scalpels (Fig. 50) have steel blades and ebony handles. These vary in regard to size and quality of material. The cheaper grades are quite as satisfactory, however, as the more expensive ones, and for general use a medium-sized blade and handle will be found most useful.

TURNTABLE