-v V Botanical Microtechnique I.on^iliHlinal scdion of kernel of \ello\\ ileiU inai/c. LT) (lavs after iJoUinalion. Ciaf III; clioxan-leniarv bulvl alcohol; plioioj^raphed al S\ with li S: L IS mm. Micro Tessar objective; reiModiued ;it UiX. So. r^ Botanical Microtechnique JOHN E. SASS Professor of Botany loiva State College SECOND EDITION THE IOWA STATE COLLEGE PRESS I f C P Press Building, Ames, Iowa Copyright, 19^1. by The loiva State College Press. All rights reserved. Composed and printed by The loum State College Press, Ames, Iowa. U.S.A. Second Edition, ig^i o^S^cXr '^^ro^s~;/>.0 ^c V ^\ 1 IBRARY )?| i;mi r ■•'».,v>>i "y"^ 4e^ R.l ACC? y4 mrt77 ti;/?o zi;or/« witJi his hands is a laborer; a man who xuorks zviih his hands and his brain is a craftsman; but a man xvho works loith his hands and his brain and his heart IS an artist. -Louis Nizer, Between You and Me (Beechhurst) Preface Permanent slides for microscopic study are indispensable in the teaching of a basic course in botany and also in specialized advanced courses. In some advanced courses, the students prepare many of the slides used in the course, but in elementary courses the slides are furnished. In the latter case, the slides either are purchased from commercial somxes or made in the departmental laboratory. Biologi- cal supply houses make excellent slides of the subjects commonh used in elementary teaching, but the quality is likely to be variable. Jobbing houses that pinxhase slides from constantly changing sources also may furnish disappointing slides at times. The relative merits of making slides and of purchasing them must be decided on the basis of local conditions. Uncertainties in the commercial supjih and the need for specialized or unlisted items necessitates the preparation of slides in the biological departments of schools. This service work often is performed by a skilled profes- sional technician with more or less supervision by the departmental staff. In other departments a member of the teaching staff, usually a morphologist, assumes this responsibility, with the aid of student assistants. Some research organizations maintain a technician for the prepa- ration of research slides. There are many types of investigation in which it is possible for the technician to prepare and place the finished slides before the in\estigator, who then carries out the study and interpretation of the material. However, in many investigations, some or all steps in the preparation recjuire an intimate knowledge of the history, structure, and orientation of the material and the aims of the study. The use of a technician who allegedly merely "turns the crank" is then less valid, and the so-called technician may in fact be a research collaborator. The in\estigator in any field of plant science is urged to utilize microtechnique as a tool, but to do so critically and intelligently and in proper fairness to the workers who contribute their skill, patience, and understanding to the furtherance of the research. It cannot be too strongly emphasized that in order to [vii] v'lii Preface have a proper appreciation of the possibilities and limitations of present-day techniques, and to utilize the services of commercial or institutional technicians to best advantage, every teacher and investi- gator in the biological sciences should be familiar with at least the elements of microtechnique. We can do no better than to quote the late Dr. Charles }. Chamberlain, the dean of American microsco- pists: "The student who has not had sufficient experience to make a first-class preparation for microscopic study cannot safely interpret slides made by others. He is in the same class with the one who claims he sees it but can't draw it; while the real trouble is not in his hand, but in his head." The term liislology is very commonly misused to imply histological methc^ds or technique. Histology means the study of the structure and development of tissues, and does not refer to the preparation of slides. A good textbook of histology need not contain a word about sectioning and staining of tissues. A person who takes an afternoon off and learns to whittle some fair freehand sections is neither a histologist nor a technician. Botanical microtechnic|ue may be defined in terms of its functions, which fall into the following overlapping categories: 1. the preparation of ])lant tissues for microscojiic stud\. 2. the skillfid use of the microscope and related equipment for the (ritical stutly and interjnetation of the material. 3. the recording and illustrating of ihe results by means of the graphic arts. In some schools microtc(hni(|ue is taught as |)art of the work in some i)ranch ol iiioi phology, ,su( li as anaionn or c\tology. That system has marked advantages. The student avIio has collected and processed his own j)lani materials, and made his slides, can \isualize the orientation ol the sections in tlic plain and inteiprci ihe lelation- ship of parts to the whole j)lant. A disadvantage of the system is that specialized courses in moi phologv are likth lo uiili/c a liiniud num- ber of methods — loi instance, (he smear nuihod in ( \ logenetics. The student inav accpiiie remarkable skill in making pi(.j)arations of one type and lunc no expeiiencc with otiui use Inl intihods. He may develojj great skill in making smear pic|)araii()ns ol |)olIen mother cells, but one cannot snuar a kernel ol corn or a pine stem. He may even ac(|uiic' disdain lor miiiiods which \cisatile and experienced possible ihc presentation ol liie luiulauKiiials ol a wide range of workers regard as indisjjinsabie lor (criain tasks. The niainu iiand' of a se])arait' (onrse in microtechni(|ue makes Preface I'x useful standard methods. Intensive training can be given in the lew processes wliich experience has shown to be the backbone of research and which ha\e long served the routine needs in teaching. A course of this type should be organized to give a systematic, graded series of exercises, each exercise pointing to some definite objective and yielding superior preparations of a given type. Student interest can be maintained by working with plants that are of interest to the student or the institution, and Avith plants that are characteristic of the region. The trend in manuals of microtechnique has been in the direction of encyclopedic works of wide scope. The extensive array of processes in the research literature and reference manuals is bewildering to beginners. Teachers have found it necessary to select and to assemble suitable material in syllabus form. This manual has evolved over a period of years in connection with the teaching of a college course in histological methods. The course and the manual were designed to meet the needs of teachers and prospective teachers of plant science, and the needs of beginners in research in the basic and applied plant sciences. Since this is primarily a training manual rather than a reference work, use is made of a graded series of assignments, beginning with subjects in which orientation is easily visualized, few sectioning difficulties are encountered, and a simple stain is used. Subsequent as- signments require greater skill in the processing, sectioning, and differential staining of cell and tissue components. A few care- fully selected processing and staining methods are presented in detail. Emphasis is placed on gaining an understanding of the aim of the undertaking and the function of every operation, rather than on memorizing and mechanically following a written outline of proce- dure. After mastering the fundamentals, the worker can readily delve into the literature of specialized fields by consulting the key references in the brief bibliography. Compilation of the author's syllabus and records into book form has been made possible by a grant from the Graduate College and by assistance from the National Youth Administration. The drawings were made by Miss Ruth McDonald. Grateful acknowledgment is made for this aid. The author's colleagues and students have given much valuable criticism, advice, and encouragement. John E. Sass Iowa State College, August, 1951 Contents Part I— General Principles and Methods 1. Iniroduction 3 2. Collecting and Subdividing Plant Materials 5 3. Killing, Fixing, and Storing Plant Tissues 12 4. Dehydration for Embedding 22 5. Infiltration and Embedding in Paraffin Wax 31 6. Microtome Sectioning of Material in Paraffin .... 40 7. Staining Paraffin Sections 55 8. The Celloidin Method 78 9. Sectioning Unembedded Tissues ^'1 10. The Preparation of Whole Mounts and Smears .... 99 11. Criteria of Successful Processing HO Part II— Specific Methods 12. Introduction 11^ 13. Vegetative Organs of Vascular Plants 121 14. Thallophyta and Bryophyta I'i^ 15. Reproductive Structures of Vascular Plants 165 16. Microscope Construction, Use, and Care 182 17. Photomicrography 202 221 Index -^riiBl; [xi] Part I General Principles and Methods /. Introduction The study of the microscopic details of the structure of plants usually requires some preparation of the material to facilitate obser- vation. Unicellular, filamentous, or other minute plants require comparatively little preparation. The material may simply be mounted on a slide in a drop of water and thus studied, even under consider- able magnification. Larger plants, or parts of plants, must ix- dissected or cut into thin slices in order to expose inner regions and to permit light to penetrate through the object. Some materials have enough natural coloration to be visible even when finely divided or sectioned. Highly transparent or colorless structures, on the other hand, must be made visible by the use of stains. Preparations that are to receive considerable handling over a period of time should have some degree of permanence. The desirable properties of microscopic jMepa- rations are, therefore, adequate thinness, coloration or retractile visibilit), and permanence. The processes used in the preparation of plant materials for microscopic study can be roughly classified in the following categories: 1. Unicellular, filamentous, and thin thalloid forms can be pro- cessed //( toto — without sectioning — and mounted as "whole mounts" to make temporary or permanent slides. 2. Some succulent tissues can be crushed or smeared into a thin layer on a slide or cover glass. The preparation is then stained and treated to make temporary or permanent slides. 3. The more complex and massive tissues are usually sliced into very thin slices, freehand or with a microtome. Materials that are not sufficiently rigid to be cut without support are embedded in a sup- porting matrix before sectioning. The sections are stained and mounted to make temporary or permanent slides. The method used for the perparation of a given subject depends on the character of the material, the use that is to be made of the [3] 4 Botanical Microtechnique slides, and such facilities as equipment, reagents, and time. The experienced worker does not overstress the merits and applicability of some one method. For example, important advances in smear methods and related processes for the study of nuclear and chromo- some details have replaced to some extent embedding and sectioning. The whole-mount method is recognized to be entirely satisfactory for many algae, fern prothalli, and similar subjects. However, micro- tome sections of embedded material must be made if we wish to stuch the undistiabed cellular organization of a tissue, the development and arrangement of organs, or the structural relationship between a fungus or insect parasite and the tissues of its host. The much- maligned celloidin method must be used to keep intact a badly decayed, fungus-infected piece of oak railroad tie for an examination of the mycelium in the wood. In order to avoid undue emphasis on any particidar method, we should recognize that each of the well- established methods has its proper sphere, in which it is the most effective and economical method of performing a given task. The sequence in which processes are arranged in this book takes cognizance of the fact that the paraffin method fiunishes by far the largest nimiber of slides produced for teaching and research. Certain operations, such as the killing of protoplasm and the preservation of fixation images, are essentially similar for smears, sectioning, and whole-mount methods. The preliminary processing of material is nuich the same in the several embedding and sectioning methods, hi \icw of these facts, the parailin method is presented with inibroken continuity of its operations. ^. Collecting and Subdividing Plant Materials The preservation ot structural details of cells and tissues is in- fluenced by the condition of the plant at the time of collecting and by the subsequent preparation for killing (fixation) . For the study of normal structure, select healthy, representative plants. Re- move the plant or the desired part with the least possible injury to the sample. If the material is to be killed at once, follow the pro- cedure outlined in Chap. 3. If the material cannot be killed prompdy, it should be stored and transported in such manner that bruising, desiccation, molding, and other injuries are minimized. Do not use material that has been obviously damaged in storage or shipment. The unsatisfactory slides obtained from such material are likely to be interpreted by uncritical observers as the result of poor technicjue. Dried herbarium specimens can be softened and sectioned to make slides in which it is possible to determine the gross features of vascular arrangement or carpellary organization (Hyland, 1941). However, such material is not suitable for detailed microscopic study. 1 he following general directions are introduced at this point for the use of readers who have selected subjects on which to work. The reader who seeks suggestions concerning suitable and tested subjects should turn to Part II and use the recommendations made therein in conjunction with the present chapter. LEAVES Remove a leaf or leaflet by cutting the petiole, without squeezing or pulling the petiole. The vascular bundles in the petioles of some plants become dislodged easily. For transportation or brief storage, place the leaves between sheets of wet toweling paper and keep in a closed container such as a tin can or a Mason jar. If the leaves appear to be wilted on arrival in the laboratory, freshen them in a moist chamber before processing. [5] 6 Botanical Microtechnique STEMS Leafy stems can be kept fresh for se\eral days by standing them in a container of water, preferably in a refrigerator. If such storage is not practicable, cut the stems into the longest pieces that will fit into the available closed container without folding or crushing. \Vrap the pieces promptly in wet paper and store in a cool place. Dormant woodv twigs, large limbs, and disks cut from logs can be kept for weeks in a refrigerator without appreciable injury. ROOTS Do not collect roots or other underground organs by pulling up the plant. The delicate cortex is easily damaged, in fact, the woody stele may be pulled out of the cortex, leaving the cortex in the ground. To collect roots without damaging them, dig up the plant, soak the mass of soil in water until thoroughly softened. Wash the soil away carefully, cut off: the desired roots and brush them gently with a camel's hair brush to remove as much soil as possible. Wrap the pieces and store as in the case of stems. FLORAL ORGANS Remove entire flowers or flower clusters and wrap in wet paper. Store in a closed container in a cool place. Large buds like those of lily can be kept in a Mason jar of water until you are ready to dissect and preserve them. Fruits may be collected and stored in a similar manner. LIVERWORTS AND MOSSES Remove groups or mats of the material with a generous quantity of the substratum. Store in a moist chamber until the plants are turgid. Saturate the substratum in order to permit the removal of complete plants without excessive damage. Dissect out the desired parts under a binocular and transfer to the preserving fluid ])romj)tly. ALGAE Collect in a tjuantity of the water in whi( h ilie plants are growing, and keep in a cool place in subdued light, ^rany filamentous forms disintegrate rapidly in the laboratory, and c\cn in the greenhouse unless the temperature and light can be carefidlv controlled. It is best to kill algae promptly after collecting. FLESHY FUNGI l"he larger fleshy fungi can i)e irausporiccl and stored. wrapj)ed Collecting and Subdividing Plant Materials 7 loosely in waxed paper. Sporulation continues and may indeed be promoted in this manner. However, since molding and disintegration take place during prolonged storage, material should ])e processed promptly. Small fungi should be wrapped in moist paper, enclosed in waxed paper, and processed as soon as possible. PATHOLOGICAL MATERIAL Particular care should be exercised to insure that the condition of the host tissues is not altered by handling, in order that abnormal structure may be properly interpreted as an histological symptom of the disease. Prevent wilting of the material, or revive it in a moist chamber, but a\()id the development of bacteria, molds, or other secondary organisms. For a pathological investigation, always collect normal, disease-free tissues of age comparable with the diseased samples. It is imperative to work out the best technique for preserving the "normal" condition of the host before attempting an authoritative interpretation of slides of pathological material. The foregoing general remarks will serve as a basis from which the worker can develop effective methods and habits of collecting and handling material in accordance with facilities and circumstances. Hold rigidly to the view that the finished slide should represent the original structure of the plant, whether that structure is presumably normal or pathological or is the result of experimental treatment. The handling of materials that are to be used for bulk specimens or whole mounts is described in Chap. 10. The preparation of perma- nent slides from microtome sections consists essentially of the following processes: 1. Selecting desired plants or parts of plants and, if necessary, subdividing into suitable pieces. 2. The killing and preservation of the contents of cells and the preserva- tion of cellular structures in a condition approximating that in the living plant. 3. Embedding in a matrix if necessary, in order to support the tissues for sectioning. See page 91 for the sectioning of unembedded tissues. 4. Sectioning of the tissues into very thin slices. 5. Staining the slices and covering with a cemented cover glass to make a permanent slide. Subdividing Material for Processing Some preliminary remarks concerning the action of reagents in the preservation of cells and tissues will aid in understanding the following description of this process. The reagents used for killing 8 Botanical Microfechnique Fig. 2.1— Methods of siilxiiN idiiif; lc;i\fs for cmbcddint;: ./ /). loiij^ iiairou leaves and iraiisvcMsc i)icccs icmovcd from siuli lca\cs: /•,". cniljccklcd piece of leaf fastened to mounting block; /•'-//, laij^e Ijioad leaf and excised pieces of l)lade and petiole: /, portion of leaf with fungus pustules: /. enlarged \ie\\ of excised aeci;i: A. em- bedded piece of leaf beating aecia. fastened to mounting block. cells contain ingredients tiiat are toxic to proioplasni. In oidei to MO)) lile |)rocesses (jtiickK and wiiliotit distortion ot structtirc, the killing fltiid nnist icacli the iinuiiiiosi (clls ol a \)'iv(.v ol tissue belore disintegration takes place. Most reagents penetrate very slowly through the cuticle or cork on the surfaces ol plant organs, btit j)enctraiion is nuuh more raj)id tlnough cm siirlaces. Therefore, Collecting and Subdividing Plant Materials 9 it is desirable to cut the organ being studied into the smallest pieces that will show the necessary relationship of parts. The subdividing of soft fresh material is best done with a razor blade, with the material placed on a sheet of wet blotting paper or held carefully against a finger. Excessive pressure against the support is likelv to ruj)ture delicate tissues as in the mesophyll of leaves (Fig. 11.1) or the chlorenchyma of a stem (Fig. 11.2). Such damage does not become visible until the sections in the ribbon are examined or possibly not until the finished slide is examined. The usual results are peeling of the epidermis and distortion of the crushed tissues. Leaves are almost invariably cut into small pieces for processing. Narrow leaves that are not much over 5 mm. wide, may be cut into complete transverse pieces measiuing 2 to 4 mm. along the rib (Fig. 2.\ A-D) . Examples of this type are bluegrass, garden pinks, hedge mustard, and some narrow-leaved milkweeds. Broad leaves should be cut into small pieces, selected to include midrib, lateral veins, fungus pustules, fern sori, or other desired structures (Fig. 2.1 F, G, I, J) . The enlarged views of the pieces of leaf (Fig. 2.1 B, D, G, I, J) and the pieces of embedded tissue mounted on blocks ready for sectioning {E and K) will aid in visualizing the orientation of pieces. Particular care should be used in subdividing pathological material (Fig. 2.1 /, /) . If it is necessary to know which is the long axis of the leaf, cut all pieces so that the shorter dimension is along the long axis of the leaf, or vice versa, and record the method in yoin- notes. Herbaceous stems, roots, petioles, and other more or less cylin- drical organs are usually cut into short sections or disks. When cut- ting out sections or subdividing pieces, do not roll or press the pieces. Keep the material moist, and work rapidly. After the final subdivision, drop the pieces into the killing fluid promptly. By means of descrip- tions and sketches, like those in Figs. 2.1 and 2.2, keep an accurate record of the part of the plant from which the pieces of tissue were obtained. Figure 2.2 gives additional suggestions for subdividing organs. A stem that does not exceed 2 mm. in diameter should be cut into sections 2 mm. long if highly cutinized, but may be as long as 10 mm. if the surface is permeable. An organ 5 mm. in diameter should be cut into 5-mm. lengths. An organ 1 cm. in diameter should be cut into disks 2 to 5 mm. thick. Stems of larger diameter are usually cut into 5-mm. disks that are halved or cjuartered longitudinally or di\ided into wedge-shaped pieces. 10 Botanical Microtechnique Fig. 2.2— Methods of sulxliv idiiig massive cyliiidiical organs: A-C, sample includes portions of all tissues in the axis; D shows the position of pieces removed from a large log; E and F, enlarged \ icws of trimmed pieces rcmo\ed from large log. Woody twigs having a diameter up to 5 mm. should be cut into 15-mm. lengths. Larger twigs should be cut into shorter pieces because the impermeable cork makes penetration by reagents difficult, except through the cut ends. Do not cut the twigs into pieces wath pruning shears or a knife. Rough handling will bruise the camlMum. phloem, the fragile primary cortex and cork cambium, resulting in the separation of the outer layers during sectioning or during stain- ing. Use a razor blade and ctit through the twig by chipping a groove deeper and deeper around \hc twig iinlil il is (tit ihioiigh. \n excellent tool tor cutting twigs into sections is a fine-toothed high- speed saw, such as a rotary dental saw or a jig saw, especially the vibrating diaphragm type. To make slides of transverse, radial, and tangential sections in the region of the (anibiinn of old trees, use tissues removed from newly felled logs or limbs having a diameter of at least 10 cm. Cut disks 2 to .S cm. thick from jjortioiis of the log that were not bruised in felling. Wrap the disks in wet burlap and take into the laboratory Collecting and Subdividing Plant Materials 7 1 at once for further trimming. Split a disk radially into pieces having uninjmed blocks of bark firmly attached to the wood. Trim off enough of the inner part of the wedge of wood to leave a block of sapwood with several annual rings and with cambium and all outer tissues intact (Fig. 2.2 D-F) . With a razor blade split a thin layer from the two radial faces, from the inner tangential surface and from the transverse faces of the block, thereby removing tissues that were compressed during the preliminary trimming. Keep the material wet during these operations. Drop the pieces into the killing Huid at once after final trimming. Wood from dead logs, dry lumber, or furniture wood requires proper trimming to establish the future cutting planes. It is usually easy to establish the radial plane by splitting the wood longitudinally, parallel to a ray. At right angles to this plane, split the block longi- tudinally along the tangential plane, and then trim in the third or transverse plane. Rough splitting can be done best with a plane bit, and rough crosscutting with a fine-toothed high-speed mechanical saw. Finally, trim all faces with a razor blade to remove surface tissues that were damaged by the rough trimming. Subsequent processing of the wood is described in the section on the preparation of hard tissues. The handling of more specialized and difficult materials such as buds, floral organs, and fruits is described to better advantage in Part II in conjunction with detailed directions for processing such materials. The handling of plant bodies and organs of the lower phyla is also described in Part II. The foregoing brief outline of methods of collecting and prepar- ing material for preservation can be modified and adapted to meet most problems. The principal preliminary operations and precautions necessary for successful processing may be summarized as follows: 1. Use fresh, normal material. 2. Remove pieces having the desired features and oriented so as to establish planes in which microtome sections are to be cut. 3. Cut into suitable pieces, with minimum bruising, compression, or desiccation. 4. Immerse the pieces promptly into the killing (fixing) fluid (Chap. 3) , and promote quick penetration of the fluid by removing air with an aspirator (Fig. 3.1) . 5. Record the necessary data concerning species, location, date, parts selected, and killing fluid used. Jj, Killing, Fixing, and Storing Plant Tissues One of the most critical operations in the processing of tissues is the kilhng of the protoplasm. The stopping of life processes within the cells should be accomplished with the minimum structural dis- turbance within the cells and minimum distortion of the arrangement of cells in the tissues. In addition to killing the j^rotojjlasm, the killing fluid or the subsecjuenl processing nuist retain or fix the undistorted structure and render the mass of material firm enough to withstand the necessary handling. No single substance has been found to meet the recjuirements of successful jireservation. The formulas used for this pmpose consist of ingredients in such proportions that there is a balance between the respective shrinking and swelling actions of the ingredients. The numerous formulas found in the literature are variations of a com- paratively few fundamental formulas, and the chemical sidxstances in the formulas are few in luunber. Any formula shoidd be regarded as a starling point for experiments to determine the proper balance of ingredients for sjK'cific subjects. 1 he formulas reconnnendrd in this chajiter ha\e been lound to i)e satisfactory for a di\ersit\ ol subjects. Preparation of Stock Solutions and Killing Formulas The followin" reauents and stock solutions are used in a wide range of killing (fixing) formulas: Glacial acetic a(i(l. 1% acetic acid (a])pr().\iinatcly) , made In adding 10 ct. ol glacial acrlit acid to 990 cc. of water. 10% acetic acid, made on ihc same basis as ilic above. Propionic acid ma\ he sul)sliuile(l for acetic acid in the al)o\e. \% chromic a( id. (10 g. cliromic anhvchide crystals per liter.) Formalin, the trade name used lor an ac]ueous soliiiioii ol loinialdi Indc. (ontainint; '^7 to lO'/r lornialdehyde i;as hv weight. I 12 1 Killing, Fixing, and Storing Plant Tissues 73 Picric acid, saturated aqueous solution. 2% osmic acid. 2 g. crystals in 100 cc. of 1% chronn'c acid, or in 100 cc. of water. Ethyl alcohol; commercial 95% grade and anhydrous grade. Bichloride of mercury (HgCL) crystals. The use of stock solutions ot 1% and lO^f acetic or propionic acid is advocateci because the error involved in measuring a small volume, say 1 cc, of glacial acetic acid is much greater than in measur- ing 10 cc. of 10% acid. Apparatus Use specimen bottles that hold a generous cjuantity of killing fluid, especially with bulky or succulent materials that may dilute tlie formula. After washing and partial dehydration, materials may be transferred to smaller bottles or vials for the remainder of the process. When the pieces of plant material are dropped into the killing fluid, the hairs, stomates, folds, and other cavities of plant organs retain air bubbles which retard penetration by reagents. If the pieces do not sink at once, attach the bottle to an aspirator, and apply suction for repeated short intervals until the pieces sink, if not to the bottom of the liquid, at least under the surface. Use a safety bottle (Fig. 3.1 A) to keep water from backing into the specimen Fig. 3.1— Aspirator setup for pumping specimens in killing fluid: A, safety bottle with finger valve or glass stop clock; B, specimen bottle or large empty bottle into Avhich specimen bottle is placed; C, pint jar used as container for large specimens 74 Botanical Microtechnique bottle. Tapping the specimen bottle gently against the sink aids in the loosening of air bubbles within or on the specimen. Highly buoyant materials should be placed into a tall vial of the killing fluid and held below the surface by means of a plug of cheesecloth. A screw-topped wide-mouthed bottle is necessary for evacuating large objects (Fig. 3.1 C). W'hen most of the pieces remain submerged after the suction is released, push any floating pieces under the surface with a matchstick, and most of them will then sink. Remo\e and discard all pieces that do not sink after pumping and submersion. Materials from which it is difficult to evacuate air do not become infiltrated readily and should be pimiped again when nearing the end of the dehydrating series, and again when in the final change of paraffin solvent, before any paraffin has been added. Connect a second safety bottle between the regular safety bottle and the speci- men. The possible entry of water vapor into the specimen bottle when the pump is shut oft is prevented by having a deep layer of calciinn chloride and a layer of cotton in the second safety bottle. The ingenious and precisely controllable vacuum apparatus of Witt- lake (1942) may be used for the killing, as well as the subsequent operations of embedding. Killing and Fixing of Tissues Killing solutions may be grouped into types on the basis of the ingredients used. Some formulas are stable and may be kept on hand ready for inmnediate use. Other formulas must be made up innnedi- ately before use. The forjnulas given on the following pages have been comj)uted so that they can be made up from the above stock solutions by volumetric measurements, llic system of letters and numbers used in this manual to designate killing fluids is explained later in this chapter. The length of time necessary to bring about killing and harden- ing of material varies greatly and is determined by the character of the fluid used, the bulk of llie indixidual pieces, and the resistance of materials to jKuetraiion by reagents. Fluids of the anhydrous type, such as Carnoy's absolute alcohol-glacial acetic acid fornuda. pene- trate small objects almost instantaneously, and killing and hardening are a matter of miniues. The chrome-acetic fluids penetrate slowlv into the interior of a piece of tissue, and have poor hardening action. Re(f)nnnendations coiucrning the duration in killing fluids are given in the description of the various fonnulas. Washing of tissues, which Killing, Fixing, and Storing Plant Tissues 75 is necessary after some killing fluids, is discussed in connection with specific formulas. One of the most useful types of killing and preserving fluid, kno^vn as FAA, is represented by the following formula: Ethyl alcohol (95%) 50 cr. Glacial acetic acid 5 cc. Formaldehyde (37-40%) 10 cc. ^Vater . . . ! 35 cc. Propionic acid may also be used, the formula is then designated FPA. Several modifications may be found in the literature. This fluid is stable, has good hardening action, and material may be stored in it for years. These properties make this formida suitable for large or impervious objects such as woody twigs, tough herbaceous stems, and old roots. The high concentration of alcohol is likely to produce shrinkage of succulent materials, although it is possible to develop a formula for some apparently tender subjects and even for filamentous algae. A balanced formula can be worked out by varying the acetic acid, which has a swelling action on protoplasm, from 2 to 6% by volume. The formaldehyde and alcohol, which have a shrinking action, should be held at the indicated concentrations. When making trials of variations from the fundamental formulas, kill a trial lot or batch of material in the formula to be tested, and a check lot in a standard formula, and carry the batches through identical process- ing simultaneously, so that differences in cellular detail will be the result of variations of formula. Pieces of thin leaf are killed and hardened in 12 hr. The actual killing of the protoplasm probably occurs in much less time. Thick leaves or pieces of small stem require at least 24 hr. Woody twigs should be kept in FAA at least a week before continuing the process- ing for embeddino. Materials do not need to be washed after FAA. The ingredients of this fluid are soluble in the dehydrating agents and are thus removed before infiltration is begun. An extensively used formula consists of FAA containing bichloride of mercury (HgCL) to saturation. This fluid penetrates and hardens tissues rapidly. It preserves bacterial zoogloea in plant tissues, thus being useful in pathological studies. The alcohol may be increased to 70%. Prolonged storage in fluids containing bichloride is undesir- able. The tissues should be transferred after 48 hr., or at most a week, to a fresh solution of the original formula which does not 16 Botanical Microtechnique TABLE 3.1 Killing Fluids of the Chrome-acetic and Flemming Type * (The numbers in the columns represent cubic centimeters of the designated reagents) Chrome-acetic Flemmi ng type Stock solution Weak I Weak II Medi- um I Medi- um II Strong Weak Medi- um Strong Cham- ber- lain 1 % chromic acid. 1% acetic acid. . . 10% acetic acid. . 30 70 50 50 50 70 97 25 10 50 75 96 10 20 10 Glacial acetic acid 3 5 20 3 2% osmic acid. . . 10 55 10 30 1 Water 40 10 * The formulas in Tables 3.1 and 3.2 have been arranged and arbitrarily num- bered, beginning with the weaker solutions. The roman numbers do not correspond with the numbers used in mimeographed editions of this manual. contain the bichloride of mercury. After foiu" or five changes of the latter solution, tissties may be stored indefinitely in the last change. Chromic acid and acetic acid are the ingredients of an important class of flinds, the chrome-acetic formtdas. These fluids are not used as extensively as some years ago. Table 3.1 gives the proportions of five formtdas. Because of widespread use of the terms weak, medium, and strong for fluids of the type given in Table 3.2, these terms are retained for the series of modifications in the table. The weaker solutions are suitable for succulent or delicate subjects, the strong solution for firm subjects. If this type of fluid is to be used for a critical study, a balanced h)rnuda shoidd be worked out by balancing the shrinking action of chromic acid and the swelling action of acetic acid. I'he above lornudas are not very satisfactory for i)idk) or woody subjects because of poor penetrating ability. Use these mixtures for filamentous and thalloici plants, root tips, floral organs, and small sections of leaves or stems. The length ol time recjuired to kill materials varies greatly. Filamentous algae are probably killed in a few minutes. Small })ieces of leaf or root tips require about 12 In. Larger pieces of ti.ssuc shoidd ha\e at least 24 hr. The j)r()gressi\e destruction of (hloroi)h\ll from the cut edges inward is a good gauge of the rate ol action. Piolonged storage in chrome-acetic produces brittleness of the tissues and nuiddy staining effects. Therefore, these fliuds are not suitable iov storage and the tissues nuist be processed after the optinuun interval necessary foi killing. Killing, Fixing, and Storing Plant Tissues 17 Materials killed in the fluids given in Table 3.1 should be washed in ruiuiing Avater. Various devices may be used tor accomplishing this prolonged washing. The simplest method is to tie a strip of cheesecloth over the wide mouth ot the bottle containing the tissues and to allow a slow stream of water to How into the bottle. More vigorous washing action can be obtained by inserting the water inlet tube to the bottom of the specimen bottle. These fluids do not have good hardening action, so it is best to avoid violent motion of the pieces. Firm materials can be washed in a vertical length of 1-in. glass tube with a stopper at the lower end, admitting a stream of water through a small tube, the waste water leaving through cheese- cloth tied over the upper end of the large tube. Osmic acid is used in a class of formulas known as the Flemming fluids. These fluids are indispensable for cytological studies but are seldom justifiable for histological work. Osmic acid is expensive, its vapors are highly irritating, and it blackens tissues, making it neces- sary to bleach sections before staining. Osmic acid preserves chromo- some details with great fidelity, but has no special virtues for the preparation of slides of such subjects as corn stem or apple leaf for anatomical or histological study. Osmic acid has poor penetrating ability and is therefore not satisfactory for bulky objects. The formu- las given in Table 3.1 will serve for preliminary tests, subject to experimental variation of proportions. Because of the blackening action and poor hardening properties of the Flemming fluids, mate- rial should be washed in water and processed immediately after kill- ing. Ihe intervals for killing are approximately those given for chrome-acetic. Table 3.2 gives several formulas based on the Nawaschin formula, containing chromic acid, acetic acid, and formaldehyde. Numerous modifications may be found in the literature. The name Craf has been coined for this widely used type of fluid. For critical work on specific subjects, experiment with variations of the formulas in the table. The acetic acid should be varied from 0.7 to 5% glacial acetic acid equivalent by volume. The optimum chromic acid and formalde- hyde concentrations for many subjects are the proportions given in formula V. The other formulas in the table, including Nawaschin's original formula, also give good results with specific subjects. The formaldehyde should be added immediately before using. If one of these formulas is to be used for making extensive collections in the field, it will be found convenient to make up the desired mixture of the chromic and acetic acids, adding the measured volume of for- 18 Botanical Microtechnique TABLE 3.2 Killing Fluids Based on the Nawaschin and Bouin Formulas (The numbers in the columns represent cubic centimeters of the designated reagents) Nawaschin type (Graf) Bouin Alien-Bouin type Stock solution 1% chromic acid. . 1 '-Vr^ acetic acid Nawaschin 75 I 20 75 II 20 III 30 IV 40 V 50 I 50 , It 50 III 25 10% acetic acid. . . 10 20 30 35 "5" 25 75 20 10 20 5 10 35 40 Glacial acetic acid . 5 20 Formaldehyde 37- 40% aqueous. .. Picric acid satur- ated aqueous . 5 .... 5 10 10 15 10 25 Water 65 40 20 maldehyde before using. A few hours after the formaldehyde is added a perceptible change of color takes place in the licpiid, and after several days the chromic acid becomes changed to an oli\e or green compound. Long before this condition is reached, killing action has been completed, and the altered fluid then serves as an excellent hardening and preserving agent. Material may be left in these fluids for as long as 5 years and yield excellent histological preparations. The effect of prolonged storage on critical cytological details deserves further study. 1 he minimum time for small masses of soft tissue is 12 hr., but it is obvious from the foregoing remarks thai several days at least can be allowed to insure hardening without danger of distor- tion or darkening ol the material. A fiuther ad\antage of the Na- waschin txjjc (hiid is that materials need not be sul)se(|uentl\ washed in water, thus axoiding the possil)le softening and pulping ol mate- rial. Boiiin's (iuid, gi\en in Table '^2, has long and deser\edl\ been a favorite. It is excellent for loot tips, especially for telophase figures, and has been used successfully for embryo sac studies. The complete mixture is stable and may be kept on hand in the hd)orator^ or carried to the field read) for use. A mininunn iiitcrxal of 12 hi-, is suggested for finely divided material. Larger ))ieces such as thick root ti])S or mature tissues should ]ia\e at least hS hi. I'lolonged storage is regarded as undesirable. Alter the optimum intei\al in the killing fluid, the material is not washed in \vatei. but is rinsed several times in 20% alcohol 01 acetone. Dehydration is then c on- tinned as descril)ed later. Killing, Fixing, and Storing Plant Tissues 79 The addition of chromic acid and urea to Bouin's Ihud makes what is known as the Allen-Bouin formula. For cytological work use the original formula, as given in the reference manuals, or one of the formulas (lacking urea) given in Table 3.2. For further trials vary the glacial acetic acid equivalent from 1 to 4% by volume. The formaldehyde should be added immediately before using. Tests have shown that tissues may be left in these solutions for several months. It is probable that hardening of the material reaches a maximum in less than a week. Dehydration and subsequent processing are carried out as with Craf. Farmer's fluid and Carnoy's fluid have limited uses in histology. These fluids kill protoplasm by rapid and probably violent dehydra- tion. Because of their ability to penetrate very rapidly, these fluids have some value for processing extremely downy, resinous, or im- permeable structures that must be preserved entire. The fluid may be used alone, followed in 1 hr. or less by the subsequent operations of the paraffin process. An alternative method consists of first im- mersing the materials in a Carnoy or Farmer formula (the time ranging from an instantaneous dip to 10 min.) and then treating in one of the more critical fluids. Two widely used formulas are as follows: 1 . Farmer's fluid Anhydrous ethyl alcohol 75 cc. Glacial acetic acid 25 cc. 2. Carnoy's fluid Anhydrous ethyl alcohol 60 cc. Glacial acetic acid 10 cc. Chloroform 30 cc. The fluids given thus far in this chapter produce an acid fixation image, preserving particularly well the chromosomes, nucleoli, and the spindle mechanism. Nucleoplasm and mitochondria are dissolved; cytoplasm is rendered in fibrillar or alveolar form. This type of image is preferred for most studies of plant structure. In certain cytological studies it is desirable to preserve mitochon- dria and allied cytoplasmic structures. In such cases a fixing fluid that produces a basic fixation image is used. Such fltuds preserve mito- chondria, nucleoplasm, and in some instances nucleoli and vacuoles. Chromatin and the spindle mechanism are dissolved. For serious studies in this field of cytology each worker must work out specific techniques based on an extensive literature. However, it is possible 20 Botanical Microtechnique to produce slides showinti; mitochondria adecjuately for teaching pur- poses, using Zirkle's modification of ErHki's fluid. Water 400 cc. Potassium bichromate 2.5 g. Ammonium bichromate 2.5 g. Cupric sulphate 2.0 g. Fix for 24 to 48 hr., wash in water, dehydrate and embed in paraffin. The desirability of wetting agents and penetrants in microtech- nic]ue has been apparent to experienced workers for many years. 1 he rapid development of numerous wetting agents in recent years has led to considerable experimentation, with the expected diverse results. The most prominent unfavorable effect of wetting agents are the peeling of cuticle and epidermis, and varying degrees of cell distor- tion. Further experimentation with the increasing number of a\ail- able substances is certainly desirable. The wetting action of a substance can be tested easily. Make a series of solutions of the substance to be tested by diluting a 1:1000 stock solution. Cut uniform pieces of a highly pubescent leaf and drop alternating pieces into distilled water and into the dilutions of the wetting agent. Note the relative time required lor the leaf pieces to sink, and use the most dilute wetting agent that will sink the tissues after brief aspirating. Determine whether the wetting agent forms a precipitate or cloudiness with the killing fluid. If a reaction occurs, do not add the wetting agent to the fixing fluid, but sink the tissues in the wetting agent, rinse with water and (()\er with the killing fluid. The final criterion is the condition of the tissues in the finished slide, compared \\ith tissues processed without the wetting agent. A workable terminology for designating killing lluid rorniulas is a great convenience for giving oral or written instructions, or making routine rccoicls. The name of the investigator who first devised a type of formula is not always a satisfactory designation because the pro- portions of the ingredients are necessarily \aried h)r (.liflerent subjects. An arbitrary number is not sufliciently des(ripti\ f, excejjt among a group of closely associated workers. Ihc ici minology pioposed here is a compromise, the t\|)e ol loi inula is inclicatcd 1)\ a name or abbievia- lion, and the |)i()p()rti()n ol ingredients 1)\ a ])(. i c cntage liguic. The proportion of a solid like chromic acid is gi\en as piecentage by weight; licpiicls like nulled glacial acetic acid aic gixcn as percentage by volume. For instance, the time-honored chrome-acetic has numer- ous \aiiants. one ol which is i'.-.\ ()..5-().r). meaning 0..5'; chromic acid Killing, Fixing, and Storing Plant Tissues 21 by weight, and 0.5' i acetic acid by \olunie. Table -5.1 gives the proportions of stock sohitions used to make 100 cc. oi mixtures in this category. A variant of the Nawaschin formula, Craf 0.20-1.0-10.0, contains 0.2% chromic acid, 1.0% acetic acid, and lO.O^o commercial form- aldehyde solution. A \ariant of the Allen-Bouin formula is designated A-B 0.20-4.0-10.0-25.0, containing in addition to the ingredients of Craf, a saturated acjueous solution of picric acid, 25.0% by volume. The foregoing system of terminology is accurate, descriptive, and convenient and has been used successfully by beginners and advanced workers. ^. Dehydration for Embedding 1 his operation removes water from the fixed and hardened tissues. Dehydration has some washing action, and makes the material firm and possibly hard and brittle. The process consists of treating the tissues with a series of solutions containing jjrogressively increasing concentrations of the dehydrating agent and decreasing concentra- tions of water. Two contrasting methods are used to dehydrate and prepare materials for infiltration. In the first method to be described, the tissues are dehydrated in a nonsolvent of paraffin and then are transferred to a solvent. In the second method, the dehydrant is also a solvent of paraffin. The first method of dehydration also is used prior to infiltration in celloidin. Dehydration by Nonsolvents of Paraffin The most commonly used dehydrating agent in this category is ethyl alcohol. This is usually pinchased in two grades, conniiercial 95% grain alcohol and absolute (anhydrous) alcohol. The solutions in the dehydrating series are made by dilming 95% alcohol with distilled water. After ascertaining the exact concentration of the alcohol j:)urchased from a given soinxe, it is casv to compute a table gi\ing the respective j)roj)ortions of alcohol and water for each solution in the scries. Hovve\er, since the series is intended to consist of a graded series of solutions rather than definite concentrations, it is (juite ade(juate to assume the 95% commercial alcohol to i)e 100% and make up a series containing (aj)])roximately) 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80%, alcohol by volume. Next in the series is the undiluted (onnnercial alcohol (actual 95*^;). followed by an- hydrous alcohol. Ihis graded series of solutions should be kept on hand in the laboratory. As discussed in the preceding cliaj)tcr. some killing fluids recjuire I 22 1 Dehydrafion for Embedding 23 more or less prolonged washing of the tissues in water; other fluids require no washing, and dehydration is begun directly alter killing or after a brief rinsing in water. Begin dehydration with a dehydrant having approximately the same percentage of water as the killing or storage fluid. For example, after FAA, begin dehydration in 50% alcohol. After weak chrome-acetic or the weaker Craf type formula, such as I and II, begin in 5 or 10% alcohol. The stronger formulas such as III, IV, and V, which have greater hardening action, make it possible to begin dehydration in 20 or 30^; alcohol. When using the Craf V (Cornell) formula for routine chromosome counts, root tips may be transferred directly from the killing fluid into 75% alcohol. After Bouins fluid, begin with 50% for firm subjects and 20% for delicate materials. Solutions in the dehydrating series are changed by decanting the liquid from the tissues and promptly flooding the material with a generous volume of the solution next in the series. A piece of fine brass-wire screen or a layer of cheesecloth is used to retain materials that tend to float out of the bottle. The volatility of the solutions high in the series demands speed in making the change to avoid drying of the tissues. The material should not be permitted to become dry even for an instant at any stage in the process. Never drain the fluid from several specimen bottles, and then look on the shelf for the next reagent, only to find that the bottle is empty. The interval in each of the solutions in the series depends on the size of the pieces, the nature of the material, and the solubility of the residual reagents left in the tissues. For root tips or small pieces of leaf use 30-min. intervals up to 70%. After a picric acid formula make each interval 1 hr. For twigs killed in FAA use 4- to 8-hr. intervals up to 709; . For large blocks of wood the interval should be about 12 hr. Beginning with 70%, double the previous interval for each grade. Change the cork for a thoroughly dry one when first changing to 100% alcohol. Make three changes of anhydrous alcohol. Plan the timing of the dehydration series so that the series is stopped at 70% for storage until you can resume the process. Some workers are inclined to make an imnecessary ritual of the time element in dehydration. It is recognized that drastic changes of concentration bring about shrinkage of protoplasm and distortion of cells. Long intervals in low concentrations of dehydrating fluid, or long washing in water, tend to make tissues soft and promote dis- organization.^Long exposure to high concentrations or anhydrous reagents shrinks tissues and causes brittleness. With these general 24 Botanical Microtechnique precautions in mind the intervals can be regarded as sufficiently flexible to conform to the demands of other duties. Isopropyl alcohol can be used in exactly the same manner as ethyl alcohol. Isopropyl alcohol can be purchased without restrictions, and the commercial grade can be dehydrated as described on page 29. Methyl alcohol has not been used extensively for dehydrating plant tissues. Its toxicity is objectionable, and the vigorous dehydrating action damages delicate structures. Acetone is an excellent dehydrant. Its jjurchase and use present no legal, administrative, or disciplinary problems, making it a desir- able substitute for ethyl alcohol. Acetone is obtainable in several grades, at jnices that vary widely with the cjuality and source. If anhydrous acetone can be purchased in drum lots at reasonable cost, only this one grade needs to be stocked and used for all the dehydrat- ing grades. Acetone of good quality, but not strictly water-free, can be obtained and used for the gradations, and the more expensive anhydrous grade used only for the final stages in the process. 1 he procedure with acetone is exactly the same as with ethyl alcohol. It is permissible to change from alcohol, or a killing fluid containing alcohol, to a grade of acetone having approximatel) the same water concentration. Acetone is highly volatile, and care should be taken not to jxrmit acetone to evaporate from tissues or slides during processing. Glycerin is used as a dehydrant, especially for algae and other delicate subjects. The high boiling point of glycerin jjermits the elimination of water by evaporation. Ihe slow, progressive dehydra- tion prevents sudden changes of concentration and minimizes plas- molysis. Material must be washed in water before using glycerin, because the evaporation process obviously does not wash residual reagents out of the tissues. Moderately firm tissues can be washed in riuining water, but delicate materials shoidd be washed by diffu- sion. Rinse the material carefully to remove the bulk of the kill- ing fluid, transfer to a 2-Cjuart jar of \vater, and allow the jar to stand undisturbed for 2 hr. Sijjhon off most of the water without agitating the material, and refill the jar with water. Repeat the re- placement of water at least twice then proceed with the <;l\cerin method. Tiansfer the material to a large volume of a 5% solution of gly((iiii in water. Use a wide-mouthed botik' oi jar and mark the level of the 5% glycerin. I he xoluiue should be so gauged that after the elimination of water the residual glycerin will more than cover Dehydration for Embedding 25 the tissues. Evaporation of water may be accomplished by several methods or combinations of methods. The most practicable are as follows: 1. In an incubator oven at 35 to 40 °C. 2. In a desiccator at room temperatures or in the above oven. 3. In a vacuum desiccator or vacuum oven. If the glycerin solution becomes colored or turbid during evap- oration it may be replaced with fresh glycerin solution of the same concentration. When the volume of the solution has been reduced by evaporation to one-half of the original volinne, the glycerin con- centration is approximately 10 7c, and the liquid may be replaced with fresh 10% glycerin and the evaporation continued. Most of the water can be removed by evaporation, especially in vacuum. After a nearly anhydrous condition is attained, the tissues are firm enough to withstand transfer directly into anhydrous alcohol. Change the alcohol at least twice, and proceed with the graded transfer to the desired paraffin solvent as described below, or proceed with one of the whole-mount methods (Chap. 10) . TRANSFER TO A SOLVENT OF PARAFFIN (CLEARING) After the use of dehydrating agents that are not solvents of paraffin, the dehydrated tissues are transferred to a solvent. The term clearing, applied to this transfer, is derived from the fact that some paraffin solvents render the tissues transparent. The clearing action is merely incidental to the function of the reagent, to serve as a solvent of paraffin. The most common solvents are xylene (xylol) and chloroform. Either reagent may be objectionable or even toxic to some workers. Xylene is inexpensive and is by far the most widely used solvent. Chloroform is more expensive, but it is less likely to be toxic. Benzene and toluene can be used, but their lower boiling points increase the fire hazard. As in the case of dehydration, a graded series is used for clearing. After dehydration in ethyl alcohol, the following absolute alcohol- xylene series is used. For critical cytological work 10 gradations have been recommended. The interval in each mixture ranges from 1/2 hr. Grade number Ethyl alcohol % Xylene % 1 2 3 4 75 50 25 0 25 50 75 100 26 Botanical Microtechnique for very small or thin pieces to 3 hr. or more for large pieces of tissue. A similar acetone-xylene series can be used. Chloroform may be substituted for xylene in a similar series, except that more abrupt changes are permissible. A practical series is as follows: (1) Vs chloroform -/i absolute alcohol (2) % chloroform Vs absolute alcohol (S) Pure chloroform, changed at least once Chloroform does not make tissues as brittle as does xylene. Trichloroethylcne is a good solvent of paraffin and may be sub- stituted for xylene in the foregoing processes. Trichloroethylcne is not inflannnable and is not toxic unless inhaled directly in large qtianti- ties. It dissolves Canada balsam but docs not affect stained sections. This reagent deserves thorough trial with a wide range of subjects. Any reagent that decreases the hazards of fire and ]:)oisoning is Avorth serious consideration. Cedar oil is an excellent clearing agent after delnchation in ethyl alcohol. The procedure is to pom a layer of cedar oil into a dry vial, then carefully ])our the anhydrous alcohol containing the material over the cedar oil. The pieces gradually sink into the oil and become strikingly clear. The alcohol is removed \\ith a ])ipette, and the cedar oil is rinsed out of the tissties \\ith several changes of xylene. Recognition of the fact that the transparency of the tissues at this stage of the j^rocess is of no value, and the widespread use of the higher alcohols for dch\xlration and as wax sohcnts. ha\c' jiractically eliminated the use of dealing oils. Following dehydration in any of the bui\l alcohols or dioxan, no clearing reagent is used, because these reagents are sohents of paraffin. They do not render the tissues appreciably transparent. Dehydration in Solvents of ParafFin THE BUTYL ALCOHOL METHOD Normal and tertiary buixl al(()li()l lia\t \kvu iniroduced into microtechni(jue in recent years and show nuuh piomise as dehy- drating and infiltrating agents. Normal l)utyl alcohol, also designated butanol, was the first of these iiigher alcohols to l)i' used extensively. Lang's careful experiments ha\e shown thai a miscibility curve of the three components of the dehydrant may be used to ascertain the Dehydration for Embedding 27 composition of solutions for an ideal dehydrating series. For critical cytological work, follow Lang's miscibility curves (Lang, 1937) in making up a series. The following series is a simplification that has been found to give excellent results in histological and anatomical work. n-Butyl alcohol Grade number (Butanol) Ethyl alcohol Water 1 10 20 70 2 15 25 60 3 25 30 45 4 40 30 30 5 55 25 20 6 70 20 10 7 85 15 0 8 100 0 0 Note that each of the first six grades consists of three ingredients. The last two grades are anhydrous. Use new anhydrous butyl alcohol for Nos. 7 and 8. After being used once, No. 8 may be used to make up any of the first six grades. After an aqueous killing fluid, wash or rinse the tissues in water, dehydrate in alcohol in the usual manner to 30%, then transfer to the abo\e reagent 1 and follow the series. After FAA or other fluids having a water content of about 50%, rinse in 2 changes of 50% alcohol and begin the n-butly series with No. 2, in which the water content is 60%. With many histological subjects good results can be obtained by dehydrating to 50% in steps of 10%, then continuing in n-butyl series 3, 5, 7, and 8. Tertiary butyl alcohol (TBA) is regarded by some workers as the most ideal dehydrating reagent of any thus far used (Johansen 1940) . Unlike the two other butyl alcohols, its odor is agreeable. The cost is at present much too high for extensive routine work. Tertiary butyl alcohol is used in accordance with the principles of dehydration described in the preceding pages. Dehydrate in ethyl alcohol to 50%, then pass through the following series: 95% ethyl Absolute ethyl Grade number alcohol alcohol TRA Water 1 50 10 40 2 50 20 30 3 50 35 15 4 50 50 5 25 75 28 Botanical Microtechnique Make three changes of anhydrous tertiar\ buiNl alc(jhol and pro- ceed with infiltration in wax. An unfa\orable factor in the use of tertiary butyl alcohol is that it solidifies at 25.5°C., a temperature that is not uncommonly attained in laboratories and stock rooms. Provisions must be made to keep this reagent fluid for immediate use. The low boiling point of 82.8°C. presents some fire hazard. The butyl alcohols have greatly extended the range of usefulness of the paraffin method by making it possible to cut materials that are rendered hard and brittle by ethyl or propyl alcohol or acetone. THE DIOXAN METHOD Dioxan, diethylqne dioxide, is becoming widely accepted as a dehydrating agent and paraffin solvent in the embedding of plant materials. This reagent is miscible with water and may therefore be progressively substituted for water in the tissues. Unlike the vigorous dehydrating action of the alcohols or acetone, the substitution of water by dioxan rs not associated with great plasmolyzing stresses. This fact permits dehydration by rapid substitution. Tissues do not become excessively brittle, and the histological details obtainable are equal to those obtained by other methods. The dioxan method requires nuich fewer separate operations than does any other method, and the operations may be at widely spaced intervals, thus reducing the burdensome routine of handling the specimens many times at frequent intervals. Kill the material in the desired formula. After the optimum fixing interval, wash in water if required by the formula. Animal tissues are said to be transferable directly from the wash water into pure dioxan, but plant cells are plasmolyzed by such treatment. Materials that were washed in water are transferred through the following three grades at 4- to 12-hr. inlerxals. Wide latitude in these intervals is permissible. (1) i/( dioxan % water (2) 2/^ dioxan 1/^ water (3) Anhydrous dioxan. Replace the cork with a jierfec tlv drv one. Make two more changes of anhydrous dioxan after intervals of 4 to 8 hr. Proceed with progressive infiltration in paraffin as described in the next chapter. Materials that were killed in FA A, or in anv fluitl tiiat is followed Dehydration for Embedding 29 by rinsing in 50% alcohol, are transferred through the following series at 4- to 12-hr. intervals. For small root tips the intervals need not be over one hour. (1) 1/2 dioxan i/, water (2) 2/^ dioxan 1/^ water (3) Two changes of anhydrous dioxan as in the previous schedule. Infiltrate in paraffin as descibed later. The following five-grade series is recommended for delicate or easily plasmolyzed material: 10% "commercial" dioxan in water, 25%, 50%, 75% dioxan at 1- to 4-hr. intervals. Change corks and make two or three changes of anhydrous dioxan at intervals of 1 to 12 hr., depending on the size of the pieces. The time intervals in this series are not critical. Anhydrous dioxan is a solvent of wax, but the rate of dissolving and infiltration can be increased by the addi- tion of 5 to 10% xylene or chloroform to the last change of dioxan. If anhydrous dioxan is difficult to obtain, a dioxan-normal butyl alcohol series may be used. The method has been tested extensively and is highly recommended. Dehydrate in the foregoing dioxan series to the commercial grade. Transfer to equal volumes of commercial dioxan and commercial butyl alcohol for 1 to 12 hr. Make two or three changes of anhydrous butyl alcohol and proceed with infiltra- tion. A similar dioxan-tertiary butyl alcohol series also is satisfactory. Regardless of the dehydrating agent and wax solvent that were used, it is desirable to evacuate any residual air that may remain in the tissues at this point. Place the uncorked specimen bottle into a dry jar, use a safety bottle between this jar and the aspirator, and evacuate until no more bubbles come out of the specimens (Fig. 3.1) . During the experimental period following the introduction of dioxan, unsatisfactory results were reported by many workers. Some lots of dioxan produced severe shrinkage; other purchases yielded acceptable, though variable, results. An inexpensive and satisfactory commercial grade dioxan can now be obtained. Dehydrating agents can be re-claimed after they have been used and have absorbed some water. Commercial grades that contain a low percentage of water can be made anhydrous. It is rarely profit- able to re-claim ethyl alcohol, or any other dehydrating agent that contains more than 5% water. If the water content is not over 5%, remove part of the water with anhydrous calcium chloride. Use a large jar with a layer of CaClo at least one-third of the volume of 30 Botanical Microtechnique the container. Pour the fluid, tor instance once-usecl anhydrous butyl alcohol, into the jar. As the alcohol accumulates, shake the jar occasionalh. Allow to stand for a day and decant the liquid onto anhydrous calcium suljihate, known commercially as "Drierite." After several hours, decant and filter the fluid and it is ready for use. If the fluid has become colored from materials extracted from the tis- sues, distill at the boiling point of the reagent being distilled. This information can be found in a chemical handbook. Wet calcium chloride can be regenerated by drying first in dry air, then in an oven at 110°C. Drierite can be regenerated by air drying and then heating in a furnace at 225 to 250°C. for two hours. 5. Infiltration and Embedding in Paraffin Wax The paraffin matrix in which tissues are embedded serves to support the tissues against the impact of the knife and to hold the parts in proper relation to each other after the sections have been cut. These functions are best performed if all cavities within the tissues are filled with the matrix and if the matrix adheres firmly to the external and internal surfaces of the material. Infiltration consists of dissolving the paraffin in the solvent containing the tissues, gradu- ally increasing the concentration of paraffin, and decreasing the con- centration of solvent. The solvent is eliminated by decantation or evaporation, or both, depending upon the character of the solvent and the process used. Properties and Preparation of Paraffin The properties of the embedding paraffin are important factors in the success or failure of sectioning. Desirable properties are as follows: 1. Constant and known melting point and appropriate hardness; the waxes used for most botanical work have melting points between 50 to 55°C., with a tolerance of 2° for a given grade. 2. Smooth, even texture, with a minimum of crystalline or grainy structvne. 3. Absence of particles of dirt, included water, and volatile or oily com- ponents. Commercial paraffins from different sources differ widely in properties and suitability for embedding. Purchases made from a given source may vary from time to time — some lots giving satis- factory results, whereas other lots, treated by indentical methods, are unsatisfactory. For these reasons paraffins from the available sources should be tested as to melting point, texture, behavior under the casting methods used, and cutting properties with familiar subjects. [31] 32 Botanical Microtechnique In most parts of the United States, the wax obtained from pe- troleum is known by the name paraffin, whereas in some areas tlie term wax is used. The tw^o terms are used indiscriminately in this manual. Most of the paraffins sold for domestic canning have excellent properties but are too soft for sectioning under ordinary room tem- peratures or for cutting very thin sections. 1 his inexpensive paraffin is satisfactory for sectioning soft materials such as fruits, if sections over 20 |.i in thickness are desired. Paraffin of excellent quality and stated melting point can be purchased from biological supply houses, but at rather high cost. Canning paraffin can be used for preliminary infiltration, and the more expensive hard paraffin used for the final embedding. Canning paraffin requires no preparation; the pieces may be put into the oven tank where melting takes place readily. Bulk paraffin can be purchased in 10-lb. slabs at low cost from petroleum refining companies. This bidk paraffin usually contains considerable dirt but it can be purified easily. Heat a quantity in a pan until it just begins to smoke, then keep over a small Hamc for at least i/^ hr. Avoid heating the paraffin to the ignition point. Pour the paraffin into a tall metal container, such as a tall cofl^ee can, and permit it to cool in a warm place. This permits particles of dirt to settle. Cool until the surface begins to s()lidif\, then decant into the oven tank. The smoking hot wax can be filtered rapidly through dry filter paper. Use a coarse filter paper and kecj) the sides of the metal funnel warm with a small bunsen flame. Each ptnchase of paraffin should be tested \)\ casting a test block in a paper boat. The paraffin test block should contain no l)ul)bles, opacjue spots, streaks or internal fractures. When the chilled block is broken, the fractine should sho^v a grainless or finely granular surface. The paraffin should slice into thin curled shavings, not into brittle granules. Keep a test block at a temjx'raturc of 30 to 35°C. for 24 hr.; bubbles and opaque crystalline spots should not a])pear. Cast blocks of good paraffin should remain free Iroiii iiittinal defects indefinitely, especially if stored at a constant, low temperature. Occasionally, one encounters old blocks thai are ahnost as clear as glass. Some such waxes have adequateh fine grain and ma\ cut \ery well. In other cases, the impact of the knife causes opaque fracturing of the wax, as when ice is struck ^vith an axe. The wax has ob\iously crystallized into a coarse texture during storage. WMien a block of such parallni is melted slowly in the o\en and recast, the new block Infiltration and Embedding in Paraffin Wax 33 is uniformly opatjuc, has a fine, even grain, and excellent cutting properties. Transparency again develops with age. It seems that desirable properties are inherent in good paraffin. The undesirable properties of some paraffins can only be minimized by the method of preparation, casting and storage. The tcxtvne and cutting properties of paraffin can be improved by the addition of rubber and beeswax, and in some formulas, a hard wax, such as ceresin wax. Hance's formula is recommended. Dissolve 20 g. of crude rubber in 100 g. of smoking hot paraffin. Cool and cast into slabs like canning wax. Make up the following mixture: Paraffin 100 g. Rubber-paraffin mixture 4—5 g. Beeswax 1 g- Heat the mixture smoking hot, filter through paper, and allow to cool until it begins to solidify before putting into the oven tank. Ceresin wax may be added to the above formula, 2 to 5% by weight. Other hard waxes need further study as hardening agents. Com- pounded rubber paraffins are available from biological supply firms. Tissuemat and Parlax are two products having excellent properties. Commercial rubber paraffin can be mixed with twice its weight of low-priced hard paraffin, greatly improving the properties of the latter. Consult the catalogues and compare descriptions and prices of trade-marked and other embedding paraffin waxes. Apparatus OVENS FOR INFILTRATION A water-jacketed copper oven with thermostat-controlled electrical heating is the most reliable type of oven. In an oven of this type, a built-in removable copper tank makes a suitable container for the supply of melted paraffin. The tank can be ecpiipped with a brass petcock, but petcocks develop leakage after little wear. A more satis- factory method is to dip out the paraffin with a spoon as needed. Debris settles to the bottom of the tank, and the clear paraffin is used from the top. An incubator oven with a reliable thermostat is satisfactory for paraffin work, but the temperature in diflierent parts of the oven is not the same and must be determined. In the latter type of oven the supply of melted paraffin may be kept in a container with removable cover and dipped out with a spoon kept hooked in the container. If it is possible to have two ovens for infiltration, use an inexpensive wooden incubator oven set for 35°C. for preliminary 34 Botanical Microtechnique infiltration and a water-jacketed oven set for 52 to 55°C. for tlie final operations. DEVICES FOR CASTING BLOCKS Several methods are in use for casting the embedded tissues into a mold. The most practical mold is a paper tray or boat made from strong glazed paper, such as herbarium mounting paper. The method of making boats is illustrated in Fig. 5.1. Fold and crease along the dotted lines; the narrower fold is slighth more than the desired depth of the boat, the wider fold is nearly twice as wide. Fold as shown in B, lapping the wide side to lock the narrow side. Small unattached objects like spores or minute seeds are cast in a pyramid mold (Fig. 5.1 C). Soak the boats in smoking hot canning wax until bubbles cease to come from the paper. Remove the boats from the wax, shake off surplus wax and cool the boats on a paper towel. A supply of wax- soaked boats of various sizes can be |)iepared in ad\ancc and used as r i 1 1 i i _ _ 1 Fig. 5.1-Mii1i(hI of laving oul (A) and folding (B) paper boats for casting paraffin blocks. C, pyramid mold. needed. It is probable that a boat formed from a llexible plastic will eventually be developed. Some form of hot plate is used to kcc'i) ilie parallin in tlu boat melted while the material is being arranged. Electric plates with thermostatic control are available. A sheet-copper table is used in many laboratories. An easily controlled heating table consists of a sheet of V2-in. boiler plate, (i to H in. wide and 18 to 24 in. long, mounted on legs or on a large ring stand (Fig. 5.2) . Fhe warm-pan method must be used foi materials that arc very small. l)uo\ani, or irans|)areiit. Vhv boat is sujjported on a wire Infiltration and Embedding in Paraffin Wax 35 triangle in a pan, which serves as an air bath, heated by a small Bunsen flame (Fig. 5.2) . Infiltration With Paraffin Wax The following infiltration procedure may be used with any of the common sohents, if the respective specific gravities of the solvent and the wax are taken into account. Paraffin wax, either in solid or melted form, floats on chloroform and also on dioxan. Therefore, either of these solvents, used alone, provides the progressive infiltra- tion that will be emphasized in this chapter. The addition of chloro- form or xylene to dioxan, as suggested previously, merely accelerates the dissolving of the wax. Paraffin sinks in xylene, but if the melted wax is poured into a bottle of cold xylene along the side of the bottle, a solidified layer of wax will remain on top of the solvent during preliminary infiltra- tion at 25 to 35°C. When the solvent is warmed above the melting temperature of the wax, the nearly pure wax will sink to the bottom and envelop the tissues. The abrupt concentration gradient between Fig. 5.2-Casting tables: A, boiler-plate table (the dotted line indicates the melting zone) ; B, warm-pan device. the wax and the tissues may be destructive to fine detail. The addition of lOVt chloroform by volume to the much cheaper xylene raises the specific gravity sufficiently to float wax chips or melted wax, and a gradual downward diffusion gradient is obtained. Normal and tertiary butyl alcohol have a lower specific gravity than paraffin wax, and the wax sinks upon the tissues. Flotation of wax can be obtained by the addition of chloroform, 15% by volume to normal butyl alcohol, and 25% to tertiary butyl alcohol, respec- tively. If chloroform is not added, the addition of wax must be made by small increments, in liquid form, and each increment must be homogenized to prevent the added wax from sinking and enveloping the tissues. 36 Botanical Microtechnique If a mixture ot two solvents is used, the bottle should be kept corked until the infiltration is well advanced, to prevent the differen- tial c\'aporation of the two solvents in the oven. Pour a teaspoonful of melted paraffin over the cold solvent, where the paraffin solidifies as a layer on toji of the solvent. Remove the stopper and place the bottle into the 35°C. oven. The layer of }Kiraf- fin does not melt, but ii gradually dissolves and diffuses downward into the tissues. If all of the paraffin dissolves at this temperature, add more melted wax. If the bottle becomes filled with solution, pour some of the solution into the waste can (not into the siuk!) . Con- tinue the addition of melted wax until a thin layer of undissolved wax remains on top of the solution. The undersurface of the layer of paraffin eventually develops a translucent, crystalline appearance. When this stage is reached, the solvent is obviously saturated with paraffin at this temperature. Tissues are not damaged by prolonging this infiltration at 35°C., therefore, this part of the process may be extended over 2 or 3 days. Transfer the specimen bottle to the 53 °C. oven, where the layer of solidified paraffin soon melts and continues to dissolve, and the infiltration initiated at the lower temperature is contintied. If the specific gravity of the solvent has not been adjusted as described above, it is best to remove the solid supernatant wax at this point and add wax by small increments at intervals as described later. If the tissues are not extremely delicate or fragile, whirl the bottle genlly until the liquid is homogeneous, as shown by the absence of refraction waves within the liquid. At intervals of 1 to 4 hr., pour off one-half of the homogenized solution into the waste can. Replace the decanted licjuid with an ecjual volume of melted soft paraffin, and replace the bottle into the oven quickly. After four or more such partial rej^lacements, poiu' off all the paraffin-xylene solution, ^\•hich now consists mostly of j)araffin. and rej^lace with pure paraffin. After 1 to 4 hr. make another complete replacement and make a hultoti test. Cast a button of paraffin about the size of a siher dollar b\ jxjur- ing some of the paraMm irom the tissues into a pan of cold water. Promptly replace the sjjecimen bottle into ilie oven. Allow the test disk to cool thorouglih. Tlie cooled test button should not be greasy. Chew a piece of this paraffin. The jjresence of e\en a slight trace of xylene or other solvent is easily detected by taste. Examine for the paraHin or connnercial casting comj)oinid at 1- to 4-hr. intervals. The defects and cjualities described on page 31. If the test j)iece indicates that all the solvent has Ijeen remoNcd, make two changes of hard material is then ready to be cast into a mold. Infiltration and Embedding in Paraffin Wax 37 The use of two, or even three grades of paraffin with different mehing points, used successively during infiltration, has been sug- gested in the literature. If a laboratory is ecjuipped with three ovens, maintained at 40, 50 and 54°C., there may be some point to the successive use of waxes having those melting points, but if the three waxes are used at the same temperature, there seems to be little basis for the procedure. It can be demonstrated easily that high- melting-point wax that contains a trace of solvent becomes low- melting-point wax. The progressive method outlined in this chapter may be used with only one grade of wax; however, the use of inex- pensive canning wax for preliminary infiltration, followed by casting in a liigh grade filtered wax or compounded formula, is economical and entirely satisfactory for most tasks. A vacuum oven is an aid in the infiltration of difficult material. Cavities that resist evacuation at low temperature can be exhausted at 50°C., when the material has progressed to approximately equal volumes of wax and solvent. The low boiling point of tertiary butyl alcohol makes the use of a vacuum oven impossible with this solvent until the solvent has been almost completely replaced by wax. Most material can be adequately evacuated by the time it is in the final solvent, and the dissolving wax will then diffuse into all spaces occupied b) the solvent. CASTING INTO A MOLD Assume that the infiltrated material is in the final change of pure paraffin. If the oven has cooled because of frequent opening, the paraffin in the specimen bottle may have congealed. Heat the neck and upper portions of the bottle in a Bunsen flame. Never heat the bottom of the bottle because the tissues resting on the bottom will be overheated and ruined. Apply only enough heat to liquefy the paraffin. Slight heating repeated at 10-min. intervals is safer than melting at one heating. If you have not yet provided means of identification, write the designation of the given lot of material on a y2-cm. square of paper and put into the bottle. Select a wax-soaked paper boat that will accommodate the pieces in the specimen bottle, without wasting space or wax. Heat one end of the casting plate with a small flame and place the empty boat at the melting zone. Pour the paraffin containing the tissues into the boat. Arrange the pieces with a bristle. A warmed needle may be used, especially to move pieces that have become frozen into unsatisfactory positions. Slide the boat over the edge of the melting zone toward the cold end of the plate as fast as a row of pieces is arranged. When the pieces are suitably 38 Botanical Microtechnique oriented, move the boat lo the cold end of the plate. Sweep the Biinsen flame rapidly over the surface of the paraffin; this permits contraction on the upper surface while the bottom of the paraf- fin is cooling, thereby preventing the formation of cavities. As soon as the paraffin is hardened enough to keep the pieces of tissue from moving, float the boat in a pan of cold water and brush with the Bunsen flame again. Allow the surface to solidify, and submerge the boat in the water, holding it under with a staining jar cover or other weight. When the paraffin is thoroughly cooled, peel off and discard the paper boat. Some purchases of paraffin wax, and occasional lots of proprietary compounded waxes, develop white areas or bubbles if kept at room temperature after casting. This can be minimized or prevented by storing the cast blocks in a refrigerator for several days. If the sjx)ts appear upon subsequent exposure to room temperature, permanent low temperature storage should be provided. When using the warm-pan method (Fig. 5.2 B) . place a paraffin- soaked boat on the triangle, and warm the bottf)m of the pan Avith a Bunsen flame of such size that the paraffin on the boat is kept just melted. Pour the material into the boat, and arrange the pieces, oc- casionally flaming the top surface of the paraffin. There is little danger of overheating the material; hence the operator need not hurry in arranging the pieces. When the pieces are arranged satis- factorily, remove the burner, and jjoiu" cold water into the ])an tmtil the water level is slightly above the bottom of the boat. The pieces become liardened into place quickly. Sweep the flame o\er the stnface of the paraffin to permit internal contiaction. (louiplete (lie hardening as in the former method. The sj:)acing of pieces in the block dej)cncls on the si/e of the pieces. Root tips and small pieces of leaf can be sj)aced 5 iinii. apart; large pieces of stem require more supporting ])arallni dining section- ing and should therefore be spaced at least 1 (in. aj)ait. Very slender loot tips, coniferous needles, and similai oljjc'cts may be blocked in groups of three or more j)ie(es laid parallel so thai they can be microtomed simultaneously. RECASTING Poor paraffin of a cast block can be replaced with good paraffin, or j)oorly arranged material can be rearranged and recast, and exces- sively large pieces can be trinnned and recast. Cut the jiieces out of the block, trim the pieces if desired, cut away excess paraffin if it is Infiltration and Embedding in Paraffin Wax 39 of bad quality, and drop the pieces into a bottle of melted [)araffin in the oven. Do not apply extra heat; the temperature should not exceed 53 to 54°C. When the old paraffin has amalgamated with the new wax, make at least one change into new casting wax and cast into blocks. REINFILTRATION Poorly infiltrated tissues can sometimes be salvaged by reinfiltra- tion. This should not be attempted if there has been excessive collapse of cells, a frequent result of poor infiltration. Cut the pieces out of the paraffin block, trim away excess paraffin, and drop the pieces into anhydrous dioxan, normal butyl or tertiary butyl alcohol, xylene or chloroform. After 24 hr. at 35 °C. transfer to the 53 to 54° oven and continue progressive infiltration. A vacuum oven may be used for such salvage operations. Cast blocks should be stored under conditions that minimize damage to the tissues and to the texture of the paraffin. I rim the edges of the cast block so that both surfaces are flat. Store in a stout manila en\elope or small cardboard box bearing adequate identifica- tion data. If several blocks are stored in one container, use thin card- board separators. Box containers should be stacked so that the blocks lie flat. Stout enxelopes support the blocks well enough to permit filing the envelopes in the vertical position in a filing cabinet. Storage temperatures should be low enough to prevent bending of paraffin blocks. 6. ^Aicrotome Sectioning of Material in Paraffin Material embedded in paraffin is almost invariably cut with a rotary microtome, in which the knife is stationary and the piece of tissue is moved up and down past the cutting edge. Cutting is ac- complished by wedge action, like the action of a chisel or a plane. After a section has been cut, and the tissue carrier has passed the knife on the upstroke, an automatic feed mechanism advances the tissue carrier forward, and another section is cut. Successi\c slices remain attached to each other, forming a ribbon of paraffin. Ihe successive sections cut from a piece of tissue are thus kept in serial order, and this older can be j^reserved throughout the processing of the slides. From serial slices of an organ of a plant it is possible to reconstruct the external or internal structure of the organ, of a tissue system, or even of a single cell. As an example of serial section- ing we may use the homely illustration of a loaf of bread, cut into slices and the slices hiid out in order. The piece of material to be sectioned is fastened to a mounting l)lock, which is clamped into the microtome. Inexpensi\e mounting blocks can be made of hard, porous wood, such as oak or ash. 1 he most useful sizes range from I by 1 by 2 cm. to 2 b) 2 by 3 cm. Soak the blocks in hot canning wax. Bakelite and other |ilastics make excellent l:)locks, preferred to wood l)(.cause j)lastic blocks do not comjjress when clamped into the miciotonu" (Fig. ().l A). Wood or plastic blocks arc satisfactory for most woik. being inexpensive and sufficiently rigid for sections over (I \y in thickness. For loutine sam- pling of material, many pieces of tissue can be mounted on separate blocks, the mounting l)Iocks miml)ered by means of string tags, and test sections made from each piece. Hie mounied blocks can be kept with the j)roj)er batches of embedded material until staining trials establish which block has the desired siaye. [40] Alfcrofome Sectioning of Material in Paraffin 41 Metal mounting disks (Fig. 6.1 B) afford greater rigidity than plastic blocks and are indispensable for cutting very thin sections, or for sectioning large pieces of firm material. Disks remain cold longer after chilling, thereby keeping the paraffin block cold for sectioning. Disks are much more expensive than homemade blocks, and most laboratories have a limited supply, making them unsuitable for the routine sampling method described above. Some microtomes have a built-in tissue-mounting disk on a ball-and-socket joint. This device is satisfactory for work in which each piece of material is used up at one cutting, thus emptying the carrier for the next piece or for the use of other workers. For class use or for work requiring much sampling of diverse materials by several workers, removable disks or blocks are much more desirable. To fasten a piece of material on a mounting block, trim the paraffin around the material so that the cutting plane is established. Fig. 6.1 — Mounting of tissues on object blocks: A. wood or plastic block with scored surface; B, metal object disk; C. method of orienting paraffin block and fastening to object block. Lay the cutting face on a clean surface. Heat the mounting block, press it firmly on the back of the specimen, and hold in contact until the wax cools (Fig. 6.1 C) . Build up a fillet of paraffin around the specimen to afford firm l^racing for the tissues (Fig. 6.2) . Cool thoroughly before sectioning. Some workers of acknowledged skill use a heavy knife for class- work, for routine preparation of teaching material, and for research. Other workers of equal ability use either a knife or a razor blade in accordance with requirements of the work in hand. For sections ranging from 8 to 15 [.i in thickness, a sharp razor blade in a suitable, rigid holder will match the work of the heavy knife. The thick type of razor blade (Enders or Christy) can be stropped, used repeatedly, and discarded when stropping no longer restores the edge. In a course in microtechnique, for the routine preparation 42 Botanical Microtechnique of teaching material, and loi many research tasks, razor blades are economical and entirely satisfactory. For cutting very thin sections, uncommonly thick or large sections, or for totigh materials, the heavy microtome knife is indispensable. The greater rigidity of a knife ]:)ermits sectioning of material Avith which a flexible razor would chatter, cutting sections of uneven thickness. Ihe sharpening of a microtome knife is a laboriotis process, best learned by obser\ing a demonstration. If a knife is badly nicked or bowed in the center it is best to send it lo the mantifacturer for grinding. A straight edge and a ne^v correct bevel of the cutting edge are thus established. W'ith a properly grotuid knife, occasional strop- ping restores the edge for a considerable time, depending on the hardness of the material being cut. A honing back is a longitudin- ally split metal cylinder that is slipjied over the thick back of the knife for honing or stropping. The diameter of the cylinder deter- mines the angle of the honed wedge. I'he metal of the cylinder is usually softer than the knife and wears away faster by honing. "When the cylinder is appreciably worn, or unevenly worn, a new one should be fitted and a new wedge angle honed on the knife. It may be necessary to hone the knife on a fine gray hone tising soap stids as a lubricant. W'hen the fine hone and strop fail to restore the edge, and it is not possible to have the knife machine-ground, a uvw -wedge and cutting edge can be established as follows. Place the cutting edge on the coarser yellow hone with the knife vertical, and make one light stroke, remoA'ing the cutting edge compkteK. I hen la\ the knife flat on the hone and stroke with long oblicpie stiokes. alternating the two sides, imtil the two sides of the new wedge meet. Examine periodically with a microscope. Hone on the fine gray stone initil the edge consists ol uniform, minute serrations. Stro]) as usual before using. Factors Influencing Sectioning Successful sectioning in paraffin depends ujkmi a nmnber of inter- acting factors, the most important of Avhidi will i)e discussed briefly. QUALITY OF THE PARAFFIN The hardness ol I lie parallm should be appiopriate for the char- acter of the tissues, the desired thickness of the sections, and for the room temperature at which the cutting is done. 1 he paraflin shoidd Microtome Sectioning of Material in Paraffin 43 have a grainless or very fine-grained texture, should be free from bubbles and opaque spots, and should contain no grit or other debris. PROPER INFILTRATION Improperly infiltrated material breaks out of the paraffin block. Examine the cut face of the material with a hand lens or a binocular dissecting microscope. Crumbling within the tissues may indicate inadequate penetration by paraffin during infiltration or may be the result of excessive hardness or brittleness of the tissues. Breaking out of entire sections from the paraffin ribbon indicates poor ad- hesion between external surfaces of the piece of tissue and the paraffin. Inadequate infiltration may be due to incomplete dehydra- tion or excessively rapid infiltration. The remedy lies in rcinfiltration. ORIENTATION OF THE MOUNTED MATERIAL The paraffin around the piece of material should be trimmed rectangular, with the material approximately centered laterally in the paraffin. If the tissue is not vertically centered in the paraffin, the thicker layer of paraffin should be at the top, affording support against the pressure of the cutting action (Fig. 6.2 F) . Trim the upper and lower edges of the wax so that the sections are close enough to each other on the slide for efficient use of the slide and cover glass. See Fig. 6.6 for efficient placement of sections. The edge which ap- proaches the knife should be parallel to the knife (Fig. 6.3 C) . For most paraffin sectioning the knife is placed at right angles to the vertical motion of the paraffin block. The other angle to be con- sidered is the declination, or the tilt of the flat face of the knife toward the tissue (Fig. 6.3 A, B) . This angle must be determined by trial. RIGIDITY OF MOUNTING The piece of tissue should be firmly attached to a mounting block or disk and supported, especially on the edge away from the knife, by a generous layer of paraihn (Fig. 6.2 E) . The mounting block, the knife, and the knife carrier must be firmly clamped into place. Inade- quate rigidity of the tissue mounting or of the knife results in alter- nate sections of unequal thickness. This can often be recognized in the ribbon but usually becomes evident during staining. The thicker sections will be more deeply stained than the alternating thin ones. In sections of a large stem there may be alternate deeply stained and lightly stained bands in each section. 44 Botanical Microtechnique TEMPERATURE FACTORS Cutting is influenced 1)\ the temperature of the paraffin block, of the knife, and of the room. If the temperature of one or more of these factors is too high, compression of the sections occurs on impact with the knife. If the temperature is too low, the sections may curl, or successive sections may not adhere and thus fail to form a ribbon. Thick sections are relatively more tolerant to higher working tem- peratures than are very thin sections. A heavy microtome knife per- mits a higher temperature than does a razor blade. If the temperature is too high for the grade of paraffin being cut, cool the mounted Fig. 6.2— Methods of oricntiiio ol)jc(ts of various shapes: A, a leaf iiioimtcd for cross sections; B, a small stem or other cylindrical organ mounted for cross sections; C, for longitudinal sections; D and E, sectors mounted for cross sections; F, sector of large herbaceous stem mounted for radial sections. paraffin block and the knife or ra/or-blade holder in a jiaii ol ice water. .Align the tissues and knife in the niicioiome cjuickh, and ciU sections until the parallm becomes too soft, when the cooling should be repeated. Knife cooling devices are described by johansen (I*) 10). If a refrigeration loom is a\ailable. j)erfe(t control is possible by setting lip the microtome in the (old looiii and warming a /one around the knife with a desk laiii|). The author's department has a cooled mic lotome loom with a floor space of 5 b\ 7 ieet antl a height of l iron alum (ferric ammonium sulphate) . Select clean, transparent, violet-colored crystals, especially avoiding crystals with a rusty coating. Discard die solution when a yellow precipitate develops in the bottle. Ihc following mordant will keep lor months (Lang) : 4% iron alum 500 cc. Acetic acid (glacial) 5 cc. HaSOi (concentrated) 0.0 cc. Some satisfactory destaining agents are: 1. Mordant dihited ^vilh an ecpial xoluiiie ol water. 2. Saturated aqueous sohition of picric acid. 3. Equal volumes of mordant :ind liic above picric acid. The stock solution of stain is a 0.5% acjtieous solution of hema- toxylin. iVIeasurc the required volinne of distilled water, add a pinch of sodium bicarbonate, about as large as a match head, to a liter of water. Bring the water to the boiling poim. remove from the heater and laid the dye. Do not boil the solution! Cool promptly and store Staining Paraffin Sections 59 in a refrigerator. Dilute the stock solution with twice its volume of water for the 4-hr. schedule and with 4 parts of water for a 12-hr. stain. Although the new stain will give satisfactory results, it improves after 2 or 3 days. 1 he stain begins to deteriorate in a few months. Another type of stock solution consists of a 5 or 10% solution of hematoxylin in absolute ethyl alcohol or 95% alcohol. Dilute to 0.5% in water as needed. Mordanting is sometimes necessary with the synthetic dyes des- cribed in the following section. The complex problem of mordanting is well summarized by Popham (1949) , and specific suggestions are made throughout the present chapter. THE COAL-TAR DYES 14ie coal-tar dyes comprise a large and highly diverse class of synthetic dyes. Their derivation, chemical composition, and properties are discussed in great detail by Conn (1936). Specify dyes that are certified by the Commission on Biological Stains (Conn, 1936) . Only the members of this group of stains that are in common use for botanical work will be presented here. Coal-tar dyes are used in a variety of solvents, and the general formulas for the most common stock solutions are as follows: (1) 0.5 to 1% solution in water, with 5% methyl alcohol optional, as a preservative. (2) 0.5 to 1% solution in ethyl alcohol, with alcohol concentrations of 50, 70, and 95% and absolute alcohol preferred by various workers. (3) Saturated solution in clove oil, or in equal volumes of clove oil and anhydrous ethyl alcohol, or in methyl Cellosolve, or equal volumes of clo^e oil, anhydrous alcohol and methyl Cello- solve. The following table shows the usual solvents (x) in which the best known coal-tar dyes are used. Dye Acid fuchsin (acid) Aniline blue (acid) ( = cotton blue) Bismarck brown Y (basic) Crystal violet (basic) Eosin Y (acid) Erythrosin (acid) Fast green FCF (acid) Orange G or gold orange (acid) . . . Safranin O (basic) Water X X Alcohol, % 70 50 70 95 95 95 100 50 to 95 Clove oil or Cellosolve X X X X 60 Botanical Microtechnique The principal botanical uses for the common stains are indicated in the following tabulation: Cellulose cell walls. Hematoxylin (self-mordanting type) . Fast green FCF. Aniline blue. Bismarck broAvn Y. Acid fuchsin. Lignified cell walls. Safranin. Crystal violet. Cutinized cell walls. Safranin. Crystal violet. Erytbrosin. Middle lamella. Iron hematoxylin. Ruthenium red (material cut fresh) . Chromosomes. Iron hematoxylin. Safranin. Crystal violet. Carmine (for acetocarmine smears) . Mitochondria. Iron hematoxylin. Achromatic figure. Crystal violet. Fast green FCF. Filamentous fungi in liost tissues. Iron hematoxylin. Safranin (). Fast green FCF. Cytoplasm. Eosin Y. Erytbrosin B. Fast green FCF. Orange G or gold orange. The above tabulations indicate some relationshii) between the acid or l)asi( character of a stain and its specificity. A basic stain is one in wbicb the (olor bancr is a basic radical; in an acid stain the color bearer is an acid radical. As a rule basic stains are selective for nuclear structtnes and. in sonic processes, lor lignified cell wall. Acid stains usually are selective h)r components ol the c\toplasni and for iMilignified cell wall. The common (baring oils (doxe oil. cedar oil, bergamot oil, and wintergreen oil) usualh aie used in (oncentraled h)ini as j)urchased Staining Paraffin Sections 61 or thinned slightly with xylene. An inexpensive and highly satis- factory clearing agent, known as cnrbol-xylene, consists ot 1 volume of melted c.p. phenol (carbolic acid) and 3 to 4 volumes of xylene. Staining Processes To meet the needs of teachers and beginners, staining processes are arranged in a graded sequence, beginning with the simplest processes, in which the variables and possibilities for errors are reduced to a minimum. The simplest type of stain is a progressive stain, in which the intensity of the color imparted to the tissues is proportional to the length of immersion in the stain. Some of the most useful stains of this type have hematoxylin as the active ingredient. In this cate- gory of self-mordanting stains, the most important are Delafield's hematoxylin, Harris' hematoxylin, and Mayer's hemalum. Many modi- fications may be found in the literature. The term "hemalum" is used in this manual to refer to any of the self-mordanting alum hema- toxylins. The choice among these stains is a matter of personal preference. HEMALUM (PROGRESSIVE) The modification of Mayer's hemalum, on which staining Chart I is based, is selective for cellulose, pectin, fungus mycelium in many cases, weakly selective for chloroplasts, strongly selective for metabolic (resting) nuclei, and moderately selective for chromosomes in some subjects. Hemalum may be used without any other stain for meriste- matic organs, for anther and ovary slides in which a critical chromo- some stain is not necessary, and for subjects having but little strongly lignified or differentiated tissues. This stain develops a metallic scum on standing. The particles of this scum adhere to the adhesive and to the sections on the slide, therefore the stain should be filtered before using. The preliminary processing of slides, prior to immersion in stain, is essentially the same regardless of the stain used. This prestaining process will now be outlined and the procedure is understood to apply when an aqueous stain is used. After the affixed sections have been dried in the 53°C. oven, the sections and adjacent parts of the slide are found to be coated with melted paraffin from the ribbon. Obviously, the first operation is to dissolve this paraffin by immersing the slide in a jar of xylene. If slides are taken directly from the oven, 62 Botanical Microtechnique the parafFin dissolves in 1 or 2 min. With cold slides it is better to allow 5 min. The slide is now in a very dilute solution of paraffin in xylene, which is removed by immersing the slide in anlnclrous alcohol. As outlined in Staining Chart I, progressive transfer to Avater is then made through the indicated grades of ethyl alcohol. Isopropyl alcohol or acetone also may be used in most of the staining charts in this chapter. The slide has been run doiaji to water, and is now ready to be stained in an aqueous dye. Transfers should be made cpiicklv so that the slides do not become dry. The inter\als can be shortened to 30 sec. by moving the slides up and down in the solution with forceps. The series of reagents in which slides are deparaffined should be replaced when the 30% alcohol becomes cloudy or when the fluid drains from the slides as if the glass were oily, indicating that paraffin and xylene have been carried down the series until the 70% and 30% cannot hold the xylene-paraffin contaminant in solution. The ad- dition of 10% rj-butyl alcohol to the anh\clrous and 95% grades pro- longs the useful life of the series. riie correct staining interval for a given subject must be deter- mined by trial. An experienced worker can make a good guess for a trial slide and make corrections for subsequent slides. One collection of lily ovary killed in Bouin's solution required only 10 min. for a brilliant stain, whereas another collection, fixed in Craf rccpiired 1 hr. A collection of lily anther in the microspore stage yielded excel- lent slides with a 30-min. stain. To determine the correct interval, stain three slides of a subject tor three intervals, i.e.. 10. 20, and 40 min., respectively. Mark the slides before staining. The sample slides may be held in distilled water and ])ut into the stain at intervals, or they may be put into the stain simultaneously and removed after the desired intervals. After staining, rinse the slides in two or more changes of distilled water, then rinse in three changes of tap water, or in running tap water for 2-5 min. Note that the color in the tissues changes from purj^le to blue after the trans- fer into tap water. Hematoxylin gives a reddish-purple color Avhen acid and a blue color when alkaline. The latter color is pre- ferred for tlif subjects recommended for this first exercise. If the city water in your ccjnnnunity does not produce the bluish tinge in tissues that have been stained in hemalum, use 0.1% sodium carbonate for the last rinse. This process may be called nlknlizing. At this stage, examine the three test slides that were stained as Staining Paraffin Sections 63 suggested above. Use a smear microscope, preferably one that has only lOx (16 mm.) and 20x (8mm.) objectives and no condenser. The magnification is adequate and the objectives have such long working distance that they are not likely to be dipped into reagents on the slide. The tissues must not become dry during this quick examination. Nuclei should be blue-black. Cellulose cell walls should be black, whereas lignified cell walls should be nearly colorless. Plastids may be pale blue to blue-black, and cytoplasm blue-gray. If the foregoing structures do not have a deep enough color, transfer the slide from the water to hemalum and give it another interval in the stain, ustially as long as the first immersion. Rinse and wash in tap water, examine again and if satisfactory, proceed with de- hydration as in Chart I. If a slide is left in hemalum longer than the optimiun period, the contents of the cell may become black, and the details of the wall and protoplast may be obscured. The slide can be destained by brief immersion in dilute acid. The preferred destaining agents are 1 to 5% acetic acid, 0.5% hydrochloric acid, or a saturated aqueous solution of picric acid. Try one minute in acid, wash, alka- lize and re-examine with a microscope. When the stain is satisfactory, proceed with dehydration according to Chart I. Staining Chart I now calls for progressive dehydration of the tissues and the surface of the slide, followed by "clearing." Consult the reference manuals for the various clearing agents in common use. An inexpensive agent is carbol-xylene, the formula of which is given on page 61. Both ingredients must be of high purity. Phenol has a great affinity for water and removes the last traces of water from the preparation. Xylene has nearly the same index of refraction as glass, thus rendering the tissues transparent. High-grade phenol and xylene should not affect the stain even after several days of im- mersion. Equal volumes of xylene and cedar oil may replace the carbol-xylene. The final operation consists of cementing a cover glass on the preparation. Have ready a supply of newly cleaned and dried cover glasses. Use a cover glass of generous, but not wasteful, size, with shape and dimensions in keeping with the material to be covered (Fig. 6.6) . Discoloration of resin and fading of stain with age proceed from the edges of the cover inward. Have a margin of at least 5 mm. between the sections and the edge of the cover glass. For mounting one section on a slide, or a few sections in a single row, use a 1/2-. %-, 64 Botanical Microtechnique STAINING CHART I Progressive Hemalum Xylene 2-5 mill, (de-waxing) i absolute (anhydrous) alcohol 2-5 min. i 95% alcohol 2-5 min. i 70% alcohol 2-5 min. I 50% alcohol 2-5 min. i 30% alcohol 2-5 min. i distilled water 1-2 min. i Hemalum 5-'^0 min. i distilled Wilier 1 min. Tap water (see page 62) resin and cover glass t xylene III 5 min. t xylene II 5 min. t xylene I 5 min. T carbol- xylene 5-10 min. t absolute alcohol II 5-10 min. absolute alcohol I 5-10 min. t 95% alcohol 5-10 min. t 70% alcohol 5-10 min. t 50% alcohol 5-10 min. t 30%) alcohol 2-5 min. * 'I he iHKiiiiicr is advised to topv each staiiiiiis chart on a hiiRC card. Bv means of colored arrows, indicate the sequence of operations used to correct overstaining or undcrstainmg. or %-in. cover glas.s. For large longitudinal sections of rectangular outline, or for covering several rows of sections on a slide, use a square or long cover glass of such size that there is a margin ol at least 5 mm. Calijicr all cover glasses, using onlv those that lull within 0.15 to 0.20 mm. in thickness. Canada balsam has been the most widely used mounting medium Staining Paraffin Sections 65 for many years. Stained sections mounted in balsam may remain in perfect condition for 25 years. However, it is nuich more likely that the stain will fade, the balsam will become dark yellow, and may even become cracked and opacjue like dried varnish. In recent years, numerous synthetic resins have been tried as mounting media. (Lillie, AVinkle and Zirkle. 1950). Further experimentation can lie expected in the future and the many possible polymers will be tested. The reader is advised to consult the catalogues of biological supply dealers for the currently recommended resins. The affixing of cover glasses should be accomplished quickly and neatly. Remove a slide from the last xylene, and place with tissue upward on a sheet of dry blotting paper. Working rapidly to avoid drying of the tissues, wipe excess xylene from around the sections, put a drop of resin on the tissues and lower a cover glass obliquely onto the resin. A black background aids in seeing and expelling bubbles. If the size of the drop of resin is correctly gauged, there should be no excess resin squeezed out around the edges or over the cover glass. Newly covered preparations must be used with care because the cover glass is easy to dislodge and the tissues may be dam- aged. Drying new slides in the 53°C. oven for one or more days hardens the resin somewhat and permits safer handling of the slides. This is a convenient point at which to discuss the repair of damaged slides. It is possible to salvage a slide that has some sound sections as well as some sections that have been damaged by misuse. Place the slide upside down under a low-power objective and locate the damaged sections. Place a mark over each broken section with India ink. Allow the ink to dry thoroughly, and drop the slide into a jar of xylene. After the cover glass has slid off, rub off the damaged section with a matchstick, rinse in xylene, and mount a new cover glass. Destaining and Restaining Slides may be examined for color at several stages in the staining process, in fact from any reagent that is not so highly volatile that the preparation becomes dry during a brief examination. See page 63 for the procedures used to increase or decrease the intensity of the color imparted by hemalum. If the slide is examined out of xylene or carbol-xylene and the stain intensity needs to be increased or decreased, transfer the slide backwards through the dehydrating series to water, and proceed with corrective measures. It may be necessary to modify the stain intensity of a finished slide 66 Botanical Microtechnique iliat has had a co\er glass affixed with balsam or synthetic resin. 1 he cover can be loosened and allowed to slide off by immersing in a jar of xylene as long as necessary. After the cover has slid off, transfer the slide to the absolute alcohol after the de-waxing xylene, run down to water and proceed Avitli the restaining or destaining process. HEMALUM (REGRESSIVE) The destaining action of acids on hematoxylin is selective- cytoplasm is destained more rapidly than cell walls, plastids, and nuclear structures. This fact makes it possible to use a self-mordanting hematoxylin as a stain that is adequately differential for many subjects. The slides are purposely overstained in Delafield's, Harris', or Mayer's hemalum, then destained in acid until the proper contrast is obtained. The foregoing single stain, using a self-mordanting hematoxylin formula, either as a progressive or regressive stain, deserves more extensive use for routine diagnostic examination of research material. An enormous amount of time and energy can be spent in ajjplying elaborate multiple stains to large numbers of slides, many of which are discarded after a moment's examination. In such a series of slides, stained with a single stain, the few slides having the desired stage can be easily restained if a more diagnostic differential stain is needed. HEMALUM WITH A "GENERAL" COUNTERSTAIN The foregoing hemalum stain can be supplemented by a counter- stain, a stain that has little specific selectivity, but furnishes optical contrast for the principal stain. A counterstain is introduced into the staining series at a place having approximately the same water concentration as the solvent of the counterstain. One of the most useful counterstains is erythrosin. llie stock solution contains 1/2% stain dissolved in 95'/( alcohol. Referring to Staining CJhart II, note that the slide, previously stained to the correct intensity in hemalum, rinsed and alkali/.ed, is put into erythrosin alter 95*^^ alcoliol. Ihe interval in erythrosin nuisi be determined hv trial and may range liom a few seconds to I In. This counterstain is remo\'ed from different types ol material in variable degree by tlie siihsc (luent dehydration. The final intensity ol ilu- pink counterstain depends on ilic tcnacilN witli Avliidi ilic tissues retain tlic stain. 11 the ])ink color is too dark, it will obscure some ol ilic details stained blue by the hematoxylin. Excess counterstain can be removed by running the slide back to 50',' alcohol. More pink can be added as sliowii on Staining Paraffin Sections 67 Staining Chart II. The same slide can be repeatedly destained or restained in the counterstain until exactly the desired effect is obtained. The hematoxylin is not affected during this manijjulation. STAINING CHART II Hemalum With "General" Counterstain Pre-Staining Operations and Intervals as in Chart I Hemalum to correct intensity i 30% alcohol i 50% alcohol i 70% alcohol i 95% alcohol erythrosin (see text) > Other common counterstains used with the above hematoxylins, are orange G, gold orange, eosin, fast green, and light green. The underlying principle for applying other counterstains is the same as for erythrosin. Counterstains may also be dissolved in clove oil and applied after the last dehydrating step, omitting carbol-xylene because clove oil is an excellent clearing agent. Counterstains may also be dissolved in water, 50% to absolute alcohol, or Cellosolve and introduced into the series at the corresponding point of dehydration. HEMALUM AND SAFRANIN After acceptable results have been obtained with the foregoing single stain and the double stain, undertake the mastery of a double stain having two selective components. One component of the next double stain to be discussed is a self-mordanting hematoxylin; the second component is safranin, which is highly selective for chromo- somes, lignin, cutin, and in some cases for hemicellulose. An important feature of this combination is that the hemalum is applied to the desired intensity and remains fixed throughout subsequent processing, whereas the safranin is applied until the material is strongly overstained and then differentialh destained. 68 Botanical Microtechnique Staining Chart III begins with a slide that has been stained in hemalum as shown in Chart I; the slide is then immersed in satranin. The interval in safranin ranges from a few minutes to 12 hr. Some collections of young corn stem require at least 1 hr. in safranin. Wood sections cut in celloidin may take up enough safranin in 5 min. to make destaining difficult. Untested material should be tried at intervals of 10, 30, and 60 min. and 8 to 12 hr. After removal from safranin and rinsing in water, all cells of the section are found to be stained deep red, the blue color of the hemalum being masked. Dehydration and differential destaining are accomplished simultane- ously by passage through the alcohol series. Safranin is removed from STAINING CHART III Hemalum and "Specific" Counterstain Pre-Staining Operations as in Chart I Hemalum to resin and correct intensity cover glass i t safranin ^ 1^^^^ III 1-12 hr. ^ i ■wash t xylene II 30% xylene I alcohol ^ i carbol- 50 7c xylene alcohol i 7iV/c alcohol t aljsolute alcohol 95% al)s(>hite auono hoi ^ alcxjliol cytoplasm aud uulignihed tissues by 50 and 70% alcohol and at a slower rate by similar grades ol acetone. Higher coudutrations of alcohol and anhydrous alcohol also dissohe the safranin. hut 90% acetone and aidiydrous adtone have slight destaining action. Acetone, therefore, ])erniits easier control ol desiaining than does alcohol. Lignified tissues, (utin. and |)laslids retain safranin tlnoughout suitably rapid dehydration. Ihe correct stain has been attained when lignihed (cll walls are a dear, transparent red and uidignified Avails are blue, with little or no reddish tinge. C^hloroplasts may be blue, violet, or red. In order to make chloi()i)lasts red enough to show up Staining Paraffin Sections 69 clearly, it may be necessary to compromise by leaving too much red in the cellulose walls. It a finished preparation is found to be un- satisfactory, the cover glass can be removed, and the material destained or restained. However, alterations in the intensity of the safranin can be made best after the slide has been examined from carbol-xylene. Carbol-xylene has a very slow destaining action on safranin. Preparations left in carbol-xylene for 4 to 12 hr. show highly critical differentiation of structures having varying degrees of lignification, such as the stratifications in the walls of xylem cells and sclerenchyma. SAFRANIN-FAST GREEN The next type of stain combination to be considered has two components, both of which are subject to differential destaining and which react upon each other during dehydration. This staining process is obviously more difficult to control than the preceding processes. As shown in Staining Chart IV, the first stain to be applied is aqueous safranin, in which the preparation is strongly overstained. One hour in safranin is occasionally enough; some woody materials stain well in 5 min. Your previous experience with the hemalum- safranin combination will indicate the safranin-holding capacity of tested materials. The safranin begins to dissolve out during passage through 30, 50, and 95% alcohol. The counterstain, fast green FCF in 95% alcohol, is now applied. Both the green stain and its solvent have a differential solvent action on the safranin, and remove it from the unlignified tissues more rapidly than from the lignin, cutin, and chromatin. 1 he interval in green is usually a matter of seconds, rarely as much as 2 min. Correct contrast has been attained when lignin, chromatin, and in some cases cutin are brilliant red, chloroplasts pink to red, and cellulose walls and cytoplasm are green. > The two stains of this combination can be manipulated until the desired contrast and intensities are obtained. If alcohol is used in the dehydrating series, the slide may be placed on a microscope, kept wet with 50% alcohol, and observed until only the lignified elements remain red. The slide is then rapidly carried through the subsequent processes. Acetone is too volatile to permit such examination. With some experience it is possible to judge when the safranin has been destained sufficiently to add the green counterstain. If the stock solution of fast green acts too rapidly for a given subject, the green color will mask or remove the red, and all cells may become stained deep green. In such cases dilute the green stain with 1 to 5 volumes of 95% ethyl alcohol. The slide may be examined best out of carbol- 70 Botanical Microtechnique xylene. If red color is still evident in cellulose walls and cytoplasm, carry the slide backward through the series to fast green, double the previous interval in fast green, run upward again to carbol-xylene and examine. This process can be repeated until the desired color contrast between chromatin, lignified walls, cellulose and cytoplasm is obtained. If the red color is too pale when the slide is examined out of carbol-xylene, transfer to the de-waxing xylene and proceed as with a new slide. The green will be removed in the down series. If the green is too intense at the carbol-xylene stage, back downward to 70% alcohol, in which the green is removed rapidly. Try 10 seconds and carry up to carbol-xylene again and examine. Several stains can be substituted for fast green in Chart IV. The most commonly used other green stains are light green and malachite green. Several excellent blue counterstains arc cotton blue, methylene blue, gentian violet (crystal violet) , and aniline blue. Any of these green or blue counterstains can be used in solution in 95% alcohol, in the sequence shown in the chart, or they may be dissolved in 50% alcohol or in clove oil and introduced at the appropriate place in the series. The above safranin-grcen or safranin-hlue combinations STAINING CHART IV Safranin-Fast Green Pre-Staining Operations and Intervals as in Chart I Aqueous resin and safranin cover glass 1-12 In. I i wlvuc J II water, change a until colorless i 30% alcohol i 50% xylene II wlenc I t carbol- alcohol ^^'''le 70% absolute alcohol alcohol TIT I (optional) 95% t alcohol absolute I alcohol II fast green in f 95% alcohol > absolute 5-30 sec. alcohol I Staining Paraffin Sections 71 serve as excellent cytological stains for many subjects, primarily for the preparation of classroom materials. THE TRIPLE STAIN (FLEAAMING) The triple stain is of considerable historical interest and is still in high favor in some laboratories. The three components are safranin, crystal (gentian) violet, and orange G (or gold orange) . Safranin is intended to stain chromatin, lignin, cutin, and in some cases chloro- plasts. Gentian violet should stain spindle fibers, nucleoli during some phases, and cellulose walls. The orange dye acts as a differentiating agent, serves as a general background stain, and stains cytoplasm and in some subjects cellulose walls. All three components are highly soluble in the reagents used in the staining process and are subject to changes of intensity and mutual interaction during most of the process. The correct balance of relative intensities is, therefore, very difficult to control. The process yields spectacularly beautiful slides from the hands of an expert. However, an attractive or gaudy poly- chrome effect is not adccjuate justification for the use of an elaborate and time-consuming process. The real test of the desirability of a multiple stain is the specific selectivity of its color components for definite morphological or chemical entities in the cell. The sphere of usefulness of the triple stain may be jndged by a consideration of the stains used in modern cytological research. It is noteworthy that the most critical modern work on chromosome structure and behavior has been done with the iron-hematoxylin stain, with the gentian violet-iodine stain, and with acetocarmine smears. The most reliable work on the spindle-fiber mechanism and spindle-fiber attachment also has been done with the first two stains. As an illustration in the field of anatomy, it will be obvious that in studies of vascular tissues a stain is required primarily to show a xylem-phloem contrast, distinguishing between lignified and un- lignified cell walls. This usually is done adequately with a two-stain combination. There is no special virtue in having a delicate orange background for a study of the organization of a vascular bundle or in a section of pine lumber. However, in many cytological problems involving the entire cell rather than merely the actively dividing; chromosomes, the triple stain is an indispensable tool. Another legiti- mate sphere is in pathological studies in which it is desirable to produce polychrome contrasts between a parasite and its host. The object of the above discussion is to emphasize again the view that any elaboration that does not serve a definite, useful function is a waste 72 Botanical Microtechnique of time. The triple stain should be kept in its proper place among the diverse tools ol the technician. The three stains used in the conventional process are the following standard stock solutions: Safranin O, aqueous, or in 50% alcohol. Crystal violet, or gentian violet, 0.5 or 1.0% in water. Orange G, or gold orange, saturated solution in clove oil. Mordanting is necessary for some subjects. After killing in fluids that contain osmic and chromic acids, mordanting is usually not necessary. For materials that do not retain the stains, mordant for 1 to 12 hr. in 1% aqueous chromic acid or in an aqueous solution containing 2% chromic acid and 0.57c osmic acid. Staining Chart V is intended primarily to show the sequence of operations in a typical schedule. The variability of the time element STAINING CHART V Triple Stain Pre-Staining Operations and Intervals as in Chart I Safranin resin and 4-'^4 hr cover glass i ^^ "" xvlene water, 3 changes 2'chan<'^es i (jars) crystal violet 'I 10 luin. to 1 hr. rinse with I xylene (pi[)ette) t water. ^ changes . ' . , " rinse with -l clove oil-xylene flood sections > drain off .inhydrous alcohol with orange G orange G in any of the operations hardly can be overemphasized. The suggested intervals merely furnish a starling point for experimentation to determine the o|)tiinuni linie schedule h)r any specilic subject. Many modihcations ol schedide have appeared in the literature. Staining Paraffin Sections 72 Variations in the composition and purit) ol the component stains ha\e necessitated revisions of schedule to suit the currently available stains. Some workers prefer to differentiate the safranin with very dilute HCl or with acidified alcohol before adding the violet. The acid must subsecjuently be thoroughly washed out with water. The gentian violet may be almost fully differentiated in clove oil containing no orange G. The orange can then be added progressively and the violet brought to final differentiation in xylene containing 1/10 to I/4 (by volume) clove oil saturated with orange G or gold orange. Quadruple-stain combinations using four coal-tar dyes are avail- able in some excellent commercial slides. Conant (Triarch) uses a combination of safranin, crystal violet, fast green, and gold orange; Johansen (California Botanical Materials Co.) uses safranin, methyl violet 2B, fast green, and orange G. These complex processes yield striking preparations but are probably unnecessarily elaborate for most tasks. The advanced worker can obtain details of procedure from the excellent service leaflets of the above manufacturers and from the Johansen manual (1940) . Staining processes using coal-tar dyes are entering a most interest- ing and important phase of development. Many new organic solvents are being produced by synthetic methods. Solvents that have been little more than chemists' curiosities are now being produced in large quantities and are available at reasonable cost. Some illustrations are the higher alcohols, such as the butyl, propyl, and amyl series, ethyl and methyl Cellosolve, trichloroethylene, and many other solvents. The stains themselves are imdergoing constant study and improve- ment. The possibilities of systematic study, or just plain dabbling, should gratify the heart of the most inveterate experimenter. IRON HEMATOXYLIN The next stain to be considered is known as Heidenhain's, or iron- alum hematoxylin. The history of this stain and the names of several investigators who have contributed to its development may be found in the literature. This stain is primarily a cytological stain, used especially for chromosome studies, but it is useful for studies on the cell wall, plastids, and in some studies in pathological histology. The formidas of the mordant (iron alum) , the stain (hematoxylin) , and the destaining agent are given in the stain formulary. The schedule advocated here and outlined in Staining Chart VI is known as the short schedule, or 4-4 schedule; i.e., 4 hr. in mordant, thorough but 74 Botanical Microtechnique quick rinsing in five changes of distilled water at 1-niin. intervals, then 4 hr. in stain. The material becomes stained solid black and must be differentially destained. The destaining solution removes the stain rapidly from cytoplasm, less rapidh from plastids, and slowly from chromatin and from the active tips of mycelium. The destaining action should be observed under a microscope and stopped by STAINING CHART VI Iron Hematoxylin Pre-Staining Operations as in Chart I resin and cover glass t xylene III t xylene II t xylene I t carl)()l- xvlene t absolute akohol II t absolute alcohol I t akohol t 70% alcohol t 50% alcohol t 30% — > akohol 4% iron alum 4 hr. i dist. water 5 changes 1 min. intervals hematoxylin 4 hr. i dist. water 3 changes destaining reagent uinil cliffcrcntiated i dist. water 3 changes i running tap water 5 min. washing first in disiilled water, then i)\ prolonged washing in luiuiing tap waler. Dehydration and subsec|uent processing lollow as shc^wn on the chart. In the linished slide, chromosomes, the chromatin of resting nuclei, middle lamella, and active mycelia should he blue- black, whereas the cell walls and cytoplasm shoidd be practically colorle-ss. A finished, covered i)rei)aration may be destained by soaking off the cover glass, nnming the slide back through the series into water, then innnersing in the destaining solution. If a preparation is Staining Paraffin Sections 75 found to be destained too much, it can be run back into water and mordanted and stained again. As in some other staining combinations, restained preparations are seldom as clear in detail as slides stained correctly the first time. THE CRYSTAL VIOLET-IODINE STAIN The next stain to be considered is another cytological stain. It is included here because it yields preparations that are valuable in the teaching of some topics. In teaching mitosis at the elementary college STAINING CHART VII Crystal (Gentian) Violet-Iodine Pre-Staining Operations as in Chart I resin and IKI 15 min. rinse in water i crystal violet 1-4 hr. i rinse i IKI 15 min. } rinse i 50% alcohol picric acid 30 sec. i 95% alcohol 10-60 sec. cover glass t xylene III t xylene II t xylene I t examine t 1/^ absolute alcohol 14 xylene [_ 1/3 cedar oil 30-60 sec. t absolute alcohol II 30 sec. t absolute alcohol I 30 sec. level, most teachers prefer longitudinal and transverse paraffin sec- tions, stained to show cell walls and tissue organization as well as the more prominent features of nuclear division. Crystal violet stains the chromosomes a brilliant blue-black, on a colorless, almost invisible, cellular background. Such preparations are a valuable supplement to the standard classroom slide. The procedure given in staining Chart VII is but one of the numerous variants of the process. Probably no other staining pro- cedure is as responsive to the individual touch of the technician, and a specific procedure should be regarded as a point of departure for developing a personal routine. 76 Botanical Microtechnique The following version recognizes that it is cheaper to rinse out stains and mordants in water than in alcohol, and also prolongs the usefulness of the dehydrating series. The stain is a 1/4 to V^% aqueous solution of crystal (gentian) ^ iolet. Ihc mordant is aqueous IKI (Chajx 9) . The picric acid solution is a saturated solution in 50% alcohol. De-waxing and running down to water are carried out in Coplin jars. However, the slides nuist be carried one by one through the dehydrating series, which must not contain other stains as contaminants. Therefore, a sjjccial dehydrating set shoidd be maintained for the violet process, preferably in wide-mouth screw- capped jars. Differentiation is accomplished in the alcohol dehydrating series. The slide shoidd be agitated in the fluids with forceps. Observe closely in the 95% alcohol, and when visible color no longer comes out of the sections, move rapidly through anhydrous alcohol. Differ- entiation shoidd be complete when the alcohol-xylene-cedar oil clear- ing solution is reached. The function of the oil is to retard evapora- tion to j^ermit examination. If the cytoj^lasm is too blue, back down to 95% alcohol. If the blue has been lost from the chromosomes, carry back to water and IKI and restain. The Tannic Acid-Ferric Chloride Stain (Foster) This stain is used lor meristematic tissues, in which it stains the thin cell walls. Because of the simplicity of the schedule, no chart is necessary. The reagents thai bring about the staining are as follows: 1. Tannic add. 1% aqueous, \vitli \% sodium benzoatc as a preservative. 2. Ferric (hloiidc. ,8% a(|ucous soluiion. The ])rocediU"e Irom \vatei' is as l()ll()^\•s: 1 . lannic ac id 10 min. 2. Wash tliorout;lily in water. 3. Ferric diloiidc. 2-.^ niin. 4. Wash ill wati'i, and cxaniiiif \\illi iiiic rose ope. Re]>eat stejjs 1 to 1 iiuhisi\c' until the (ell walls are sharply outlined. Xiulci nia\ be stained in safranin il desired, using atjueous safranin, or safranin in M)' i alcohol. Variations of the fundamental method are described by Northen (19,Sr)) . riiis stain is likely to undergo further modification and will probabK become one of our most useliil histological stains. I he loic'going outline ol the elements ol staining jMoccsses is likely to be ade(|uate for liie a\erage needs of sludenis and teachers Staining Paraffin Sections 77 and lor many research problems. The beginner is warned not to dabble in a wide variety of processes but to gain a mastery of a few funcfcimental methods. Ability to analyze and remedy difficulties should be cultivated. The advanced worker who finds that a research problem requires more specialized methods should ttnn to the literatme and search out methods that ha\e l)een used for similar investigations. O. The Celloidin Method Infiltration The celloidin process is used for subjects that are tough, brittle, or friable (crumbly) . Paraffin does not afford adequate support for sectioning such materials. One example of a subject for which the celloidin process must be used is a graft union (Fig. 13.9 a) , in which the incompletely united members must be kept intact before and after sectioning. Another illustration is the preparation of pathologi- cal materials which may be in such disintegrated, fragile (ondition that the sections would fall apart without the celloidin matrix. Sectors from large trees having wood, cambium and bark tissues cannot be kept intact withoiu embedding in celloidin (Fig. 13.9 b, r) . Unembedded small twigs are difficult to hold and orient for longi- tudinal sectioning without some matrix. A twig or sector from a tree can be embedded in celloidin, blocked as shown in Fig. 8.2 D-F, and sectioned accurately in any desired plane. A properly selected piece of material may yield 100 sections, uniform in thickness, orientation, and staining jjroperties. Permanent slides can thus be made by the hundreds at low cost. In some laboratories the celloidin method is neglected, or e\en scorned, but tlie above illustrations sh(n\' that the process has its place in any well-eciuipped, versatile laboratory. The matrix for the celloidin process is a form of nitrocellulose, know^n by several trade names — celloidin, collodion, Parlodion, and some less common names. Ihis product is sold in the loi in of shreds or chips, packed dry or in distilled water. The latter method retards the development of a yellow color. Celloidin shoidd be dried thor- oughly before being dissolved lor use. Ihe most (oninionh used solvent consists of approximately ecjual volumes of ether and methyl alcohol. These reagents nnist be of the best qualii\ and auliydrous. Five stock solutions of celloidin are usually used. These solutions contain, respectively, 2, 4, 6, 8, anil 10 g. of dried celk)idin per 100 cc. of solvent. Fhese solutions are designated for convenience as 2% celloidin, etc. I 78] The Celloidin Method 79 Infiltration in celloidin consists ot transferring the previously killed and dehydrated tissues into a dilute solution of celloidin, con- centrating the celloidin, and finally molding the thickened celloidin into blocks containing the material. Concentrating the matrix may be accomplished by one of the following processes or by combinations of processes. 1. Transfer the tissues thorugh a graded series of celloidin solu- tions of increasing concentration. 2. Add chips of dry celloidin at intervals to the initial 2% solution. 3. E\aporate the solvent from a large ^•olume of a 2% solution. METHOD 1 Transfer the dehydrated tissues from the dehydrant into the solvent. After one to several hr., transfer to 2% celloidin, covering the material with at least five times its volume of celloidin. Fasten a dry, rolled cork into the bottle by means of wire loops (Fig. 8.1 A, B) . Put the bottle into the 53°C. oven. The interval in the oven varies widely. For sections of twigs having a diameter of 3 to 5 mm., 24 hr. in 2% celloidin may be enough. For larger pieces and for dense materials increase the time to 2 days or more. After the interval in 2% celloidin, cool the bottle, remove the stopper, and pour the celloidin into a dry pan (not into the sink!) . Keep away from flames or sparks. Cover the tissues im- mediately with 4% celloidin. Reseal the bottle, and repeat the interval under pressure in the oven. Repeat this operation with 6, 8, and 10% celloidin. Following the last treatment, continue to thicken the celloidin by adding a chip of dry celloidin every 24 hr. When the celloidin is so thick that it just flows at room temperature, the mate- rial is ready to be hardened as described on page 81. To determine whether the consistency of the celloidin is correct for blocking, dip a thoroughly dried matchstick into the celloidin, lift out a mass of celloidin, and immerse in chloroform for 1 hr. The celloidin should become hardened into a clear, firm mass that can be sliced easily with a razor blade. A comparison of samples taken during successive stages of infiltration will show the progressive increase in firmness and improved cutting properties. METHOD 2 The material is first given at least 48 hr. in 2% celloidin in a sealed bottle in the oven. At intervals of several days the bottle is cooled and unsealed, a chip of dried celloidin added, and the bottle 80 Botanical Microtechnique sealed and returned to the oven for the next inter\al. When working with deHcate material, the celloidin chip should not be dropped onto the material but tied into a bag ot dry cheesecloth, which is then suspended in the bottle so that the celloidin is just immersed in the solution (Fig. 8.1 C) . 1 he periodic addition of celloidin is continued Fig. 8.1— v4, Specimen Ijottlc tor infiltration with celloidin under pressure, with stopper fastened by wire loops: B. detail of wire loops for fastening cork; C, cheese- cloth-bag method of thickening celloidin. until the solution is thickened to the degree described in the pre- ceding method. METHOD 3 This method is \cry slow but yields superior results. The material is started in a large volume of 2*^ celloidin, at least foin times the depth occupied by the material. Mark the initial \v\c\ of the solution. Cork the bottle loosely, but wire the cork so that it cannot be pushed out. Keep the bottle in a warm place, away from llanies or sparks. Slow evaporation takes place, and. when the Noliniie is one-hall the original, the sohnion is in aj)j)roximately 1', celloidin. Add new 4% celloidin tcj make up the original volinne. If the celloidin has become colored, replace with new 4% sohnion. Continue the process of slow evaporation until the material is in thick celloidin. An objec- tionable featuie of method -5 was jiointed out by \\^alls (1936). If the evaporation rate is too rapid, ii seems that the ether of the solvent evaporates nioie lapidly than the meih\l alcohol, and the celloidin jells before adecjiiate thickening is obtained. Ihis can be remedied by adding a small c|uantily of j)ure ether and coin inning infiltration until the j)ro|)er \iscosit\ is attained. The Celloidin Method 81 A low-viscosity nitrocellulose has been recommended for rapid infiltration of firm materials (Davenport and Swank 1934; Koneff and Lyons 1937). Ihis inexpensive celloidin forms a firm matrix, and its solutions in ether-alcohol tolerate 6% water, thus minimizing the extreme brittleness produced in woody materials by total dehydration. The above references give complete details of procedure. Hardening and Blocking In the celloidin process the solvent is not eliminated completely during infiltration. The thickened celloidin solution is hardened by immersion in chloroform. Remove an infiltrated piece of material and a mass of enveloping celloidin and immerse in chloroform. The celloidin loses its stickiness at once and soon becomes hardened throughout. It is best to leave the material in chloroform for 12 hr. to harden the celloidin in the innermost cells of the material. Trans- fer the hardened material into a mixture of approximately equal volumes of db'^i ethyl alcohol and glycerin, in which the material may be stored indefinitely. Large pieces of embedded wood may be removed from the glycerin- alcohol, clamped directly into the microtome, and sectioned. Subjects having easily separable soft tissues are often damaged by compression in the clamp. Such materials are sectioned best by mounting them on blocks of wood or plastic. The mounting block may then be clamped rigidly into the microtome clamp without damaging the tissues. A twig or other long slender object should be mounted into a plastic tube or a wood block having a hole of suitable size drilled lengthwise through the mounting block (Fig. 8.2 A-C) . Prepare mounting blocks by drying them thoroughly in a 110°C. oven, soak in anhydrous methyl alcohol, then store in waste 4% celloidin until needed. When the material being infiltrated is put into 8'^( celloidin, drop a prepared mounting block into the specimen bottle and continue the infiltration. To mount twigs for cutting transverse sections, remove the desired twig and a stutable drilled mounting block from the thickened celloidin. and push the twdg into the hole, leaving 6 to 10 mm. of the twig protruding. Fill any space remaining around the twig by pushing slivers of matchstick into the hole from below. AVrap a generous mass of thick celloidin around the twig and mounting block, and harden in chloroform (Fig. 8.2 C) . For longitudinal sections of a twig, lay the infiltrated twig on a large, undrilled block, wrap well with additional thick celloidin, and harden in chloroform. 82 Bofanical Microtechnique When the surface of the celloidin is hard (2 niin.) , press the twig gently until it is flat on the mounting block, thus affording firmer support for sectioning. A batch of embedded material usually con- tains more pieces than are needed for immediate sectioning, therefore, only a few pieces need to be mounted on blocks. Most of the pieces are merely removed from the thick celloidin, hardened in chloroform, and stored in glycerin-alcohol. The pieces can be blocked at any future time bv the method to be described later. Fk.. 8.2— Methods of mounliiig tissues on blocks for sectioning in celloidin: A, cutaway view of drilled block for holding long object; B and C, cutaway views of drilled bk)ck containing twig or other long object enveloped in hardened celloidin; D-F, infdlrated blocks of wood and bark embedded in hardened celloiciin. oriented on mounting blocks to cut transverse, radial, and tangential sedions. respectively. Sectors from large limbs usually must be fastened on mounting blocks for sectioning. II I he j)ieces ha\e cambium and other tissues of the bark, these tissues may peel off when the piece is compressed in the microtome clamp. Mount three pieces from each subject, on separate i)lock.s, so that transverse, radial, and tangential sections can be cut (Fig. ^.2 D-f) . A generous wrapping of celloidin should en- velop part of the mounting block as in Fig. 8.2. The rigidity of the moiuuing of such material can be improved by mounting the material in a recess that has been drilled about l/Ki in. deep into the end of the mounting block. Blocking of pic'\ ioiisly hardened tinbttlded material is a simple operation. Remove the desired pieces fiom the glycerin-alcohol storage (liiid and soak in anhydrous ethyl alcohol, (-hange the alcohol t\\i(c a! 1- to S-hr. iutcix ;ils. This rciiKncs the small ainoiuit of water The Celloidin Method 83 left from the storage fluid, softens the celloidin, l)ut does not dissolve an appreciable amount. Transfer the pieces into thick celloidin of a consistency suitable for casting. Also put a supply of mounting blocks into this thick celloidin. After at least 24 hr. in thick celloidin, mount and harden as previously described. It is sometimes necessary to trim pieces of tissue prior to reblocking in order to establish the correct cutting planes. Trimming should be done when the pieces are removed from glycerin-alcohol. The glycerin prevents drying and shrinkage of the tissues during trimming. Waste celloidin from various stages in the process can be salvaged by evaporating in a shallow pan in a place free from dust and open flames. The dried sheet is cut into shreds, dried at 53°C., and used to make solution for treating motmting blocks and to make 2 and 4% celloidin for method 1. Celloidin solution that is too discolored to be salvaged is most easily disposed of by pouring it into a pan of cold water. The celloidin hardens into a crust which can be lifted out and discarded. Cellosolve is the trade name used for two synthetic organic com- pounds, ethylene-glycol-monoethyl ether and its methyl homologue. These fluids are solvents of celloidin and may ultimately replace the inflammable alcohol-ether solvent used heretofore. These solvents are not inflammable at ordinary working temperatures, therefore the entire process may be carried out in open or loosely stoppered bottles. The evaporation rate is very slow at 50 to 55°C. Methyl Cellosolve, which boils at 124.3°C., evaporates slightly faster than ethyl Cel- losolve, which boils at 135.1°C. The latter solvent is preferred for fragile subjects which tend to collapse if the celloidin concentration is increased too rapidh . An inexpensive method of using Cellosolve is to dehydrate the tissues in the appropriate grades of alcohol and to transfer to a 2% solution of celloidin in Cellosolve. Subsequent infiltration may be accomplished by successive treatment in 4, 6, 8, and 10% solutions at 50 to 55 °C., or by beginning with 2% and periodically adding celloidin chips by the cheesecloth-bag method. The interval in each grade ranges from 24 hr. for small or porous pieces to a week for large blocks of wood. Cellosolve may also be used as the dehydrating agent. Materials in which the preservation of the protoplast is not important may be transferred, after killing and washing, directly into Cellosolve. Make two or three changes of Cellosolve before beginning the infiltration. However, if the material requires thorough or gradual dehydration 84 Botanical Microtechnique to insure adhesion of the celloidin, it is better to dehydrate in ethyl alcohol or acetone and to use tlie much more expensive Cellosolve as the infiltration solvent. Special Treatment of Hard Woods The foregoing methods of infiltration yield excellent results ^vith some soft woods such as willow, poplar, basswood, white pine, and many other woods. These can be infiltrated in celloidin without special preliminary treatment, but oak, hickory, walnut, the yellow pines, and other woods are too hard to section by the regular process. Such materials can be softened by treating with hydrofluoric acid (HF) . This highly corrosive reagent is pmchased in wax bottles and should be used in wax or wax-lined or plastic containers. Because of the corrosive action of the licjuid and vapor on glass, metals, and the skin, HF siiould be used in an isolated part of the premises, away from valuable instruments. The staining and microchemical reactions of tissues are materially altered by this treatment. Twigs having li\ing bark tissues are first killed as usual and trans- ferred to HF. Dry woods arc prepared for treatment in HF bv alternate boiling in water and exhausting in an aspirator in cold water until the pieces are saturated. The safest concentration of HF for most subjects is commercial acid diluted with approximately twice its volume of water. The duration of treatment in HF varies greatly with the hardness of the material, the size of the pieces, and f)ther factors. As a trial, treat a hard wood such as oak for T) days, wash in running water lor at least 1 hr. to make it safe to handle the pieces, and try to cut thin slices with a sharp razor blade. An alternative mciliod of testing is to wash the pieces in running water for 4 hr., clamj) a piece into the sHding microtome, and list its ciuting properties. After making a test, wash and \\'i])e the danij) and the knile thoroughly. If the material is too hard to tut rtaihh litlicr freehand or with the microtome, return to the HF for another ■\- to 5-day inter\al, and test again. When the wood seems to cut satis- factorily, wash for at least 4fasm is fixed aii(f hardened. For sections of woody twigs oi firm lu'rl)aceous stems, I'AA is recom- mended. Foi critical studies try Craf II or III (C^hap. 3) or an experimentally determined modification. Ihe c|uality of preservation produced by a fornuda can be ascertained b) examining sections in a drop of the fluid. Finn sections are killed and hardened almost Sectioning Unembedded Tissues 93 instantly. After 10 niin. in the llitid, rinse the sections in water, and proceed with the staining. The preceding methods permit the stndy of living cells, or cells in which protoplasmic details were fixed by reagents. If these methods of sectioning unembedded material fail to give satisfactory results, the materials must be treated by methods which may distort or destroy fine protoplasmic details. Nevertheless, the following methods are useful, within the stated limitations. Dry lumber of soft woods such as white pine, basswood, or willow can be sectioned successfully without embeddding. Trim the wood carefully into blocks measuring approximately 1 by 1 by 2 cm., and prepare the blocks by alternately boiling in water and pumping in cold water, until the pieces are thoroughly saturated and sink. Hard woods that cannot be cut after this treatment may be sectioned by one of the following methods. The live-steam method of sectioning wood is based on the principle used in the manufacture of veneer. If a jet of superheated steam is directed upon the surface of a block of wood, the surface becomes soft enough to permit the cutting of a thin section. Steam can be generated in a flask, but a safer device is a small steam generator, provided with a pressure gauge, water level gauge, safety valve, and a water inlet. Such generators are obtainable from dealers in chemical apparatus. The steam from the generator passes through a copper tube, in which there is a superheating coil heated by a Bunsen burner, and the superheated steam emerges through the small orifice of a nozzle which can be adjusted over the specimen. Successive sections are cut at experimentally determined intervals of steaming. For additional details and variations of the apparatus refer to Crowell (1930) and Davis and Stover (1936). - Jeffrey's vulcanization method makes possible the sectioning of extremely hard materials, such as walnut shells. Materials are sealed in a chamber made from a section of pipe, and heated in a dental vulcanizer at approximately 160°C. for 1 to 5 hr. This is followed by treatment in hydrofluoric acid. Materials may be cut unembedded or in celloidin. Because of the special equipment necessary, the procedure is not described here, and interested workers are referred to Jeffrey (1928). Sectioning With the Freezing Microtome Unembedded tissues that are too soft or fragile to stand up under the impact of the knife can in some cases be frozen and then 94 Botanical Microtechnique sectioned. The device used on the sliding microtome for this purpose is known as a freezing attachment. The various freezing attachments are described in the catalogues of the manufacturers of microtomes. The device replaces the usual tissue clamp or object carrier of the microtome. Freezing is accomplished by the evaporation or expansion of a freezing agent — ether, CO^ gas, or solid COo (dry ice) . The piece of tissue to be frozen is usually enveloped in a fluid or semifluid medium, which on freezing affords additional sup})ort. Gum arable is probably the best-known supporting medium. Make an aqueous solution of gum arable of thick, sirupy consistency. Add a few crystals of carbolic acid. Dip the specimen to be cut into the gum. Freeze a 2- to 3-mm. layer of gum on the supporting disk of the freezer. Place the specimen on the disk, wrapping a generous quantity of the gum around the specimen. Turn on the freezer, and, as the gum begins to congeal, wrap more gum on the specimen until the material is well supported. Proceed with the sectioning. Gelatin is another satisfactory supporting medium. Make a gelatin solution that is semifluid at room temperatures. Add 0.1 per cent carbolic acid as a preservative. Warm on a water bath for use. and use in the manner described above for gum arable. A semifluid solution of agar is another excellent medium, used like gelatin. Freehand or microtome sections can be mounted in a drop of water or 50% glycerin and studied with the microscope. If glycerin is used, the water can be evaporated and the cover glass sealed with lacquer or paraffin, making a semipermanent mount. The mounting media described on ])age 102 also can be used. Sections of dark- colored woods, or other materials having adequate coloration, can thus be made into scmij)ermancnt or jxTmancnt slides without staining. Staining The staining of sections of uiKinbeddcd tissues is essentially the same as the staining of celloidin sections. I'he worker \\li() has had previous experience with paraffin sections can foll()\\- tlie staining charts in Chap. 7, making the necessary modifications. For the benefit of workers who wish to use the present chaj)ter without having had previous experience, an outline of some simple, practical processes is offered. Sections cut in alcohol should be progressi\c]\ transferred or run down to water before staining. Add an e(iual Nolume of water to the alcohol containing the sections. Mix genily. jK)ur off half of the liquid, and add an equal volume of water. Pour off all the liquid, Sectioning Unembedded Tissues 95 and rinse the sections Avith two changes of water. Proceed with the staining process. The most easily controlled stain combination consists of a self- mordanting hematoxylin (Chap. 7) followed by an aniline dye. Assume that sections of a firm woody subject are to be stained. Drain off the water in which the sections are floating in a watch glass, and flood the sections with hemalum or similar stain. After 5 min. remove a section with a brush, rinse in distilled water and then in tap water and examine with a microscope. Lignified cells, such as tracheids and phloem sclerenchyma, should be practically colorless. Soft tissues like pith, cortex, and cambium should have blue-stained walls. Nuclei should be blue-black. If the sections are overstained the entire protoplasts become blackened, obscuring cellular detail, and the walls of bonified cells become stained. Sections can be destained with 1/2% HCl and thoroughly washed in tap water. When the correct intensity of blue is attained, cover the sections with safranin. Try an interval of 15 min. in safranin. Locate this point in the staining schedule given in Staining Chart III. Rinse the sections in water, and cover with 50''/ alcohol. The safranin will dissolve out of the nonlignified tissues faster than out of lignified cell walls. Slower destaining can be obtained with 50% acetone. When the nonlignified cells are still deep red, rinse the sections quickly in anhydrous alcohol or acetone. Acetone stops destaining action better than does alcohol. Complete the process as shown in Staining Chart III. Mount the sections on slides as described in Chap. 7. Having gained some familiarity with the above stain and with the use of staining schedules, study the discussion of staining in the paraffin method (Chap. 7) and the celloidin method described earlier in this chapter, and try some of other stain combinations described in those chapters. Sectioning by Encasing in Water-Soluble Waxes These little-known methods are intermediate between embedding methods and sectioning without embedding. As a matrix, use one of the water-soluble wax-like synthetics, such as glycerol monostearate, which melts at 55°C. The living or fixed material is transferred directly from water to the melted matrix, which is then hardened, fastened to a wood mounting block, and sectioned. Very little infiltration occurs, but the material is encased and held with sufficient rigidity to make fairly good sections. This method has been used successfully with nearly mature clover seeds and shows much promise 96 Botanical Microtechnique for similar subjects. Other synthetic stearates should be tried (Johansen 1940, McLane 1951). Microchemical Tests One of the important advantages of sectioning fresh initreated material is the avoidance of the chemical and physical alterations that are undoubtedly produced by the processing necessary for embedding. Although the protoplasm is probably changed by the handling incident to sectioning and moiniting fresh material, the nonliving constituents of cells and tissues probably are not markedly changed chemically. This makes possible the use of microchemical tests that reveal with more or less acciaacy the chemical nature of important structures. Although the science of chemical microscopy is highly developed, it occupies a minor place or is \irtually ignored in many botanical curricula. However, certain chemical tests are generally regarded as indispensable in even an elementary stud) and are therefore included here. (Loomis and Shull, 1937.) Starch; Iodine-Potassium Iodide Test (IKI) Water 100 cc. Potassium iodide 1 g. Iodine 1 g. Place a drop of the reagent directly upon the specimen. Most starches give a blue-black color. Waxy starch, found in some genetic stocks of maize, turns yellow or brown. By using a very dilute solution of the reagent and impart- ing only a trace of color to the starch, the laminations in the granules may be observed with the microscope. When testing entire living cells such as those of S})irogyra or leaves like those of Elodca. the aqueous reagent reacts very slowly, and a reagent made with 70'/c alcohol shoukl l)e used. Sugars; Osazone Test Sohilioii A Cilv(erin (\\arm) 10 cc. PlKiiyllndra/iiK-iiydrochloride ... 1.0 g. Solution B (.l)(.eriii 10 cc. Sodium acetate h() g. Mix a drop ol each sohuion on a slide, float the sections in the mixture, place the slide over the moiuh ol a wide-mouthed flask containing boiling water, and heat for 10 to 15 niin. Glucose and fructose produce fascicles of yellowisii needles: m;illose |)r()cluces fan-sha])ecl chisters of llaltened needles. After .^0 to 60 niin. of heating, sucrose becomes hydroh/ecl and reacts to form needles like those produced bv glucose. Sectioning Unennbedded Tissues 97 Reducing Sugars; Fehling's Solution Test Although this is not a microtest. it is included because it is essential for a systematic examination oi the prominent chemical constituents of cells and tissues. Solution A Water 11. Copper sulphate 79.28 g. Solution B Water 1 1. Sodium potassium tartrate 346 g. Sodium hydroxide 100 g. Mix equal volumes of A and B in a test tube, add a quantity of the finely pulverized materials to be tested, heat to boiling. A brick-red precipitate indicates reducing sugars. If a negative or slight test is obtained for reducing sugars, a test for sucrose can be made by first hydrolyzing the sucrose. Add 1 cc. concentrated HCl to 10 cc. of the extract to be tested. Heat in a water bath at 70°C. for 5 min. Cool and neiuralize with sodium carbonate, and test for the resulting reducing sugar with Fehling solution. Lignin; Phloroglucin Test Solution A Phloroglucin, 1% to 2% in 95% alcohol Solution B Hydrochloric acid (try concentrated acid, as well as acid diluted with 1 to 3 volumes of water.) Float the sections in a drop of phloroglucin on a slide, and cover with a cover glass. Place a small drop of the acid at one edge of the cover glass. Examine with a microscope. Lignified walls become violet-red. Cellulose; Iodine-Sulphuric Acid Test Mount sections or crushed fragments in IKI. Observe with the microscope, and locate blue-stained starch. Place a drop of 75 per cent H2SO4 at one side of the cover glass. As the acid diffuses in, note that cellulose walls swell and become blue. Cellulose; Chloriodide of Zinc Test Water 14 cc. Zinc chloride 30 g. Potassiimr iodide 5 g. Iodine 0-9 g. Mount thin sections in a drop of the reagent. Cellulose becomes blue. Proteins; Millon's Reagent Test Concentrated nitric acid 9 cc. Mercury 1 g. 98 Botanical Microtechnique When dissolved, dilute with an equal volume of water. Plate the speci- men on a slide, drain or blot off excess water, put on just enough reagent to cover the material, and heat with a small flame. Proteins give a brick-red color. This is not a highly satisfactory reagent. Futhcrmore, it is highly corrosive and must be used with care. Do not permit inexperienced students to use this reagent on a microscope. The instructor should set up a demonstration microscope, after draining excess reagent frcmi under the cover glass. Fats and Oils; Sudan III Alcohol (80%) 100 cc. Sudan III 0.5 g. Cut very thin sections or smear a fragment of the material on a slide, flood with the dye, and cover with a cover glass. After 10 to 20 min. the microscopic globules of fat should assume the bright, clear color of the dye. Cotyledons of the soybean and peanut are good subjects. 10, The Preparation of Whole Mounts and Smears Gross preparations may be roughly classified as follows: 1. Dry preparations: herbarium sheets, Riker mounts, and bulk speci- mens. These methods are not within the scope of this manuaL 2. Wet preparations: museum-jar preparations; bulk material for dissec- tion: whole mounts, smears, and macerations for microscopic study. Wet preservation may be used for a wide range of subjects in all major categories of the plant kingdom. Many subjects, such as the algae, can be preserved for critical study only by wet preservation. These methods can be adapted to furnish material for gross specimens, for dissection, and for microscopic preparations. Entire plants or parts such as leaves, flowers, and fruits can be preserved in fluids that kill the cells, prevent decay, preserve the material in firm condition, and possibly preserve the natural colors. The best-known preserving fluid is ethyl alcohol, usually used at a concentration of approximately 70 7^. This fluid preserves even the bulkiest objects. Considerable brittleness is produced, but preserved material can be made flexible for dissection by soaking it in water. Cells are plasmolyzed by alcohol, but this is not objectionable in dissections or freehand sections, in which the condition of the proto- plasm is not important. Ethyl alcohol is difficult to obtain in some institutions, its use at field stations may be undesirable, and shipment of materials in alcohol involves legal technicalities. Regardless of these objections, ethyl alcohol is firmly established as a preserving fluid. Formaldehyde is an excellent preservative. This reagent is obtained as an aqueous solution containing 37 to 40% formaldehyde gas by weight. The U.S. P. (United States Pharmacopoeia) grade is adequate for preserving bulk materials. The most useful concentration for bulk preservation contains 5 parts formaldehyde solution in 95 parts of water. For massive objects the concentration must be doubled. At these concentrations the material does not become brittle, and some materials become pulpy after prolonged storage. Formaldehyde [99] 100 Botanical Microfechnique vapor and the solution are higlily irritating and poisonous. j)ioducing persistent skin and pulmonary disorders. Materials that ha\e been stored in strong formaldehyde should be rinsed in Avater if used for prolonged study. An improvement over formaldehyde for the preservation of algae is the following: Water 93 cc. Formaldehyde (U.S.P.) 5 cc. Glacial acetic acid 2 cc. Hydrodictyon and Spirogyra stored in this fluid for 5 years were found to be in excellent condition for whole mounts in Avater. A further improvement is to add glycerin. Water 72 cc. Formaldehyde (U.S.P.) 5 cc. Glacial acetic acid 8 cc. Glycerin 20 cc. This is one of the best preservatives for unicellular, filamentous, and even the larger bulky algae, for fleshy fungi, for liverworts, and for mosses. A trace of fast green dye imparts enough color to minute or transparent subjects to make them more readily visible under the microscope. The material should be mounted on a slide in a drop of the preservative. Fhe volatile ingredients soon e\aporate, but the glycerin prevents drying of the preparation during a long period oi study. Liverwort thalli can be removed from the iluid and placed in a watch glass for study. The thalli of Marchantia, for example, are so firm that the gametangial disks stand upright in a normal position. The glycerin keeps the material moist for hours, and specimens can be returned to the stock bottle uninjured. The natural colors of plants can be ])reserved in one of several fornudas. The simi)lest formula for green plants consists of one of the lAA formulas with (()pj)er sulphate added (lilaydes 19:^7). Ihe following formula is usually satisfactory: Water .85 cc. C;oi)])er sulplialc 0.2 g. When completely dissohed, add Glacial acetic acid 5 cc. Formaldehyde (U.S.P.) 10 cc. Flhvl alcohol (95'/f) .50 ^x. The Preparation of Whole Mounts and Smears 101 Exhausting the aii' Irom the submerged specimens with an aspir- ator aids penetration. li the materials can withstand briel boiling in the fluid on a water bath, penetration and fixation of the color are hastened. Materials preserved in this formula can be subsequently embedded and sectioned, provided that the pieces are small enough to insure quick penetration of the preservative and satisfactory preser- vation of cellular details. Keefe's formula is one of the best and shcnild be used if the expensive vnanium salt is available. 50% alcohol 90 cc. Formaldehyde (U.S. P.) 5 cc. Glycerin 2.5 cc. Glacial acetic acid 2.5 cc. Copper chloride 10 g- Uranium nitrate 1-5 g. Delicate subjects may be ready to use in 48 hr., but most materials require 3 to 10 days for complete fixation of the color. Leafy plants can be treated and then mounted as herbarium specimens, in which the color will persist for many months. This formula does not pre- serve the colors of flowers, nor is it satisfactory for gymnosperms. The red and yellow coloration of fruits can be preserved in the following formula (Hessler) : Water 1 1. Zinc chloride (dissolve in boiling water and filter) . . 50 g. Formaldehyde (U.S.P.) '. 25 cc. Glycerin 25 cc. Allow to settle and decant the clear liquid for use as needed. With the introduction of dioxan into microtechnique, several wprkers independently developed the idea of using this reagent in killing and preserving fluids. McWhorter and Weier (1936) , for example, devised the following fornuila: Dioxan 50 cc. Formalin 6 cc. Acetic acid 5 cc. Water 50 cc. This solution preserves unicellular and filamentous algae, fungi, and other delicate subjects. Temporary mounts for microscopic study can be made on a slide in a drop of this fluid, or permanent slides can be made. 702 Botanical Microtechnique Temporary and Semipermanent Slides The torcgoino outline of methods of preserving ijulk materials leads to a consideration of methods of preparing mounts for micro- scopic study. The simplest method ob\iously consists of mounting a small quantity of the material in a drop of water. However, water mounts dry out during prolonged study, and it is better to mount the material in 10% glycerin. As the water evaporates, introduce more glycerin solution under the cover glass at intervals, until no further evaporation takes place. Such preparations can be kept almost indefinitely if stored flat and handled with care. The preservative and swelling action of lactic acid and phenol (carbolic acid) is utilized in an important class of formulas. More or less durable slides of algae, fungi, fern prothalli, sections, and other small objects can be made by mounting in one of these lactophenol solutions, with or without added dye. The following selected formulas are taken from Maneval's valuable compilation of these methods. Aman's Lactophenol Phenol (melted) 20 cc. Lactic acid 20 cc. Glycerin 40 cc. Water 20 cc. Phenol-Glycerin Phenol (melted) 20 cc. Glycerin 40 cc. Water 40 cc. If a staining effect is desired, add a \% aqueous solution of either cotton blue, aniline blue, or acid fuchsin as follows: Lactophenol 1 00 cc. Glacial acetic acid 0 to 20 cc. Dye solution 1 to 5 cc. The optinuun concentration of glacial acetic acid is that which produces no (()llaj)se oi bursting of cells or filaments. Try the above formida and dilute wiih lactophenol uinil tlie best ])roportions are established. Glycerin-jelly preparations are a finther advance toward perma- nent slides and are preferretl to glyceiin mounts if the slides must withstand considerable use. The moinuing mediiun is made as follows: Gelatin 5 g. Water 80 cc. Glycerin 85 cc. Phenol (dissolved in 10 drops water) 5 g. The Preparation of Whole Mounts and Sniears 103 Dissolve the gelatin in the water at 35°C.; then add the other ingredients. Filter while warm through fine silk or coarse filter paper. This mounting medium keeps well. Materials to be mounted in glycerin jelly are first stained (if necessary) , then dehydrated by the glycerin evaporation process. For filamentous algae and fungi the most satisfactory stains are the self-mordanting hematoxylins and iron hematoxylin. Staining trials can be made with small quantities of the plant material until a satisfactory schedule is worked out. Then stain a large batch, and dehydrate by the glycerin method. To make a slide from the dehydrated material, place a piece of glycerin jelly about as large as a match head on a clean, dry slide, and warm until melted. Remove a quantity of the plant material from the pure glycerin, draw off excess glycerin with filter paper, and put the plant material into the warmed jelly. Lower a cover glass carefully over the material. If the material is not excessively fragile, a lead weight on the cover glass will squeeze out excess jelly and make a thinner mount. AVhen the jelly is cool, clean off any excess around the cover glass and seal around the edge of the cover glass with a quick-drying lacquer such as Duco. Sealed preparations will keep for several years, but it is well to remember that the mounting medium is soft. Such preparations are not desirable for use by elementary students. Permanent Slides of Whole Mounts Permanent stained slides in a hard, durable mounting medium are much more satisfactory than soft, easily damaged, temporary or semipermanent slides. Modern methods make possible the rapid, quantitv production of permanent slides almost as quickly and easily as the making of old-fashioned semipermanent slides. Filamentous algae and delicate objects may be killed in any of the formulas de- scribed in the foregoing pages. Wash out the killing or storage fluid with water and apply an appropriate stain. Any of the self-mordanting hematoxylins will give excellent results. It is best to overstain strongly, leaving the material in the stain for 1/2 to 1 hr. Wash in distilled water until the rinse water is no longer tinted. Destain in 1/10' < HCl. Cover the material with the acid in a shallow dish and agitate gently. After 1 to 2 min. drain, rinse in tap water, and examine with a microscope. Repeat the treatment in acid until only the nuclei and pyrenoids remain blue-black, then wash thoroughly in tap water. Iron hematoxylin will give the most critical staining of nuclear structures and pyrenoids. Mordant algae for 1 to 2 hr. Rinse quickly but thoroughly in distilled water and stain for 2 to 8 hr. Destain by 704 Botanical Microtechnique immersing in the destaining agent for 1 to 2 min., rinse in distilled water, examine with the microscope, and repeat the brief immersion in ahrni until the nuclei and pyrenoids are sharply differentiated. After the last rinsing in distilled water, wash thoroughly in tap water. Dehydration and mounting may be accomplished by one of the several methods outlined below. THE VENETIAN TURPENTINE METHOD The Venetian tmpentine method fields excellent preparations, but this method is likely to be supplanted by modern methods using a variety of synthetic organic solvents and synthetic resins. Therefore, only a brief otitline of the method will be given, and the interested reader is referred to Chamberlain for details (1932) . Kill and stain the material, and dehydrate by the glycerin evaporation method. Rinse otit the glycerin in 95% alcohol, then complete the dehydration in at least three changes of absolute alcohol. Transfer to 10% Vene- tian turpentine in absolute alcohol. Eliminate the alcohol by evapora- tion. Wlicn the material is in thickened turpentine, mount the de- sired amount of the material in a drop of that medium. Dry the slides in a horizontal position. Preparations made by this method are durable and the stains arc permanent. THE BUTYL ALCOHOL-RESIN METHOD 1 he term resin is used here in the broad sense to include Canada balsam as well as the increasing ninnber of synthetic resins. Test the solubility of the currcnth available mounting resins in (niJixdyoiis normal and tertiary butyl alcohol and in dioxan. and use the solvent- resin combination that has the highest transparency and least color. The following tertiary biuyl alcohol - Canada balsam j^rocedure serves as an example of the series of operations in a whole-moiuit method. Stain and wash as before. Transfer a small quant it\ of the material into a watch glass of 50%, alcohol, and obser\e with the microscope. If there is nuich distortion, try 20% alcohol on another batch. Well-hardened material can Avithstand 50',. W'luii tiu' proj^er starting ]X)int lor dehydration is established, carrv tlic material iu steps of 20 to approximately 70% alcohol. Add to the 70% a few drops of sto(k solutiou ol (ouiucistaiii — eosiu ^. erythrosin B, or fast green, saturated solution in al)s<)liue akohol or in melhvl Cello- solve. Lea\e in the coiuuerstain until slightly o\erstained. This may recpiire 1 to 12 hi. Rinse in 70%, ahohol. and transfer thiough the r()ll()\vin!; sii ies at 1/2- to l-hr. inter^■als: The Preparation of Whole Mounts and Smears 105 3 parts alcohol to 1 part TBA (anhydrous tertiary butyl alcohol) 2 parts alcohol to 2 parts TBA 1 part alcohol to 3 parts TBA Pure TBA; change twice at 15-min. intervals Transfer to a large volume of 5%. solution of balsam or synthetic resin in TBA in a short wide-mouthed bottle. Allow the TBA to evaporate slowly at a temperature of about 35 °C. When' the balsam is slightly more fluid than that used for covering sections, mount the material. Remove a suitable quantity of the plant inaterial with its enveloping balsam, place on a dry, clean slide and lower a dry cover glass carefully so as not to produce bubbles or to push the plants too close to the edges. Dry the finished slides in a horizontal position. (Johansen, 1940.) THE DIOXAN-BAISAM METHOD This is the most promising of the newer methods of making whole mounts. The method was worked out in almost identical form inde- pendently by McWhorter and AV^eier (1936), Johansen (1937), and the present writer. It is probable that numerous other workers had developed similar schedules. Stain filamentous or other delicate materials as in the preceding methods. Pass through a series of aqueous solutions of dioxan, con- taining the following percentages of dioxan: 20, 40, 60, 80. 90, then three or more changes of anhydrous, chemically pure dioxan. The interval in each should be 1 to 2 hr. Examine a few filainents under a microscope, mounted in the last fitiid. If the material is in good condition, transfer to a 10% solution of balsam or resin in dioxan. Use a wide-mouthed bottle and gauge the volume of the liquid so that the material does not become exposed as the dioxan evaporates. Place the uncovered container into an oven or a dust-free place at a temperature of approximately 35 °C. The dioxan evaporates in 2 to 8 hr., leaving the material in thick balsam in which it is mounted. Extremely fragile materials, such as Volvox or I'aiicheria, should be started in 5% resin and evaporated very slowly by keeping the con- tainer loosely covered. This process is so rapid that it is worth the time to carry small trial lots of the material through the process at different speeds. The condition of the material can be examined at various points in the process. When the optinuim or shortest safe schedule is found, the main batch of material can be carried throiiph. W6 Botanical Microtechnique ACETOCARMINE SMEARS The aceLocarmine method has become so commonplace that it may well be included in an elementary manual. This rapid method combines killing, fixing, and staining. The freshly-made preparations can be used in nonpermanent form for counting chromosomes, de- termining their association, and studying intimate details of structure. 71ie slides can be made permanent if desired. The stain is prepared by dissohing 1 g. carmine in 100 cc. of boiling 455( acetic acid (or propionic acid) . Cool and decant. Add 2 drops of a saturated aqueous solution of ferric acetate, and allow to stand for 12 hr. Filter and store the main stock in a refrigerator, keeping a small cjuantity in a dropper bottle in the laboratory for immediate use. Some workers omit the iron salt and incorporate the correct amount of iron from the reaction between steel dissecting needles and the acetic acid in tlie dye. This requires considerable experience. Excessive iron prevents clear differentiation and may produce a dark grantdar precipitate. This can be minimized by clean- ing the steel needles in -ib'/i acetic acid periodically during dissection. If iron is added to the dye formula, use nickel or chromium plated needles or pyrex needles drawn to an abrupt, fine point. The simplest method of using acetocarmine consists of macerating or smearing a fresh anther in a drop of the stain. Small anthers may be dropped into the dye entire, then dissected under a binocular, discarding pieces of anther wall and leaving only the masses of sporo- cytes. Large anthers shoidd be sliced into the thiiniest possible slices on a sheet of wet l^lotiing paper, the fragments transferred to a drop of dve and macerated as above. Lower a cover glass over the drop of dye containing the sporocvtes, and press or tap gently. A carefid sliding motion on the (()\er glass sometimes aids in smearing the cells into a thin film. Pass tlie slide qincklv o\er an alcohol lamp llame several times. Ihe amount of heatinu iiuisi be determined bv trial, l)ut do not luai lo the boilin" j)oini. Diain oil excess stain, and seal tlu' edges ol the coxer glass will paraffin. Examine the slides with a microscope, and stcire satis- factory ones in a refrigerator. I'lic coloi impioxcs in a lew clays, readies maxiimmi intensiix. then begins to dctc i ioi ate. Eresh anthers are not available at all times, and it is often neces- sary to make extensive collections in the field lot subsecjuent study. Kill entire anthers, or grass spikelets, or e\cii large portions of an inlloicscciuc in Farmer's oi- (".arno\"s lluid. Mai/e cytc^logists prefer an alccjhol-acid latio ol j:l, but ratios ol 1:1, 2:1 etc. may The Preparation of Whole Mounts and Smears 107 be tested until the optimum fixation is obtained for a given species or stage. Plant materials can be stored in these fluids for months at 0°C., depending on specific response. Most workers transfer the tissues after 1 or 2 days of fixation to 70% alcohol, in which tissues can be stored for many months in a refrigerator. Make the smears as with fresh material. Root tip smears are being used extensively for chromosome counts and for the study of the morphology of metaphase chromosomes. The principal problem is to obtain separation and flattening of cells. Some large root tips, such as onion, may be squashed flat easily, whereas the small root tips of sweet clover resist crushing. Fix the root tips in Farmer's or Carnoy's fluid for at least one day. Smear directly from the fixing fluid, or transfer to 70% alcohol for prolonged storage. The responses of a species to the fixing formula, the fixing interval and the permissible storage period in fixing fluid or 70% alcohol, must be determined by experimentation. The piece of root tip that is fixed is usually at least three times as long as the most actively meristematic region. Cut off: this active end into a drop of acetocarmine, and macerate into linear strips with needles. Some materials are benefited by some heating at this stage. Cover and heat carefully. Press or smear the cover until the cells are sufficiently separated and flattened. Chromosome groups tend to become clumped with the above simple treatment. This defeats the main goal of the smear method, the rapid and positive counting of chromosome complements. Clump- ing can be prevented, and some morphological details accentuated by pre-treatment of the living root tips. Soak the tips in a saturated aqueous solution of paradichlorobenzene for 1 to 4 hours and fix in the preferred formula. Methyl alcohol, in concentrations of 1% to .3% in water has also been used for pre-treatment and found to yield spread-out chromosome complements (Dr. J. G. O'Mara) . There are unlimited opportunities for testing other benzene derivati\es. and unrelated compounds in the smear method. The separation of cells can be improved by hydrolyzing the middle lamella with 5 to 10%, HCl. The acid may be in water, or 70% alcohol, or in Farmer's fluid. After immersion in the acid for 5 to 30 min., return the roots to fixative, which should be changed at least once. Enzymes also have been suggested for digesting the middle lamella. Several readily available plants, especially Rhoeo and Trades- cant ia. yield excellent sporocyte smears that show the coiled chromo- 708 Botanical Microtechnique nemata (Sax and Humphrey) . Make an acetocarniine smear of one anther ot a flower to determine the stage ot meiosis. \\'hen the desired stage is found, smear the sporocytes from a fresh anther on a per- fectly clean slide. Immerse the slide, or flood the smear with ammonia pre-treatment solution. This contains 1 % (concentrated) ammonium hydroxide in S()^'( alcohol. Try pre-treatments of 10 to 30 seconds. Drain, add a drop of acetocarniine and proceed as ^vith ordinary smears. Smear preparations can be made permanent. If the cells were smeared on clean glass, they will adhere during the subsequent treat- ment. Many versions of the method may be foimd in the literature. Essentially, the process consists of dehydrating the smear and mount- ing in balsam or resin. Ihe schedule used by Sears is one of the simplest. Slip the cover glass by immersing the slide in equal Aolmnes of acetic acid and alcohol. One effective method is to in\ert the slide in a Petri dish, with one end of the slide held up on a piece of glass rod. The coxer glass slides off gently without dislodging the smear. Pass the slide and its cover glass through the following fluids at 2-5 min. intervals: (1) eqiiiil volumes of etliyl and tertiary i)ulyl akoliol (2) tertiary butyl alcohol, 3 changes Place the slide with the smear upward on blotting paper, place a drop of thin balsam or resin on the smear, and lower the cover glass carefidly. Place a lead weight on the cover glass. A similar acetic acid-ethyl alcohol series can be used (McClintock) but more intermediate gradations must be used to prevent collapse of the sporocytes. The advanced xvorker can (ind additional deiails in ihe (oin- prehensive review of acetocarniine methods by Smith (1947) , Avliose bibli()gra])hy also gives citations of the workers xvhose names appear in thr ])rcs(ni chapter. MACERATION Whole inounis or sections of tissue fie(]ueiuly do not convey an adequate tlnce-dinunsional iinpicssion ol (cll sirutiure. A xaluablc and much-neglected type of preparation is made by isolating com- plete individual cells from a mass of tisstie. This is accomplished by chemical and medianical sejiaration oi cells. 1 he separation takes place along the middle lamella. Preliminai \ treatment of the tissues is the same, regardless of the subseijuent maceration method. If the The Preparation of Whole Mounts and Smears 109 material is dry, boil it in water containing a wetting agent uniil thor- oughly saturated, jiiniiping with an aspirator it necessary. Divide the material into slivers no thicker than a toothpick. Treat with one oi the following macerating processes. Schultze's Metliod.~Co\er the material with concentrated nitric acid. Add a few crystals of potassium chlorate. Heat gently on a sand bath, in a closed h(K>d, until the material is bleached white. Wash thoroughly, and shake with glass l^eads until the material dis- integrates. increase or decrease the duration of heating until entire unbroken cells can be isolated. Jeffrey's Metliod.— The macerating fluid consists of equal volumes of 10% chromic acid and 10%- nitric acid. Treat for 1 to 2 days at 30 to 40°C. Wash and shake with glass beads. Harlow's Method.— Treat the subdivided and lx)iled material in chlorine water for 2 hr. Wash in water. Boil in 3% sodium sulphite for 15 min. Wash and macerate. Repeat chlorination and the sulphite bath if necessary. Following any of these maceration treatments, wash the pulp thor- oughly by decantation. A centrifuge is an aid in washing. Unstained material may be mounted in water or glycerin for study. The cells may be lightly stained in aqueous safranin, washed by decantation or centrifuging, and mounted in water. Tlie mounting media de- scribed on page 102 may be used to make semipermanent slides, or the dioxan or butyl alcohol methods can be used to make permanent slides. //. Criteria of Successful Processing W'c ha\c carried to completion the processing of some plant ma- terials and have the finished slides before us. Our notebook contains a record of the entire process, from collecting the living plants to the completion of staining. We can now examine the slides critically and consider the criteria by which we may judge the success or failure of the processing. The severity of our scrutiny depends on the objectives of the stud) for which the preparations were made. We may be primarily interested in the distribution of tissue systems or the position of organs, j^aying little attention to the protoplasm. Rapid and convenient methods that preserve the desired structures with adequate accuracy need not be regarded as slovenly. Another study may require the preservation of the constituents of the proto- plasm in their normal structure and position. The use of more elaborate, time-consuming jjrocesses is then justified. It is axiomatic that slides for elementary students must be nuich more perfect than for advanced workers. Beginners waste much time puzzling over imperfections. They will draw faithfully a break pro- duced in microtoming, the gap between the cell wall and the col- lapsed plasma membrane, or a sjjeck of debris in the tissues. Tlie j)athologist should be especially critical. Control slides of noinial tissues nuist show cellular details with almost diagrammatic perfection in order to furnish a basis for interpreting pathological conditions. Studies in physiology, (violog). and experimental mor- phology also demand careful control of processing and critical exam- ination of slides on the basis of some useful criteria. The following ilhisi rat ions Avere selected Irom well-known and widely used subjects. Leaves of different species exhibit different re- actions to killing fluids. The quality of the fixation can be determined easily by the condition of the j^alisade cells. The outlines of the cells should not be wrinkled, and the chloro})lasts should line the walls. Figure ll.l a shows the excellent preservation of cellular and tissue detaiK in soybean leaf, a highly spongy and therefore dilficult subject, processed as oiulined in the legend. [ 110] Criteria of Successful Processing 111 fQS I Li:' J LA'S! ^ 1 ©\^^l^^^# ^rw Fig. 11.1— Comparison of fixation in leaves: a, soybean, Ciaf III, acetone-tertiary butvi alcohol: h, cherrv, turgid condition, Craf II, alcohol-xylene; r, cherry, plas- molyzed, lAA, alcohol-x)lene; d, red ash, plasmolyzcd, FAA, alcohol-xylene. . A Striking illustration of the effects of different killing fluids is afforded by experiments with young leaves of cherry. Figure 11.1 c shows the severe plasmolysis obtained by killing in FAA. Figure 11.1 h shows the turgid, normal condition of the palisade and spongy parenchyma, following killing in a Nawaschin type formula, Craf II. After killing, both lots were processed simultaneously by identical methods, an alcohol-xylene series and careful infiltration in paraffin. Stems are subject to the same defects as the leaves described above. Study Fig. 11.2 a, a cross section of alfalfa stem. Note that the deli- cate hypodermal chlorenchyma is intact, the cells have smooth, rounded outlines, and there is no marked cleavage between cells. Within each chlorenchyma cell the plasma membrane and the chloro- 112 Botanical Microtechnique Fig. 1 1 .li— C;oinj);ii isoii ol lixalion in alfalfa stems: a, good fixation with C.raf 0.20- 1.0-10.0, dioxan process; b, plasmolyzcd cells in cortical region, Craf 0.30-1.0-10.0, acetonc-7!-biitvl alcohol process; c, good fixation, hnt with peeling epidermis. ])rocess as in a. plasts are appre.ssed aj^aiiist the cell wall, iiulitatin^ ahsciue ol jilasmolysis. The physical ajjpearaiuf ol these cells may be regarded as approximating the stnuiurf ol li\ing ttlls. ()bser\e liiiilur that the epidermis is not |)eeled a\\;i\ liotii the iiiiui tissues. These features ai( iiidicati\e ol siiccesslid processing. in comparison -with tlu' ;il)ove case, examine Fig. 11.2/^ also a cross section ol allaUa stem, killed in FAA and carried through an alcohol-xylene series. Ihere is marked plasmolysis; tlu- massed chloro- plasts and cytoplasm absorb the stain and cannot be destained diller- Criteria of Successful Processing 713 cndally, producing harsh stainino; effects. Note especially the collapsed plasma membranes in the epidermis and chlorenchyma. This em- bedded material was brittle and only the pieces from the upper three internodes could be cut without serious tearing. Figure 11.2r is from a third batch of alfalfa stem which was proc- essed exactly like the material from which Fig. 11.2a was obtained. The structure of the deeper cortical cells is well preserved, but the epidermis shows extensive peeling away from the chlorenchyma. The condition of the protoplasts indicates that the killing fluid and sub- sequent processing were not at fault. It is probable that the peeling of the epidermis was caused by compression of the extremely soft hypodermal tissues when the fresh stem was cut into pieces for killing. Roots are somewhat more difficult to judge than organs containing chloroplasts. Root cells that contain leucoplasts should be examined for the position of these plastids. If the plastids are inconspicuous, the condition of the thin plasma membrane will indicate whether plas- molysis has occurred. The critic must be much more lenient in the examination of sec- tions of wood. Boiling the wood in water, desilicification with hydro- fluoric acid, and infiltration under pressure for long periods are not conducive to preservation of protoplasmic or even cell-wall details. Mechanical tearing can be easily recognized. In the nonliving elements (tracheae and tracheids) the concentric layers of the thick wall should not be separated. The innermost layer is often found to be completely separated in poor preparations. Parenchymatous elements, such as wood rays, diffuse wood parenchyma, and epithelial cells of resin canals, are subject to plasmolysis. Perfect fixation of these paren- chymatous cells shotdd not be expected. The embryo sac of lily is a difficult subject that tests the effective- ness of a method and the skill of the worker. The young megasporo- cyte of lily is comparatively easy to preserve in good condition. Such fluids as chrome-acetic and Bouin's, followed by the traditional alco- hol-xylene series, yield excellent preparations and a good ratio of well-preserved sporocytes. The sporocyte and integument initials in Fig. 11.3 are preserved very well indeed for routine class material. With continued enlargement of the sporocyte the cytoplasm prob- ably becomes highly fluid, the integuments elongate as thin sheets of tissue, and these structures become increasingly subject to damage. Look for evidence of plasmolysis of the sporocyte and wrinkling of the rims of the integuments. In Fig. 11.4rt the finely granular cyto- plasm is obviously not shrunken, and the rounded rims of the integu- 7 74 Botanical Microtechnique Fi(.. 1 1.3— C()iii|);ii isc.ii of fixalion in \()iing oxiilcs of f. ilium: a. I.. sl>cri().siiiii . chroiiie- acftic (().5-0.'i) IS hr., akoluilwlciie: h. L. tiiiini . Allcn-Bouin III, 1 davs. ihice- gradc (lioxan; c, L. speciosiDii. cliromc-arelic (0.3-0.")) IS In., aholiol-wiciK': .. V«S^ £»w;^vfto ■■■ /•-'-■ ' -J ■.*•■, -''V 'Xty ■^•v^-- m-y^ WTt' S-&^J ■v:. V-7-. <-. -( A ,i.N 5^^ Fig. 13.2-Root tip meristems and histogens. a, b, Zea mays: c, Melilotus alba: d. Lotus tenuis, chromosome complement, iion-hematoxvlin. A good dicotyledonous stem growing point can be obtained from young seedlings of Lima beans, kidney beans, soybeans, flax and peas, sprouted in sphagnum or sand. Lima and kidney beans have a large epicotyl of simple structure at the postdormant stage, after the seed coat has been ruptured but the plumule has not yet emerged. Peel off the seed coat, separate the cotyledons, and remove the epicotyl and radicle (Fig. 13.1 5, C) . Good fixation can be obtained with Graf II 126 Botanical Microtechnique loUowccl b\ the acetone-xylene series. Cut the jmraffin sections at right angles to the flat, overlapping phniuile leaves. Stain as recommended lor corn seedling. The median section will show a ■- I '% li(.. l.S.:?-Vc'<4Clati\c and ll<«ial apices ot sttins; a. /.id. \ri;clali\c. 1 neck alUM ciiKM-c'iur: h. V.va. lassrl pi iuu.ulia. 7 weeks allcr enuMgence; r. Bromus incini.s. \])iil; (/. liiniiii. plimuilai hiul of sccdlinj^f. inllorcscciuf pninoKlia in Vegetative Organs of Vascular Plants 127 broad apical meristeiii, two siiiall leal pi iinordia, and tragmcnts ol the folded plumule leaves. The radicle can be used for histological or cytological preparations of the growing point. Peas show a more advanced condition at a corresponding stage of germination. The pea bud is perfectly glabrous. Sprouts showing axillary bud primordia are obtained when the sprout has emerged from the seed in the form of a loop (Fig. \S.\ D) . Increasing complexity de\elops rapidly as the sprout becomes straight. The epicotyl in sprouting soybean is more advanced in organi- zation than in beans or peas. Extract the soybean bud from the burst seed. For an older stage, permit the epicotyl to elongate until the tips of the plumule leaves just protrude beyond the cotyledons. Remove the cotyledons, pull the plumule leaves apart, and remove the entire bud (Fig. 13.1 A-G). Soybean buds are pubescent and must be pumped with an aspirator until they sink in the killing fluid. Large multicellular hairs in the axils of the leaf primordia are easily mistaken for axillary buds by elementary students. The bud is a desirable item for advanced teaching (Fig. 13.4^). The growing point of the flax seedling is glabrous and very simple in organization (Fig. 13.3 d) . When the appressed cotyledons of the seedling have begun to diverge, make a transverse cut 1 nnn. below the cotyledonary node and another cut 2 mm. above the node. The cotyledons serve as a guide to orientation in the microtome. Section at right angles to the flat sides of the cotyledons. Axillary buds of Coleiis, tomato, and other herbaceous plants or the buds from potato eyes also are desirable subjects. Before dropping buds of this type into' the killing fluid, it is best to dissect away some of the outer bud scales. Uniformly good fixation of buds of the above legumes and other recommended herbaceous plants has been obtained with Craf II and the dioxan method, the ethyl-normal butyl series or the dioxan-normal butyl series. Some good results, with occasional unexplained failures, have been obtained with dioxan alone. Buds of trees and shrubs collected at different seasons show the initiation of leaves and flowers, or the dormant condition. Expanding spring buds of the maples, basswood {Tilia glabra) , and tulip poplar (Liriodendron tulipifera) are recommended. In the shrubs, lilac (Syringa) , honeysuckle [Lome era) , and elderberry (Sambiicus) are excellent subjects. Remove the buds from the twig as shown in Fig. 13.1 H-J. Slice off a longitudinal slice from each side, peel off some of the tougher outer scales under a magnifier, drop into the killing 128 Botanical Microtechnique 0-; Fig. 13.4— Growing poinis of .stems: i(la: h. siciii of alfalfa. Mcdicaoo sathui. The Stem J he tc(liiiic|iKs usee! loi ihc proccssint^ ot sttnis ian<;c Ironi the foiei^oint^ iiuthocls used lor ihe delitaie nieristeiuai i( li]) to the rather drastic and api^arentK ertide nieihods necessary to make slides of seasoned hiniber. The ])()riion ol stem to he selected lor sectioning depends on the decree ol dilkixiitial ion ihat is lo he ileinonstrated. e*^ '^S.Cb ' Fig. 13.7-fl. Stem of alsike do\er. TrifGlium Inbrkhnn: b, stem of tomato, Lycopcrsicu tu esculeiitinn. Vegetative Organs of Vascular Plants 133 MONOCOTYLEDONOUS STEMS It is convenient to discuss first the monocotyledonous stem because these stems reach a climax of differentiation in one growing season and do not present the problems raised by the secondary growth of dicotyledonous and gymnosperm stems. Maize may well be used as the standard subject for the grass stem. Complete transverse pieces of seedlings will show the overlapping whorls of leaves encircling the stem. Nodal pieces show the axillary buds, the potential ears. From the older plants use only the internodal pieces of stem, stripping away the leaves. A pot-grown plant will become fairly well lignified and yet be so small that a complete cross section, or at least a quarter sector, can be placed on a slide. However, such plants give an inaccurate picture of the number and structure of the bundles. To show the well-developed and lignified bundle sheath and cortex or rind, use large, field-grown plants at about the time of pollination. Cut the stem into short disks, and divide each disk longitudinally. Young stems collected before the internodes have become exposed should be killed in a mild fluid like Craf II. Mature stems must be killed in FAA and pumped until they sink. The dry, air-filled pith is difficult to infiltrate; it is therefore desirable to exhaust again in the anhydrous stage of dehydration. The use of normal or tertiary butyl alcohol permits paraffin embedding of all but the toughest stems, which must be cut in celloidin (Fig. 13.5) . Transverse and longi- tudinal sections of corn stem take a brilliant safranin-fast green stain. The hemalum-safranin combination is the second choice. Iron hematoxylin-safranin is used only if the middle lamella is to be emphasized. Other important plants that illustrate the large grass type of stem are sugar cane and sorghum. Wheat, oats, and other small grains and field grasses illustrate the small hollow culm. The most easily available grass rhizome is that of quack grass, Agropyron repens. The hydro- phytic monocots have interesting culms and rhizomes. Species of Carex having triangular stems, as well as round-stemmed species, and the cat tail, Typha, should not be overlooked. These subjects can be prepared by the methods outlined for maize. Monocot stems of the nongramineous type may be obtained from several easily available plants. The trailing Zebrina grown in green- houses has a soft stem that can be sectioned in parafi:in. Asparagus Fig.. 13.8— a. Stem of hemp. Cannabis saliva; b, stem of basswood, Tilia. 134 Botanical Microtechnique sprengeri, an important j^lani in the florist trade, has a thin woody stem. The younger portions near the tip can be cut in paraffin, the old woody stems must be cut in celloidin. AVild species of Smilax have a woody stem. Kill the woody stems of Asparagus and Smilax in FA A, and cut in celloidin only if a sample embedded in paraffin cannot be cut. DICOTYLEDONOUS AND CONIFEROUS STEMS The apical meristcmatic regions of dicot stems have been discussed in the section on apical meristems. The tissue systems of these stems differentiate very rapidly close to the apex, and the first few inter- nodes below the terminal bud show the fully developed primary tissues and the initiation of secondary activity. A convenient though artificial and arbitrary classification of stem types is in common use. Herbaceous stems develop comparatively little secondai7 wood, and, if a complete cylinder of wood is produced, it is laid down late in the growth period. Woody stems begin the formation of a complete cylinder of secondary wood early in the season and produce an extensive cylinder of highly lignified xylem. Every possible gradation of woodiness between these two types may be found in the plants about us. The following examples are recommended either because they are of economic importance or because they present some structural feattire of fundamental importance. Plants that can be grown quickly in pots are convenient subjects for the herbaceous stem. Plants of kidney beans, peas, and soybeans attain usable size in a short time. Actively growing field materials are the best source for sweet clover, alsike clover, and alfalfa (Fig. 13.7) . Any of these legume stems can be killed in Craf III. The softer internodes can be carried through an accione-xylene or alcohol-xylcne series. The harder stems, esiiecially soybean, cut better after dioxan or the butyl alcohols (Fig. 13.6). The cultivated sunflower, Hclidiilhtis,, and Chrysanilirmtiiii are good representatives of the Compositae. The common fleabane, Erigeron, is a suitable native subject in iliis family. The above stems seem to withstand the dehydrating action of FAA without marked plasmolysis, and a strong Nawaschin modification like Craf I\' or V is satisfactory. The butyl alcohol process is reconnninded for these rather tough stems. Bicollateral bundles are characteristic of the Cucurbitaceae and Solanaccae (Fig. 13.7). Important members of these families can be obtained easily. Seedlings of scjuash, pumpkin, or melon grow rapidly Vegefafive Organs of Vascular Plants 135 and furnish long hypocotyls as well as epicotyl materials. Do not use FAA; kill in Craf II, and use alcohol-xylene for tender stems and butyl alcohol for tough ones. Tomato and tobacco seedlings grow slowly, but they are almost indispensable subjects. Potato plants are easily grown from tubers. Stems of these plants are not killed properly by FAA but are preserved with excellent cellular detail in Craf II. Old, tough stems of tomato and tobacco must be processed in TBA or sectioned in celloidin. Small potato tubers, 3 to 6 mm. in diameter are easy to section. Kill in Craf I, and embed in paraffin by a slow, closely graded process. Longitudinal sections show that the tuber is a stem with an apical meristem which produces leaf primordia. Medullary bundles and anomalous cambial activity occur in the Chenopodiaceae. The common weed Chenopodium album is probably the most readily available representative. Several related weeds are equally interesting. Kill in FAA or Craf III, and process in butyl alcohol or dioxan. The foregoing methods recommended for specific herbaceous stems can be used with an extensive range of plants in many species of economic importance or academic interest. For instance, commercial fibers of primary and secondary derivation can be illustrated with the stem of Cannabis saliva (Fig. 13.8). As a broad general recommen- dation, use a mild chrome-acetic-formalin on tender materials, and process in alcohol-xylene or acetone-xylene. For moderately hard stems use Craf III, and for very hard stems use FAA, followed by dehydration and infiltration in dioxan or butyl alcohol. The bush fruits like raspberry, blackberry, currant, gooseberry, and other plants having similar semiwoody stems may be handled like herbaceous stems while in the tender growing stages, but they eventually become too hard to process by the foregoing methods. Such hard materials usually must be handled like woody stems, as described in the following pages. For the study of twigs of woody plants, material collected during the winter has some advantages. The previous season's xylem is fully lignified, secondary phloem is fully matured and firm, the cambium is clearly distinguishable as a layer immediately adjacent to the wood, and the cambium does not slip readily. However, if the development of cambial derivatives is to be studied, stems must be collected at intervals during the growing season. Such materials must be processed with greater care than dormant stems. Twigs should be taken to the laboratory promptly and cut into short pieces for killing as described in Chap. 2. 736 Botanical Microtechnique Many species of forest, orchard, and shade trees make excellent preparations for the study of young woody stems. The basswood, Tilia (Fig. 13.8 b) , has become a great favorite, but there is no advan- tage in studying basswood in a region where it is not native. Species of Populiis, Fraxinus, and Acer are easily sectioned. The apple and other fruit trees have been neglected as class materials, although they are easy to section. Tougher woods like oak, hickory, or locust are much more difficult to cut, and complete j^erfect sections are not obtained with such certainty. The standard coniferous subjects are the white pines, Finns strobus in the east, and F. lambertiana or F. flexilis and several other five-needle pines in the west. These are representative of the five-needle or soft pines. For the hard pine type many more species are a\ ailable, such as several species of yellow pine, the scrub pines, and jack pines. There is not much choice among the numerous two- and three-needle hard pines. The principal American genera of conifers should be represented in a comprehensive stock of slides. Some of these trees are used as ornamentals, the commonest ones being Abies, Larix, Tsiiga, Thuja, Ficea, Fseudotsuga, and Jnniperus. Shrubby conifers are among the commonest ornamentals, and specimens of shrubby species in the genera Juniperus, Thuja, and Taxus are readily available. The methods of handling the woody dicots and coniferous stems are decidedly stereotyped. The subdividing of such materials is illustrated in Fig. 2.2. The impermeability of the cork on woody twigs necessitates the use of a fluid of good penetrating powers, and FAA has long been the standard fluid. Stems may be left in this fluid for years. Preserved stems can be rinsed in several changes of 70% alcohol at 1-day intervals and sectioned without embedding. The celloidin method is recommended because of the ease and certainty of attaining high productivity by (juantity production methods. Woody stems having bark tissues are usually stained with the combinations recommended for herbaceous stems. Hemalum-safranin, safranin-fast green, and safranin-aniline blue have become standard stains. The method of handling sections and the staining jirocesses are described in Chap. 8. Transverse, radial, and tangential sections of the cambial region of woody i)lants make instructive j)rej)arations that are indispensable for a critical study of the three-dimensional aspects of cambium, the mechanism of abscission, and the structure of developing and mature elements of the xylem and piiloem. 1 he excessive use of transverse sections and the neglect of longitudinal sections build up an in- Vegetative Organs of Vascular Plants 137 complete or even incorrect picture of the woody stem in the mind of the student. Twigs are not satisfactory for making longitudinal sections in quantities. Unembedded twigs cannot be held in the microtome horizontally for longitudinal sections. If an embedded and blocked twig is sectioned longitudinally, only the outermost sections are strictly tangential, and only a few slices from the center are true radial sections, cut parallel to a ray. For first-class preparations cut accurately on the three desired planes, use blocks of wood and attached bark removed from living trees as illustrated in Fig. 2.2. Sectioning of such blocks is quite impossible without embedding in celloidin; whereas, with the celloidin method, perfect sections can be produced in quantities (Fig. 13.9 6, c). Collect the material in the winter when the cambium is firm. Soft wood like basswood, white pine, apple, or silver maple can be cut without special softening. Kill in FAA, and embed in celloidin. Hard woods like oak or locust must be treated with hydrofluoric acid after killing and hardening in FAA. The protoplasts cannot be expected to be in perfect condition after treatment in HF. The process is described in Chap. 8. Fig. 13.9— Illustrations of material cut l)y the celloidin method: a, cross section of apple graft union; b, sapwood region of sector from 20-year-old trunk of Tilia, sections in three planes; c, sections from approximately 40-year-old trunk of apple tree. All subjects killed in FAA. The Tilia stem was infiltrated in Cellosolve solution of celloidin. 138 Botanical Microtechnique The desirability of using choice sections showing the bark in three planes cannot be overemphasized. In addition to serving as supple- mentary class material for studying the structure and development of the stem, such preparations serve as reference material for research, especially for pathological studies. Even in wood technology, in w^hich the work is largely confined to the microscopic structure of the ^\ood, preparations showing the cambium, phloem, cortex, and periderm are a valuable supplement. Seasoned lumber is frequently used as a source of material for slides, and excellent preparations can be made from such material. However, parenchymatous elements such as xylem parenchyma and the epithelial cells of resin canals are collapsed and distorted. The preparations are adequate for diagnostic purposes and for the study of nonliving elements of the xylem. For best results, use properly seasoned wood and prepare the blocks so that sections can be cut accurately along the three conventional planes as described in Chap. 2. The specialized sectioning methods necessary for dry or hard -woods are described on ))ages 84-85. The Root The processing of roots of seed plants lor anatomical study is similar to the methods used for stems. The meristematic root tip is usually prepared by careful cytological methods; sections max then be stained either with a cytological stain for nuclear struct ines or stained with some histological (ombination. Batches of rcjot tips that do not have abundant mitotic figures are usually set aside for histological preparations. The methods of obtaining root tips are described in Chap. 9. 1 lu' histogens of roots are evident at the root ti]), especially if the preparation is stained to show cell walls as xvcll as nuclei. Ihe primary tissues are evident at the beginning of the root-hair zone, where the emerging root hairs can be delected \viih a hand lens. Initiation of lateral root primordia can be demonstrated at tlie upper limits of the root-hair /one. wluie the old tool haiis arc beginning to collapse. At this icxcl tlie j)rimary tissues are usually clearly differentiated, without being excessively woody. Favorable subjects h)i ilhisii atiiig the monocot root are maize and /is JHiitii^iis i)fii< inali.s. [Uv garden asparagus. Germinate corn ni Fir,. 13.10— fl. Transverse section of root of Aspnrni^ii.s ofjirinalis showing initiation of laicra! root: /', i)racc root of Zca. 140 Botanical Microtechnique sphagnum (not in sand!) , and remove pieces of root from the desired region. Roots grown in a moist chamber or water culture have excessively spongy, fragile cortical parenchyma, unlike the structure found in plants in a more normal environment. Tissues of the tip, the hair zone, and for some distance above, are fixed well in Craf II; the older tough roots must be killed in FA A. The brace roots from field-grown plants may also be used (Fig. 13.10) . Roots of sugar cane, sorghum, and the small grains are processed by the above methods. Asparagus roots are obtainable readily from volunteer seedlings that occur in the vicinity of asparagus beds. The softer portions of the root, within 3 cm. of the tip, are killed in Craf II, but older roots that have an impermeable hypodermis and endodemiis must be killed in FAA. The butyl alcohol method is suggested for the harder pieces (Fig. 13.10). Acorns calamus is almost a classical subject for the monocot root. This water plant is abundant in suitable locations, but material is often inaccessible, and the root has no advantages over Asparagus. The processing methods are identical for these two plants. Srnilax hispida root has a remarkably thickened endodcrmis, Avith laminated cell walls impregnated with brown coloring matter. Kill the roots in FAA, and try a batch with the butyl alcohol method, using celloidin if sections cannot be cut in paraflin. Safranin-fast green gives a brilliant contrast in which the prominent endodermis is reddish brown. Young dicotyledonous roots are obtained readily from the large- seeded legumes, beans, peas, soybeans, and especially the horse bean, Vicia faba. The early stages, including the emergence of lateral roots, can be obtained from roots grown in a moist chamber of sphagnum, l^hese roots do not become excessively spongy when grown in this manner. The older roots showing extensive secondary growth must be taken from plants grown in soil. Soybean and horse bean shoidd be killed in Craf III. Young roots of the apple are particularly interesting because of the proniiiunt Casparian strips in the endodermis. Material is not so easy to obtain as with plants that can be grown (luicklv from seed. Volunteer seedlings ol aj)ple can be dug uj) (arelully and abundant roots ol \arious ages obtained. Kill the yoinigest roots in Ci;ir ill and woody loots in F.Lt. Trial batches of older roots may Tu.. nil-Tiansvcise sections of leaf of Zea: a. photoG;rnphcd at 1 IX with 18mm. Micro lessar. leprodiiced at 2S\: b, detail of uicliome and blade. 742 Botanical Microtechnique well be processed with tertiary butyl alcohol, and any age classes that cannot be cut in paraflin must be cut unembedded or in celloidin. Flax root has a simple diarch stele. Roots can be obtained easily by germinating seeds in blotting paper. Radish, mustard, cabbage, and many other roots also may be grown in this manner to adequate size for primary tissues and processed by the methods given for apple. Ranuncidiis root has long been popular as an example of the dicot root. The large fleshy roots of the buttercups, R. septe^itrionalis and R. fascicularis, are easy to obtain and to process, using the methods given for apple. The buttercup root is less likely to be of interest to the student than the roots of economic plants. Large taproots like those of alfalfa, Medicago, and sweet clover, Melilotus, are handled like the older semiwoody roots of apple. The tough, woody stems, rhizomes, and roots of ferns, horsetails and club mosses are most con\ eniently discussed at this point because they are handled like other woody materials. Collect the rhizomes of ferns in the spring, just after the fronds ha\e fidly expanded. Acceptable preservation of rhizomes can be obtained with FAA, and it is not improbable that for investigational work this formula could be adjusted to give good fixation with given species. For routine preparation of many species, uniformly good results have been obtained with Graf II. The subjects become very brittle after xylene, but very large rhizomes can be cut readily after 7?-butyl or tertiary butyl alcohols. It has been customary to embed hard rhizomes like those of Pteris aquHina in celloidin. However, the TBA process is satisfactory for portions of the rhizome that are not excessively hard, but have the woody structures adequately lignified to show the mature condition of tissues. The most brilliant and satisfactory stain is safranin with fast green. The presence of yellow deposits in the cells prodiues undesirable staining effects with the hematoxylins. The fleshy root of Botrychium is recommended. 1 csts with B. virginianum have shown iliai (.lal 1 gives much better fixation than does FAA. A very striking color contrast is obtained with safranin-fast green. Roots of the Boston fern and of available native ferns are processed as above. Species of Lycopodium occur in a])undance in some regions, and some species, especially troj)ical ones, are culti\ated in conservatories. Stems may be fixed in FAA and carried through tertiary butyl alcohol to paraffin. It is usually necessary to soak the nu)unied specimen in warm water before sectioning. Roots are easy to process successfully by the same methods. Vegetative Organs of Vascular Plants 743 SclagineUa has highly localized distribution, but excellent preserved material is obtainable from dealers, and several species are extensively cultivated in greenhouses. The processing is the same as for Lyco podium, the infiltration must be slow and thorough, because the stele is literally suspended in a highly parenchymatous cylinder and is easily torn in cutting. The vegetative organs of Isoetes are studied only in advanced work in anatomy, and there is comparatively little demand for slides. Roots should be severed and divided into short pieces. The compact stem and rhizophore may be processed entire or quartered. The methods used for Lycopodiiim are satisfactory. The highly silicified stem of Equisetiim has long been a problem for technicians. Penetration is diflicult with an aqueous killing fluid, but FAA is satisfactory. The older stems must be desilicified by treating with hydrofluoric acid. Transfer directly from FAA to the acid diluted with twice its volume of 95 'v alcohol. After 2 days in acid, wash in 50% alcohol, making at least five changes at 4-hr. intervals. Observe the precautions concerning the use of HF given in Chap. 8. Dehydrate in TEA, and infiltrate slowly and thoroughly. Rhizomes and roots do not need to be desilicified, otherwise the processing is the same as for aerial stems. The Leaf The mesophytic dicotyledonous broad leaf is the type most commonly used for the study of the so-called typical leaf. Some general directions apply for the handling of most types of leaves. Leaves are easily damaged during processing by apparently minor mishaps. It is therefore desirable to kill duplicate batches in each of the formulas used, keeping one batch in the preserving fluid while the other one is embedded and tested. Consult Fig. 2.1 concerning the usual methods of subdividing leaves. Good results can be obtained with many leaves by killing in FAA. Soft leaves having small veins can be dehydrated in acetone or ethyl alcohol, whereas leathery leaves, or leaves with thick or wiry veins should be processed in butyl alcohol or dioxan. One batch of each subject may well be killed in FAA and another batch in one of the fluids given in the following specific recommendations. The firm leaves of the trees and shrubs are represented by apple, cherry, rose, lilac, and privet. Leaves of apple and related plans may be killed in FAA, but occasional batches exhibit considerable plasmolysis (Fig. 11.1). Consistently good results can be obtained 744 Botanical Microtechnique with Craf II and TBA dehydration. The latter reagent minimizes the brittleness of these subjects. A disadvantage of the rosaceous leaf is the presence of excessive brown pigmentation in the cell walls and masses of yellow gummy materials in the cells. The embedded pieces of leaf in the paraffin block are decidedly dark, and the staining effects tend to be muddy, especially with the hematoxylins. The use of a safranin-fast green or safranin-aniline blue combination makes slides with fairly clean color contrasts. Lilac and privet leaves can be processed by the above methods. Many other trees and shrubs have leaves in this firm-textured category. Geranium leaf is firm and easy to process. Craf V gives excellent results. Do not use pieces with large veins unless TBA is used for dehydration. Leaves of softer character than the foregoing are illustrated by various easily obtainable legumes. Kidney bean, soybean, clovers, and alfalfa have more or less pubescent leaves; peas and horse bean have practically glabrous leaves. All of these leaves have been killed success- fully in FAA, but failure occurs often enough to justify more critical methods. Excellent preservation of alfalfa and soybean leaf has been obtained with Craf III followed by an acetone- T5/J series (Fig. 11.1) . The thinness of the cell walls requires a stain of good contrast, such as hemalum, followed by safranin, the xylem stain. The coal-tar dye counterstains are likely to yield weakly stained parenchyma and barely visible plastids. 1 he leaves of the Solanaceae and Cucurbitaceae represent the very tender type of broad leaf. Subdividing of fresh leaves must be done with the greatest care because of the open and fragile construction of the parenchyma. The glandular hairs should also be preserved intact. 1 he best killing is obtained with a mild fluid, such as Craf I. Practically perfect preservation of tobacco leaf has been obtained consistently with this fluid. Although the blade is soft in lea\es of this type, the veins are large and firm, justifying the use of TBA. Begonia leaf is an interesting tender leaf. I'he epidermal cells on both sides are enormous, the two layers occupying more than two- thirds of the thickness of ihe leaf. Ihe narro\v interior layer consists of poorly defined palisade and extremely loose spongy parenchyma. All these interior cells contain chlor()j)hyll; each cell has relati\ely few, but very large chlor()j)lasis. A leaf of this type is obviously difficult to preserve. Good killing lias been obtained with chrome- acetic 0.5-0.5, washed by diffusion in a large volume of water, followed by the ethyl alcohol-xylene series. Cut 15 ^i thick in order to keep the large epidermal cells intact. Coleus is another common Vegetative Organs of Vascular Plants 745 greenhouse plant with soft leaves. They are preserved well by FA A and particularly well by Craf II. The stereotyped construction of the mesophytic leaf permits the use of innumerable species to illustrate the type, making it possible to utilize plants that are readily accessible and characteristic of the region rather than use some classical species as if it had special virtues. Most broad leaves are distinctly dorsiventral, the columnar pali- sade cells being on the upper or ventral side. Leaves that normally assume a vertical position do not have such distinctive palisade cells, the upper and lower tissue zones are nearly alike, and the dorsiven- tralitv is obscured. The garden beet and sugar beet are good examples. These leaves can be fixed successfully in FA A or Craf III. The study of the dicotyledonous leaf would be far from complete without a study of deviations from the typical mesophyte. Perhaps the most striking variations are the xerophytic adaptations. The leaves of species of Dianthus show a range from the relatively large, flat leaves of the greenhouse carnation to the waxy, narrow, rolled leaves of the rock garden species. These easily obtainable leaves are well preserved by Craf II. The tough cuticle becomes brittle after xylene but cuts well after TBA or dioxan. A brilliant stain is obtained with safranin-fast green. Nereum oleander has leaves of unique xerophytic structure. The lower surface is indented with globose cavities or infoldings of the epidermis. Each pocket is lined with numerous hairs and contains many stomates. The upper epidermis is firm and highly cutinized. There are two to three layers of tough thick-walled hypodermal cells below the epidermis, and the deep-seated j)alisade cells are long and narrow. Killing fluids penetrate with difliiculty. FAA is the most rapid of the satisfactory formulas but may cause slight plasmolysis. If the pieces cut transversely out of the fresh leaf are very narrow, not over 1 mm. wide along the linear dimension of the midrib, good penetra- tion and fixation are obtained with Craf 0.30-1.0-5.0. Brittleness in paraffin is minimized by the use of butyl alcohol or dioxan. Safranin- fast green gives a brilliant and highly differential stain. Leaves of citrus fruits are also of the leathery type and have an added interesting feature, the pear-shaped oil glands in the epidermis. The spongy parenchyma is compact and firm, and the palisade cells are small and closely spaced. The impervious character of the surface and compactness of the interior necessitate the use of FAA, which produces acceptable results. If the pieces of leaf are cut very narrow, Craf III produces excellent fixation. 746 Botanical Microtechnique Hedera helix is a remarkably efficient xerophyte that can with- stand severe drought. However, the leaf has no striking structural adaptations; its tissue organization is that of a stereotyped mesophyte. This very fact makes the leaf an interesting subject for comparative studies. Kill in Craf II. The leaf of either of the common rubber plants, Ficus elastica or F. pandurata, is an interesting leathery, latex-bearing leaf. The small, compact epidermal cells are overlaid by a very thick cuticle. Under the upper epidermis there are two layers of large water-storage cells, under which there are two layers of small, short palisade cells. Two layers of compact hypodermal cells occur adjacent to the lower epi- dermis. The spongy parenchyma is ver)' open and is transversed by the prominent latex vessels. The latex does not seem to be preserved in stainable form by FAA as well as by the chromic acid fluids. Excel- lent results are obtainable with chrome-acetic 0.5-0.5 or Craf I. Other illustrations of lactiferous leaves are easily obtainable. The leaf of the ubiquitous dandelion can be preserved in perfect condition by Craf I. Leaves of the common cultivated poinsettia and of the cultivated and native Euphorbias are well preserved by Craf I. These leaves are not brittle and may therefore be put through an akohol- xylene or acetone-xylene series. The succulents have very fleshy leaves that can be preserved well in Craf I and dehydrated carefully in normal butyl alcohol. The tissues are liighly susceptible to damage, and a procedine that pro- duces severe distortion should not be condemned without a repetition of the process. The gramineous leaf is represented by mai/e, sugar cane, sorghum, foxtail, and bluegrass. Corn illustrates well the border ])arcnch\ina of the vascular bundles (Fig. 13.11), but sorghum, and especially sugar cane, have more striking motor cells. Bluegrass is a good repre- sentative of tlu' narrow type with prominent bulUfoyni motor cells along the midrib. Foxtail is intermediate between the very broad and \(ry narrow types. Acceptable j)reservation can be obtained witli FAA, but lor nearly perfect fixation use Craf III. Ihis procedure has l)een repeated many times with corn, ^vlil1 iiniforndy good results. Prior to the introduction ol ilic butyl alcohols and dioxan the older midribs of corn and oilur grass leaves were dillicult to section Avithout considerable breakage, but the use of these reagents has minimized the diduulty. Monocotvledonou^. lea\c^ other than the iirainiutous l\pe shoidd be induded in a comprehensive collection. The leaves of lily repre- Vegetative Organs of Vascular Plants 147 sent the broad flat type. The extremely large stomates are the standard subject for studying sectional views of the stomatc. Kill in Craf II and process by the alcohol-xylene method. The thinness of the cell walls necessitates a contrasting wall stain like hemalum. The stomates are showm with almost diagrammatic clarity. Leaves of Zebrina, Rhoeo, Tradescantia, Polygonatuni hiflonim, and Smilacina race- mosa can be prepared by the above method. The tender, cylindrical, hollow leaf of onion is preserved in excellent condition in Craf I and processed like those of lily. The touoh leaf of Iris is difficult to section. Fair cellular fixation can be obtained with FAA and excellent preservation with Craf I if the pieces are very narrow. The TBA process minimizes brittleness in paraffin. The favorite subjects for the study of coniferous leaves are Pimis strobus, a soft pine, and P. laricio austriaca, P. sylvestris, or other hard pine. Needles collected in the late fall and in winter are very hard and become extremely brittle in the paraffin. The cells contain much granular resinous material which remains in the finished prepa- ration. In July the needles are full grown, with all structural features fully developed, but they are still sufficiently soft to cut readily, and do not have excessive deposits in the cells. Kill in Craf III, and pro- cess in TBA; safranin-fast green yields a beautiful preparation. Longitudinal as w^ell as cross sections should be made. Needles of the spruces (Picea) are also in the tough, wiry category and should be processed like those of pine. The flat type of needle is represented by Douglas fir {Pseudotsuga taxi f olio) , the hemlock (Tsuga) , or the fir (Abies). These may be killed in the fluids recommended above. Although these relatively soft needles can be processed through xylene, dioxan and butyl alco- hol improve the cutting properties. The broad leaf of Ginkgo biloba should not be omitted from a study of the gymnosperm leaf. Collect leaves in July, and kill in Craf III. The leaves of cycads are difficult subjects because of their tough, xerophytic features. Select pinnules that are not fully matured and toughened. Cut transversely into narrow pieces and kill in FAA. The nonalcoholic fluids penetrate poorly, but excellent fixation in small pieces is obtained with Craf II. Xylene renders the tissues very brittle, but tertiary butyl alcohol permits satisfactory sectioning. Fern leaves are readily obtainable from the common Boston fern. Use pinnules that have expanded to maximum size but are still bright. 148 Botanical Microtechnique shiny green. Old leaves contain discoloring deposits in the cells. Kill in FA A or Craf II. Other conservatory or native ferns may be pre- pared by the same methods. The foregoing recommendations dealt with matme leaves. Ad- vanced students are invariably interested in the development of the leaf. The place and mode of origin of leaf primordia and the early stages of leaf development are evident at the growing points of stems, and the processing of suitable materials is discussed in the section dealing with the stem. 1^. Thallophyta and Bryophyta This chapter brings together plants and plant organs that require essentially similar treatment. For instance, a segment o£ mushroom cap, a liverwort thallus and a moss gametophyte may well be prepared for sectioning by identical methods. Filamentous algae, free-floating filamentous fungi and moss prothalli also present similar problems. Fungi that live within tissues of higher plants will be discussed in terms of the responses of the fungus and the host to processing. Algae The most satisfactory method of studying algae is by the use of fresh living material in conjunction with well-preserved bulk mate- rial. Except for some critical cytological features, most of the life history can be worked out without stained preparations. Stages that have short duration must be preserved when available and subse- cjuently studied from temporary mounts. However, permanent stained slides are indispensable for research and have a legitimate place in teaching to supplement bulk material. The methods of processing the most commonly used algae are outlined briefly in this chapter, with frequent references to the whole-mount methods in Chap. 10. GREEN ALGAE These plants exhibit a wide range of size, complexity of organiza- tion, and habitat. The following simple precautions should be ob- served in collecting, transporting, and storing plants: 1. Keep the plants in their natural substratum (water, soil, bark of tree) until the moment of killing. 2. Avoid subjecting the plants to excessive heat or to desiccation during storage or transportation. 3. Unless culture methods have been carefully worked out, kill the plants as soon as possible after collecting. 4. Subdivide or spread out large masses of material to promote rapid killing and hardening (fixing) . 5. Keep intact the organization of the filament, or other type of colony. [ 149 ] 150 Botanical Microtechnique The preservation of green algae for bulk material and for perma- nent stained slides is treated at some length in the chapter on whole- mount methods. The advanced worker Avill find further details in Johansen's (1940) comprehensive treatment of culture methods and processing of this group. BLUE-GREEN ALGAE These algae have such simple cellular and colony organization and are so easy to study in temporary whole mounts that the use of pre- pared slides is less jtistifiable than with any other group of thallo- phytes. Fresh cultures are easily found in a wide range of habitats; in stagnant pools, tanks, barrels, and crocks, on potted plants with stale soil, on damp, poorly drained soil, and innumerable other places. Some forms like Oscilhitorid, Rivularia, Xostnc, and Gloeocapsa may be found in masses that are practically pure cultures. Collections of such materials are easy to preserve. Because of the dense undifferen- tiated character of the protoplast the crudest methods of prcscr\ation, such as 5% formalin, may be used. If the reagents are a\ailablc, one of the fluids containing glycerin shotdd be used. Temporary or perma- nent whole mounts can be made as described in Chap. 10. THE MARINE BROWN AND RED ALGAE I'he algae in these groups are available in fresh condition for a very limited nmiibcr of schools. Large qiumtities of these plants are used by schools that arc totally dependent on outside sources for their materials. Therefore, a detailed discussion of methods of collecting and preserving these plants woidd have but limited usefidness. For occasional casual collecting on one's ira\els, the simplest preservative is 5 to 10% formalin in sea water. Further rcfincnunts are the addi- tion of 5 to 10% glycerin and 1/2 teaspoonful of borax to 1 liter of fluid. For more critical picservatiou. nothing has l)ciii louiul to excel chrome-acetic, with or wilhoui addition of osmic acid. One of the best formidas is the Chamberlain foi inula ( Fable 3.1) made up with sea water. Subsequent processing of filamentous loinis lor wliole mounts is outlined in C.hap. 10. If materials are purchased from collectors, the purchaser shoidd indicate whether the material is to be used for temj)orary slides or to be processed for permanent j)ieparaii()ns. Sexcial relial)le collectors will furnish material in specified stages ol the life history, carefidly fixed in a suitable lluid determined by the collector or specified by Thallophyta and Bryophyta 151 the purchaser. Such materials will yield excellent preparations by the methods recommended in Chap. 10. Few of the algae are microtomed for making slides. Some selected items that are customarily microtomed are discussed briefly. CHARA AND NITELLA The growing points of CJiara and the sex organs of mature plants must be sectioned to show cellular organization and nuclear structures. Kill in medium I chrome-acetic or Craf II. These fluids contain enough acid to remove much of the troublesome incrustation. The condition of the material after 1 week in the fluid can be easily ascertained by examining a whole mount. If abundant material is available, several variations of these formulas should be tried, and the batch having the best fixation used for embedding. Older oogonia and zygotes are not readily penetrated by the above fluids; FAA should be used. These plants become very brittle in xylene, but they section satis- factorily after the butyl alcohol process. Examine small samples dur- ing the process, thereby saving further work if a batch has undergone plasmolysis. Infiltration should be gradual, with the time interval in the oven reduced to 2 days or less. The staining of different batches is highly variable. Try iron hematoxylin and safranin-fast green. Fucus and similar bulky forms are usually sectioned to show game- tanaia. Kill in medium II or strong chrome-acetic made up with sea water. Dissect out some of the gametangia to ascertain which fluid preserves them best at the given stage. Process in TBA or dioxan. Sections are difficult to affix to the slide. It may be necessary to use an alcoholic bulk stain with some batches. Brilliant staining of imma- ture sperms in the antheridia of Fucus has been obtained with iron hematoxylin; sharp staining of nuclei during cleavage in the oogon- ium is very difficult. The more massive Rhodophyceae that cannot be satisfactorily made into whole mounts may be sectioned by the method given for Funis. Since the great majority of readers do not have access to fresh plants, the purchase of carefully preserved material from reliable col- lectors is recommended. Fungi The processing of fungi involves many problems that are com- mon to other categories of previously described subject matter. For example, in processing a fungus parasitic on a leaf, the tissues of the 152 Botanical Microfechnique host must be preserved unchanged; the fungus, with an entirely different chemical and physical make-up, perhaps an alga-like si- phonaceous plant body, also must be preserved intact. Another task may involve cutting a tough piece of wood bearing a delicate Plas- modium, presenting a conflict between the need for drastic methods and refined methods. In order to minimize duplication of procedures in this chapter, it is proposed to use extensive cross references to ap- propriate sections of the text and to give detailed directions for procedures that are not adequately covered elsewhere in the manual. SCHIZOMYCETES The preparation of slides from cultures of bacteria is described in detail in textbooks of bacteriology. The bacteria are discussed in this manual only in conjunction with a host plant. A few typical examples of plant tissues and their bacterial invaders will illustrate the general methods of processing. Bacterium steioartii invades the vascular system of corn, forming a shiny yellow mass in the xylem elements. Because of the virulence and ease of dissemination of the disease, it is unwise, in regions where the disease is not normally -" V V •-> ill '-aK > ^SsH t- . '- * ^^ :Z^ ^ "^' M^ \ Fk;. 14.1— Vasculai huiullr of Zen, witli haclciial mass (liaclerium steu'arlii) in the prolo\^lcIll. Thallophyta and Bryophyta 153 present, to infect plants to obtain diseased tissues. Preserved tissues can be purchased from the supply houses. The most satisfactory killing fluids are FAA and FJ^-bichloride of mercury (Chap. 3) . Chromic acid seems to become fixed in the gelatinous bacterial slime and interferes with clear staining. Process the corn stem or leaf as desCTibed in the section dealing with vegetative organs of seed plants (Chap. 13) . Iron hematoxylin gi\cs a brilliant differentiation of the bacteria. The xylem may be lightly stained with safranin or gentian violet, but the slime between the bacteria must be thoroughly destained (Fig. 14.1) . The above methods are satisfactory for the preparations of cucimi- ber stems infected with the wilt organism, Enoinia tracheiphila, and succulent leaves and twigs of apple or pear infected with E. arnylovora, the fire blight organism. MYXOMYCETES The slime molds are customarily studied from living cultures of the slimy plasmodium and from dried specimens of the fructification. These spore cases are exceedingly delicate and beautiful objects. Sporangia that are nearly mature can be mounted into permanent slides. Transfer directly into 95% alcohol for 10 min. Pass through three grades of anhydrous alcohol-dioxan at 10-min. intervals, then into pure dioxan, and mount in thin dioxan-balsam. A similar butyl alcohol series may also be used. The nuclei are exceedingly small, and microtoming and staining are tasks for the experienced cytologist. PHYCOMYCETES The saprophytic members of this group should be studied in culture whenever possible, and the use of prepared slides should be discouraged. Stages that are of short duration or difficult to obtain can be preserved, for either bulk material or permanent slides. The representatives of this group presented below are in common use for teaching, and the process for each plant has been thoroughly tested and may be regarded as type processes applicable for similar subjects. Strict taxonomic sequence is not maintained in the following discussion; organisms that are processed by similar technitjues may be discussed simultaneously. Zygomycetes.— T\\t order Mucorales contains the best-known members of this group. Species of Rhizopus and Miicor are easily grown in culture and studied to best advantage from whole mounts. Developing and mature zygospores can be preserved by cutting out 754 Botanical Microtechnique selected pieces of the culture agar and kUlin^ in FAA, which also serves as a storage fluid. Such material can be used for mounts in water or lactophenol or for excellent stained permanent preparations of whole mounts (Chap. 10) . Cytological preparations reqture such highly specialized and almost specific methods that the ambitious student shoidd study the research jiublications of a given species for details of procedine. Odtnycetes.—PlasinodiojjIioKi brdssicae is parasitic in the roots of cabbage and related plants. The plasmodium can be demonstrated in young roots that are just beginning to undergo distortion. Stages of cleavage and spore formation are obtained from increasingly gnarled and distorted roots. FAA gives good fixation, but Craf III is superior. A simple hemahmi-safranin stain is adequate for most purposes, safranin-fast green is more contrasty, and iron hematoxylin gives the most brilliant differentiation of the parasite nuclei. Syncliytriiim decipiens, parasitic on the hog peanut, and related jiarasites yield striking preparations, but poor fixation is frequent, and sectioning is improductive, making the slides somewhat expen- sive. Craf II was found to give excellent fixation. Iron hematox)lni is by far the most satisfactory stain. Saprolegnia and allied water molds are readily obtained and easily cultured, furnishing abundant vegetative and sexual material for stud\ in tire living condition. The best soinces are dead fish and Avatcr insects, or steam sterilized house flies placed into a large crock of pond water. Whole mounts can be piepared h\ the general methods given for filamentous plants (Chap. 10) . Ascertain the correct killing formula lor the species being studied by testing small masses in a weak chrome-acetic or Craf I and manijiulating the acetic (or jji()j)ionic) acid (ontent. W'hole-niount and sectioning methods are used for cytological study, and the reader is referred to research publications for these highly specialized and dilficult methods. Pythium and Pliytoplitliora are most effectively studied in culture, 1)111 preparations (an l)e made by the methods suggested for the water molds. Albugo (Cyslopiis) . the "white rust," is an indispensable subject in teaching. Several species octm on (onunon crops and \\eeds. For the conidial (zoosj)orangial) stage, select jnistides that ha\e just ruptured. Fully openetl pustules Avill have most of the spores washed out dining processing. Fhe sexual stages arise after the conidial stage is on the decline, and the host tissues show evidence of hypertrophy. A. Candida and A. hliti are fixed in perfect condition in Craf II. Thallophyta and Bryophyta 155 'f^B^'*' mP^ 0 Fig. 14.2— Portion of pustule of Cystopus candidus on stem of Capsclla, Ciaf 0.30- 1.0-10.0, acetone-tertiary butyl alcohol. Safranin-fast green and hemalum-safranin are both excellent combinations for elementary use, and iron hematoxylin is a good nuclear stain (Fig. 14.2) . Mature oospores are abundant in the older, hypertrophied stems and fruits of the host, therefore, a more vigorous kiUing Ikiid is needed; FAA and standard Nawaschin are both satisfactory. As might be expected, the host cells are in a distorted condition at this stage. Peronospora parasitica, a common parasite on crucifers, yields excellent preparations by the methods described for Albugo. The best compromise fluid for preserving both the host cells and the fungus is Craf II. Safranin-fast green differentiates the nuclei of the fungus, but not so sharply as iron hematoxylin. ASCOMYCETES The mold members of this group, such as Aspergillus and Penicillium, are so easy to culture and study from wet mounts that permanent slides are seldom necessary. Permanent slides can be made bv the common whole-mount methods. Such slides are useful for 156 Botanical Microtechnique quick reference rather than for detailed study. The production of the ascigerous stage is highly uncertain, and best studied from whole mounts. 1 he Erysiphales are of interest because many members are parasites of considerable economic importance. Erysiphe graminis occurs on many grasses, from which the conidial stage is easily obtained in abundance. The conidia are studied best by freshening detached leaves in a moist chamber and examining the surface under moderate magnification, wath oblique surface illumination. Microtome sections are indispensable, however. Longitudinal sections of the leaf show the clusters of long, finger-like haustoria in the long epidermal cells. Select leaves that are young and soft, kill in Craf III, and process like any young grass leaf. Iron hematoxylin is the most desirable stain, although a good triple stain is indeed beautiful. Other interesting or important species are Erysiphe polygoni on the common weed Polygonum aviculare, E. hiimuli on the rose, Podosphaera oxycanthae on cherry, Microsphaera aini on lilac, and Unciniila salicis on willow. For the best slides of haustoria, collect in the conidial stage. Development of the perithecia can be studied from successive collection up to the stage in which the perithecia begin to turn gray. Kill in Craf III if the host cells are to be preserved, or in Craf I for good preservation of the young perithecium. The latter lluid does not seem to serve so well for the host cells. Stain as reconnnended for Erysiphe graminis. Mature perithecia are very brittle and difficidt to section. Furthermore, this stage is studied to best advantage from dissections and macerations of bulk material preserved in one of the fluids in Chap. 10. The sectioning of the decayed, brittle, overwintered host leaves is a thankless and pointless task except for research. Members of the Pezizales are of considerable cytological interest as well as economic importance. Scleroiiuia fnictigena occurs on cherries and plums. The conidial stage need not be sectioned, and sections of the hard sclerotia are not particularly interesting. The delicate goblet-like apothecia yield excellent sections. Very early in the spring look for the apothecia arising from nunnmified fruits. Collect cups of various sizes and preserve each in a sej)arate \ial of Bouins fluid or Craf I. Successive stages of ascospore formation will be obtained in this way. Longitudinal sections through the center of the cup show numerous perfectly aligned asci and ascospores. Use iron hematoxylin for nuclear details, but safranin-fast green shows both nuclei and trania \erv well. Thallophyta and Bryophyta 157 Pyronema conjlneus is common on slcam-sterilized soil in the greenhouse. Cytological preparations ol the sex organs are a task lor the skilled investigator, and the reader is referred to the research literatiue. The apothecia are processed like those of Sclcroiinia. Pscudopeziza medicaginis is parasitic in the leaves of alfalfa and other legumes. Collect material when the pustules are just opening and the apothecia are bursting through the ejMdermis. Excellent preservation is obtainable with FAA, and staining presents no difficulties. Even the simple hemalum stain differentiates the ascospores. SarcoscypJia coccinea has a brilliant red, dainty, cup-like ascocarp that can be killed entire and processed exactly like Sclerotinia. The larger cups like those of Peziza repanda, Urmila, and the familiar ascocarp of Morchella, the sponge mushroom, should be suital^ly subdivided and processed as above. Fleshy portions of the fructifications of other Ascomycetes are handled like the foregoing types. Interesting slides are obtainable from Hypojnyces, a parasite on mushrooms; Cordyceps, parasitic on insects; the fruiting head of Claviceps, the ergot fungus; the saprophytic Neurospora. Species of Nectria in which the stroma is moderately soft can be sectioned. Remove the stroma down to the wood, subdivide vertically into narrow strips, kill in FAA or Craf II, and process in TBA. Always examine freehand sections or smears of fleshy Ascomycetes to determine whether the desired stage of ascus formation is present. In the Taphrinales (Exoascales) only the genus Taphrina (Exoascus) is of importance. Taphrina deformans, the casual organism of peach leaf curl, is very abtmdant in some localities. The malformed succulent leaves are Avell preserved by Bouin's solution or Craf II. Sectioning and staining present no difficulties. Ventiiria inaequalis, the apple scab organism, is widely distributed and easily obtainable in the conidial stage on the leaf. A vigorous fluid like FAA or Craf V is necessary. A simple stain such as hemalum- safranin is adequate. The perithecia mature in early spring on last year's decayed leaves. Such material can be studied well from newly gathered soaked leaves or bulk-preserved leaves. Such material yields permanent slides of decidedly ragged appearance, and microtoming is therefore to be discouraged. BASIDIOMYCETES This group contains a great diversity of forms and involves a wide range of techniques. We are again confronted with saprophytes that 158 Botanical Microtechnique can be detached from the substratum and processed easily, ^vliereas the parasitic members require adequate preservation of both parasite and host. In the following discussion the taxonomic order is sub- ordinated to methods of preparing the material. Us tila ginnles .—These parasites occur on a wide range of hosts, but the most interesting members occur on important crop plants. Ustilago zeae, the corn smut, is found on all aerial parts of the corn plant. Smut galls on stems, leaves, and ovaries should be collected. Very young smut galls, that ha\e not markedly distorted the organ being attacked, show the host cells in good condition, and contain acti\e and rather sparse mycelium. Kill this stage in Craf III. Older galls having a milky white interior contain a great mass of mycelium and distorted host cells. Small pockets of chlamydospores occur in the white mass. This stage, which can be ascertained by freehand sections, is the latest useful stage. Kill these older galls in FA A. Ihe mycelium of corn smut has a strong affinity for hcmalum, ami a simple hemalum-safranin stain shows the hyphae stained blue-black, chlamydospores stained red, the thin walls of the host cells stained blue, and the lignified elements stained red. Use iron hematoxylin for nuclear studies. Other common smuts, such as Ustilago levis, U. Iiordei, U. m'cune, and the bunts, like Tilletid Iritici, can be processed by the abo\e methods. The chlamydospores of many smuts and bunts germinate readily in water or carrot decoction. The promycelia and sjioridia are studied best from wet mounts from cultiue, but the material c an be made into permanent mounts by the dioxan or butyl alcohol methods (Chap. 10). Uredinales.— The rusts rank among the most destructive and wick- spread ])lant pests, and class materials illustrating ilu' important phases of the life cycle of the rusts are indispensable. The story of wheat rtist has been so well publicized that the organism may well be the standard item representing this groiq). Puccinia gxiininis has its red uredinial and black telial stages on wheat and many other grasses, rhe red summer-spore stage occurs on young leaves and is therefore easy to section. I'he black winter-sj)ore l)usiules occur on older leaves and on the stems, both of which are diliicuh to section without tearing. Use the youngest leaf showing the telial stage, avoiding the use of stem material if possible. Kill in 1.1 A and process like any leaf parasite. The pycnial (spermogonial) and aecial stages on barberry occur on young, tender leaves that are Thallophyta and Bryophyta 159 preserved fairly well by FAA, but Craf I followed by careful embedding yields superior results. For the most critical cytological requirements, use the Flemming modifications as described in research papers. Many stain combinations give excellent results for class material, safranin-fast green is particularly good, but iron hematoxylin is by far the best as a nuclear stain. P. coronata, the crown rust, is probably second to wheat rust in importance. The uredinia and telia on Avena and other grasses and the pycnia and aecia on Rhamnus (buckthorn) are treated like wheat rust. Two common species of Gymnosporangium have the telial stage on J II ui penis, producing woody galls of stem tissue in which the mycelium is perennial. The younger galls are soft enough to be sectioned in paraffin. Divide into wedge-shaped pieces, kill in FAA, and process in butyl alcohol. The pycnia and aecia of Gymnospornn- giiirn juniperi-virginianae occur on Pyrus, and those of G. globosuui on Crataegus. Treat like the aecial stage of wheat rust. Melampsora is very common on willows and poplars. The bright yellow uredinia, which may entirely cover the leaf, are handled like other leaf rusts. The coal-tar dyes do not seem to be so selective for nuclei as iron hematoxylin. The telial stage on the old leaves is a difficult problem, the host cells become very brittle in paraffin, and the nuclear staining is selective only with iron hematoxylin. A great diversity of host tissues in which rusts are found necessitates more or less specific adjustment of the killing fluid for each problem. The foregoing recommendations are based on success- ful preparations and will serve as a guide for other problems in this group. Tretnellales.-The order is characterized by the small, gelatinous fructifications. Septation of the basidium differs in the several families, and some authors regard as orders some of the families incorporated here. The delicate fruit bodies must be collected in an absolutely fresh condition or the time spent in processing them is wasted. The portion near the substratum is of no interest; remove the substratum completely, and kill the entire or subdivided fruit body in weak chrome-acetic, or in Craf I. Exercise extreme care during dehydration and embedding. The best stain is iron hematoxylin, with safranin-fast green as second choice. A garicales.— The primary consideration in the processing of this group is to maintain intact the more or less exposed, delicate basidia and especially the exceedingly fragile sterigmata on which the 760 Botanical Microtechnique basidiospores are borne. The texture ot the trama of the fruit body ranges from the soft, fragile pileus of a small Copnnns to the "woody" perennial pileus of Fomes. The softer members are difficult to preserve in normal condition but are easy to section, whereas the leathery fructifications can withstand processing but are very difficult to section. The basidia of many species of Agaricaceae have been successfulh fixed in weak chrome-acetic, in Craf III, or in Allen-Bouin II and III. The last is particularly good for cytological details. Bouin's solution has given good results, but it is rather erratic. Dehydrate in alcohol or acetone, beginning with 5*;; and using steps of 5<^; at 15- to 30-min. intervals. Iron hematoxylin and gentian violet-iodine are excellent for nuclear details. Safranin-fast green stains the nuclei well enough and also shows the gill and trama structure. Softer members of the Clavariaceae, Hydnaceae, and Polyporaceae are processed as above; the leathery and woody forms must be dehydrated in butyl alcohol or dioxan. Fortunately, basidia mature in the soft new growth in even the toughest perennials. Exobasidium occurs on Vaccinium, Rhododendroyi, and other members of the heath family. Kill in FAA or Craf III. Because of the U'athery texture of the host the use of butyl alcohol is advisable. FUNGI IMPERFECT! This category includes fungi for which the perfect or sexual stage has not yet been found. The perfect stage, w^hen discovered, is found to Ijc a basidial or ascigcrous stage, and the organism is ihcii transferred to the ajjpropriate group. Sporulation is l)v conidia. produced either at random on tlie mycehum or in ck)sed pycnidia. The vegetative mycelium may be a sujK'rlicial saj)r()pliytc. a saprophyte within dead tissues, or a parasite within tissues. Mycelium and conidia from cultines can l)e prepared as whole mounts by the general methods outlined in Chap. 10. Parasitic species are handled in accordance with the properties of the organ on \\hi( h they occur. Leaf parasites are the easiest to handle. I'he h)llowing illustrations are selected from successful j)reparations of important fimgi. Diphxlid zeae grows readily in agar culture and jn-oduces abundant pycnidia. Can out small pieces of agar bearing ihe pycnidia, fix in Craf I, and embed in paraffin. A heavy overstain in hemalum, slightly dillerentiated in HC:i. stains the hvaline portions of the fungus \erv well. The pvcnidia and the mature spores ha\e consideral)le pigmentation. Thallophyta and Bryophyta 161 Cercospora beticola is common on garden and sugar beets. Excise the youngest lesions to obtain sections embracing healthy tissues as well as diseased areas. If material must be killed in the field where an aspirator is not available, use FAA, which gives adequate fixation. Excellent preservation can be obtained with Craf III. Iron hema- toxylin and safranin-fast green are the preferred stains. The wood- and bark-inhabiting pycnidia are handled like perithecia of similar habitats. Such resistant subjects must be killed in FAA, and butyl alcohol is the preferred dehydrant. LICHENS The lichens are found in a wide range of habitats, from the mist- soaked rocks under a waterfall to the sun-baked face of a boulder. Collections should include a portion of the substratum whenever possible. Specimens usually are dried, and stored in containers that prevent breaking of the fragile dry plant. If wet preservation is preferred, use one of the fluids given in Chap. 10. Microtome sections of the vegetative thallus have little justification. The association of the green algal cells and the fungal mycelium is shown best by dissections and freehand sections of fresh or preserved material. The ascocarps should be preserved in fluid, examined with a hand lens for general organization, and teased apart for examination of bits of the hymenium under a microscope. Microtoming of the ascocarp is a vexing problem with most species. The gelatin in the plant body becomes dry and brittle, and the sections fail to ribbon and do not adhere well to the slide. Select a species with a small, shallow cup-like apothecium. Kill in FAA and dehydrate in butyl alcohol. Soak the embedded blocks in warm water before sectioning. Staining presents no difficulties if selectivity for the diverse components is not demanded. Safranin-fast green is probably the best simple combination. Bryophyta The liverworts and mosses have such wide distribution and range of habitat that some representative member of the group is usually available for study. The most common liverworts are the aquatic Riccia, the well-known Marchantia, and two rock-inhabiting species, Conocephalum conicuni and RebonUa hemisphaerica. Anthoceros seems to be less common, but it is easily overlooked if sporophytes are not present. Large and conspicuous mosses are usually preferred, the best-known ones are in the genera Polytrichiwi, Mnium, Catherinia, Funaria, Rhodobrywn, and Sphagnum. Liverwort and moss species 762 Botanical Microtechnique that are not locally axailable can be purchased from supply houses, preserved either for bulk specimens or for sectioning, as specified by the purchaser. These fragile plants must be collected and handled ^vith care, taking precautions to keep the plants moist and undamaged until the time of killing. Entire plants preserved in fluid are indispensable for teaching. Fhe most useful bulk preservatives are described in Chap. 10. Preservation and processing for embedding must be carried out with j^ainstaking care, approaching cytological methods. Hepaticae The following recommendations, based on Marchantia, ^\■ill apply to a wide range of liverworts. 1 he young, actively growing thallus is usually sectioned to show the construction of the pores, the highly spongy chlorenchyma, and the gemmae. Cut out 4-mm. squares of tissue. Genmia cups should be excised with a small scjuare of thallus. Antheridial and archegonial receptacles should be collected when they are just beginning to be elevated above the thallus. The game- tangia are at their best at this stage. When the archegonial disk has been fully elevated, make a collection lor the developing sjK)roph}tes. A complete series of developmental stages may be obtained by collect- ing at intervals. Kill in weak chrome-acetic or Craf I. A closely graded alcohol-xylene series is recommended. The thickness of sections can be judged best at the time of microtoming. Examine a few trial sections by melting the ribbon on a slide, and decide whether the trial thickness includes the desired structmes and is sulficiently thin to show internal detail. Sections will range from (i ^i for a careful examination of young antheridia, to 15 [.I for maturing capsules. A simple hemalum stain, with perhaps a light (ouiUerstain of erythrosin, sets oil all essential structures very well. A nutltifolor slain combination is cpiite pointless. Iron hema- (ox\lin is the uhimaie dioice for cytological details. Gennnae can be studied conveniently by dissecting them from genmia cups of fresli or preserved thalH. Permanent \\]i()le-iH()unt slides of gennnae are of little value, but such mounts can be made by the methods outlined for ])repaiing filamentous green algae. Microtome sections aie ne(essai\ to show the initiation and deMloj)- ment of genmiae. Riccia and AnI/kx cro.s are somewhat moie dilficult than the foregoing type, because the sex organs are sunken in the thallus. Skillful freehand sectioning reveals the presence of sex organs and Thallophyta and Bryophyta 163 eliminates the fruitless sectioning in paraffin ol many vegetative thalli. Ihe developing sporophytes ot Riccia are visible within the thallus, and \arious stages can be classified roughly by size. Remove enough of the thallus with these organs to show some of the envelojiing cells. Fruiting thalli of Anthoceros should be killed entire, in a vacuum jar (Fig. 3.1) , and the sporophytes dissected away with a section of thallus after hardening in the fluid for several days. Both transverse and longittidinal sections of the sporophyte should be made. The leafy liverworts are easily overlooked on collecting trips, and therefore do not receive adecjuate attention. Pellia and Porella are most commonly used to illustrate this group. They can be processed like the mosses as outlined below. Musci These plants are readily obtainable in fresh condition during the greater part of the year in all but the most severe climates, and they can be grown easily. They make usable dried specimens and can be preserved in excellent condition in the fluids given in Chap. 10. Gemmae, fidly developed sex organs, and most featm-es of the capsule can be studied from dissections. Prepared slides are needed principally for studying young sex organs, gametes, and some features of the developing sporophyte. For the study of sex organs the large and more common species of Mnium, Polytrichum, and Rhodobryitm are recommended. The proper killing fluid for sex organs and gemmae of mosses and leafy liverworts can be determined quickly. Obtain fresh turgid plants, dissect out a few short pieces of the shoot bearing the sex organs, and immerse in the fluid that is to be tried. Exhaust the air that adheres tenaciously among the leaves. After 1 hr. in the killing fluid dissect out a few gametangia, mount in a drop of the fluid, and examine with a microscope under at least 400X- If plasmolysis has occurred, adjust the formula. It is a good practice to try FAA and FPA (page 15) . If these cause excessive shrinkage, try Craf I, an excellent formula. Adjustments in this formula are made by increasing the ratio of acid until no marked plasmolysis occurs. Use the stains recommended for liverworts. Capsules of mosses are a vexingly difficult subject. Young green capsules of Mnium cuspidatiim and Funaria hygyometrica are penetrated by Craf I, but for older, coloring capsules, FAA or FPA must be used. However, the interesting stages of sporogenesis take 164 Botanical Microfechnique place long before the capsules become brittle, and there is little need for slides of old capsides. The dehydrating must be gradual, and TBA is preferred. The embedded capsules shotdd be oriented carefidly in the microtome, and both longitudinal and trans\'erse sections are desirable. The capsule has enough internal differentiation to justify the use of a triple stain; however, the simple combinations given on Staining Charts II and III are usually adequate. The sporulating capsules are studied to best advantage either from fresh plants, wet-preserved plants, or dried specimens from which they can be removed and thoroughly soaked in water or lactophenol (Chap. 9) . Spores can be germinated readily and the jirotonema held at any stage by refrigeration under weak illumination. W^ith a little planning by the instructor there is little excuse for using permanent prepared slides of protonema, although these can be made by the methods used for delicate algae. 15o Reproductive Structures of Vascular Plants The preparation of vegetative organs of the highly diverse mem- bers of the phylum Tracheophyta, the vascular plants, is discussed in a separate chapter because such organs require similar techniques (Chap. 13). Similarly, the reproductive organs of the Tracheophyta present common problems of processing and staining and are there- fore brought together in the present chapter. Orders as well as Classes ar used as major headings. Lycopodiales Ihe organization of the strobilus of the club mosses should certainly be studied by dissection, and there is no point in embedding entire strobili. Ascertain the stage of sporogenesis in each cone by dissecting out a sporangium and crushing out the contents. Mature sporangia containing dry, hard, brittle spores should not be embedded unless a cytological study is to be made. Subdivide the cone transversely, and kill in FAA, medium chrome-acetic, or Craf III. Sections should be stained in safranin-fast green or iron hematoxylin. The gametophytes of these plants are exceedingly rare, although they are said to occur in abundance in localized areas. Gametophytes may be purchased preserved in FAA. The soft thallus is easily sectioned in paraffin and stained. Selaginellales There is very little justification for making sections of the strobili of these plants because dissections under a binocular reveal so much more of the orderly organization of the cone. Dissected and crushed sporangia likewise present a three-dimensional picture that is lacking in sections. The study of nuclear details of sporogenesis and the de- velopment of gametophytes within the spores is a task for the experi- enced investigator. [165] 166 Botanical Microtechnique Isoetales Young sporangia of Isoetes arise on small sporophylls closely apjjrcssed to the rhizophore. Dissect away the sporophylls under a binocular, trim off much of the sporophyll, and kill the sporangia in medium chrome-acetic or FA A. The large sporophylls and mature sporangia are inadequately represented by sections. To section mature spores, cut the sporangia away from the sporophylls, drop the ruptured sporangia into a centrifuging tube of FA A. Process in butyl alcohol, centrifuging the mass after each change. Much tearing of the spore wall can be expected during sectioning. The preparation of gametophytes should be undertaken only after a study of research literature in which methods are given for germinating and processing the material. Equisetales Equisetian cones differentiate underground during the late siunmer and contain mature spores when they emerge from the ground the following spring. Young strobili should be dissected away from the rhizome, thoroughly washed, divided into several pieces, and killed in medium chrome-acetic or FAA. Both transverse and longitudinal sections should be made. There is little excuse for sectioning strobili containing mature spores. Compared w'ith a dissection under a binocular, a section presents an lUterly inadequate pictiue of the interesting organization of the cone. Mature spores should be studied in a wet mount, which is subsequently uncovered and permitted to dry, bringing about the uiKoiling of the elaters. Gameto{)hytes can be grown by sowing ne^vh shed spores on sterilized sphagnum. Excellent ])reserved gametophvtes also can be purchased. Embedding and sectioning arc carried oui as with other soft, delicate subjects. Ophioglossales Bolryclniun is the easiest member of this order to use for the study of reproduction. The sporoj)hylls can be teased aj)ai t and crushed to determine the stage of sporogenesis. Subdivide the fertile frond into small pieces, and kill in medium chrome-acetic or Craf II. A wide \ariety of stains will produce brilliant preparations. Collectors find gametophytes to be extremely abundant in localized areas dining favorable seasons. Preserved game lophytes can be purchased and are easy to process. Reproductive Structures of Vascular Plants 167 Filicales The position and construction of the sporogenous area or sorus and the character ol the sporophyll differ in the numerous genera. Aspleiiiuin )iidus-(n>i.s. tlie bird's nest fern, bears sori on the large, leathery, entire vegetative leaves, whereas Onoclea struthiopteris, the ostrich fern, bears the sporangia in the tightly infolded, pod-like pinnules of special fertile fronds. The preparation of the diverse subjects is practically identical. Select young sori, and examine a dissected portion of a sorus, using stages up to and including young thin-walled spores. Excise small portions of leaf tissue bearing sori, and kill in medium chrome-acetic or Craf II. Species having soft leaves are more economically dehydrated in alcohol or acetone, but butyl alcohol is advisable for the tougher types. Stain in iron hematoxylin to obtain the best nuclear details and in safranin-fast green for general use. Do not waste time embedding mature sporangia. The contents of the sporangium, the construction of the annulus, and the character of the wall of the mature spore are shown far better in a wet mount of fresh or preserved material. Some of the cultivated ferns have a high ratio of shriveled, undeveloped spores in the mature sporangium; sections of such material are disappointing. Gametophytes of native ferns can be found in great abundance by an experienced collector. Such materials are useful for gross study, but the presence of soil particles among the rhizoids makes sectioning difficult and unsatisfactory. Gametophytes can be grown on nutrient agar cultures, or on porous clay flowerpots in a moist chamber. Remove a few gametophytes for examination at intervals, kill desirable specimens in medium chrome-acetic or Craf I, and prepare whole mounts (Chap. 10) or embed very carefully for microtome sections. Iron hematoxylin and safranin-fast green yield beautiful preparations. There is no need to section thalli bearing sporophytes, and permanent whole mounts are not so desirable as wet mounts that can be handled and viewed from all angles. Gymnospermae Members of the common genera of the Coniferales are well-known trees of great economic importance, and abundant material is easily available. The life history of the pine is probably the most widely used subject, therefore, the present discussion will be centered around reproduction in the pine. The reader should consult Chamberlain 768 Botanical Microtechnique (1935) for the morphology and seasonal sequence of the reproductive cycle in other genera and orders, and adapt the methods described here to other subjects. Staminate cones of Pin us are differentiated during the season prior to the shedding of pollen. Cones can be dissected from buds and the stage of microsporogenesis ascertained by means of acetocarmine smears. Several species of Pin us undergo meiosis early in May, in the Chicago region. Killing fluids do not penetrate readily into large masses of highly resinous tissues. It is therefore necessary to subdivide all but the very smallest cones. Kill in FAA for general morphological studies and in a Nawaschin type, such as Craf II, for more critical details. Nuclei of microspores and mature pollen grains are stained adecpiately in hemalum-erythrosin. For the first gamctophytic somatic mitosis, which takes place in the microspores before they are shed, use iron hematoxylin or safranin-fast green. Preparations of the ovule history are much more difficult and time-consuming to make than the pollen history. The time of occurrence of interesting and important states varies with the species, the locality, and probably in a given locality in accordance with the weather conditions. In the Chicago area the megasporocyte of Pimis hirido is evident when the cones emerge from the I)ud. Fertilization has been found toward the end of June, while early embryo stages are obtainable during July (Chamberlain 1935) . The deep-seated megasporocyte is not reached readily by killing fluids, necessitating the use of vigorous fluids that produce distortion. The very young cones may be fixed entire in strong chrome-acetic, FAA, or /'Vi/i-bichloride of mercur). Such preparations are of interest principally to the student of developmental morphology. It may be preferable to cut away the young ovules from the sporophyll and strive to }jreserve the sporogenous and gamctophytic feature. Strong chrome-acetic seems to ha\c gi\en the best results lor most students of this group, riie Nawaschin modifications and Alkn-liouin modi- fications deserve further study. Staining of ovulate structures is particularly difficult. Resinous materials in the cells tend to make the jMeparations unsightlv, although the essential nuclei may l)c ckarlv differentiated. Safranin- fast green meets the requirements for all but research needs. After the first lew di\isions of the zygote, microtome sections are no longer adequate lor the siiul\ of emt)ryology. Ihe development of dissection methods has facilitated great progress in such studies. Reproductive Structures of Vascular Plants 169 A detailed discussion of the morphology and tcchnicjiies applicable to other orders of gymnosperms is given by johansen (1940) . Angiospermae The angiosperms are usually the central feature of the study of reproduction in plants, representing the climax in the development of reproductive organs. Floral types and features of floral organs are studied best by dissection and whole mounts of fresh or preserved material. THE FLOWER Microtome sections are indispensable for the study of vasculation and histogenesis of floral organs. Each species is virtually a problem in itself; therefore, this discussion will be limited to the methods used for the successful preparation of a few useful subjects. Buds of lily and tulip are among the most satisfactory subjects for entire flower buds. The very young buds are large and easy to handle. Embedded buds can be accurately oriented for sectioning, and the parts are so large that elementary students can locate and recognize the parts on the slide. Lily buds are available over a considerable period, beginning ^vith Lilluiu utubellatum and L. elegans in May, to L. tigrijiinii in August. ^Vell-de^ eloped floral parts are shown in buds that are less than 5 mm. long (Fig. 15.4) . Cut off at the base of the perianth, and remove successive slices from the tip until the tips of the anthers have been cut off. Drop into the killing fluid and pump vigorously. Fair fixation is obtained in FA A, but superior results are obtainable with Allen-Bouin II. Sectioning and staining are delight- fully easy. Begin sectioning at the base of the flower, discard the ribbon until the sections include anthers and ovary, and discard the block when ovules are no longer present in the apical portion of the ovary. Buds of tulip for entire sections of young flower buds are ob- tained from bulbs during late fall. Many varieties of Darwin tulips are in a suitable stage from mid-October to early November. Meiosis was found to occur in several Darwin \'arieties in October. Kill in Allen-Bouin II and carry through an alcohol-xylene, dioxan, or butanol series for entire young flower buds; for an older ovary follow the recommendations for the lily. Cut open the bulb, and dissect out the complete flower bud. Trim and kill as with lily. Maithiola, the common garden stock or gillyflower, furnishes a suitable dicotyledonous flower for complete sections. Remo\e indi- 770 Botanical Microtechnique vidual flowers, trim the end of the closed perianth, and kill in FAA for gross study or in the fluids recommended for lily. Flowers of tomato also are excellent for advanced workers. THE ANTHER AND OVARY Microsporogenesis can be studied satisfactorily in the lily. The structure of the anther and sporogenous tissues are also shown well (Fig. 15.2). Whether meiosis in the anther is demonstrated with microtome sections or smears depends on facilities for the production of enough slides for class use. Slides of adequate quality for elemen- tary classes can be produced in quantity by sectioning (Fig. 15.3 «), but smears are far superior for critical details (Fig. 15.3 /;) . For elementary work, the essential and more ob\ious features of meiosis can be demonstrated with paraffin sections from a series of anthers beginning with anthers 2 mm. long up to anthers that are just beginning to show color. Ascertain the stage by means of acetocarmine smears and handle the successive age classes in separate bottles. This saves much time in locating desired stages for sectioning. Subdivide young premeiotic anthers transversely into pieces not over 2 mm. long (Fig. 15.1 A, B) . The excellence of fixation is influenced h\ the degree of subdivision. Good fixation can be obtained by slicing anthers into disks less than 1 mm. thick while holding them under the killing fluid, Allen-Bouin II. This fluid preser^'es the sporocytes and meiotic chromosomes well enough for elementary teaching (Figs. 15.2, 15.3 rt). The anther pieces cannot be cut nuich shorter than 2 to 3 mm. because the sporocytes are loose in the anther at this stage. The chromosomes are superbly stained by iron hematoxylin, gentian violet-iodine, and safranin-fast green. The advanced worker who wishes lo demonstrate ihe intimate stiuduic ol the chromosome during meiosis should explore the raj)idly exjianding literature on smear methods, select a species on Avliidi to work, and strive to perfect his techni(|ue until he can demonstrate the structures described by investigators of the subject (Fig. 15.3 /;). The dyad condition and second or ecjuational division are of very short duration in lily, and will i)e lound in material selected and j)repared by the loicgoing methods. The quartet (tctiad) and micro- spore stages are of long duration, present during liu- long period of expansion of the llowci bud. until the aiuhers begin to color. For general purposes it is adequate to kill the eniiie anther; FAA yields surprisingly good results. Test each species by means of whole mounts before making a {olUctioii lot this stage. Man\ (uliixatcd lilies, Reproductive Structures of Vascular Plants 777 Fig. 15.1— Subdividing of reproductive organs: A and B, anther of lily; C and D, ovary of lily; E and F, mounted embedded blocks of anther and ovary, respec- tively; G, transverse disk sliced from young fruit of small-fruited variety of tomato; H and /, silique of Matthiola; J, kernel of corn sliced longitudinally; A', center piece of kernel containing essential parts of embryo; L, embedded kernel mounted for sectioning longitudinally. Trimmed edge of paraffin block produces a notched ribbon as in Fig. 6AA. especially the Easter lilies as well as L. speciosum and L. iimbellatum, have extremely high pollen sterility, and the finished preparations show both nicely preserved pollen grains and shriveled microspores. However, such preparations are useful for illustrating pollen abortion. Lilium regale, L. tenuifoliiim, and L. tigrinum are particularly recom- mended for the study of pollen formation. The first two species have a high ratio of normal pollen, whereas only some strains of the last species are satisfactory. For more critical fixation of microspore and pollen nuclei than is afforded by FA A, use the methods recommended for prophases. The somatic division of the microspore nucleus occurs over a brief 772 Bofanicai Microtechnique period and is seldom encountered. The monoploid (hai)loid) chromo- some complement is intercstinj^ and deserves carelul staining when found. Lily ovary is by far the most commonh used subject for teaching the development of the ovule and female gametophyte. The objection to lily is that the nuclear history of the embryo sac differs from the condition in corn, the legumes, and other common crop plants. How- ever, lily ovary and its parts are large, the parallel seriation of the numerous ovules makes sectioning productive, and slides ol the earlier stages, up to cjuartet formation, can be made economically in quantities (Figs. 11.4, 15.4, 15.5). Chrome-acetic has long been a favorite fluid for this subject, and formulas 0.5-0.5 and 0.3-0.70 are excellent for the smaller sporocytes (Fig. 11.3), but the results are rather uncertain with fully expanded sporocytes and subsequent stages. Bouin's solution has been used extensively, but the results are extremely variable. Figure 11.4^ shows a typical Rouin image that is all too common. The rims of the integuments often show a highly wrinkled and collapsed condition. The condition of the sporocyte and integuments after embedding can l^e determined accuratelv in a melted strip of paraffin ribbon. The proportions of ingredients in the original Bouin formula have been rather rigidly accepted by most users, but it is not improbable that superior results could be obtained with carefully determined variants of the formula. 14ie author has obtained some excellent results by using propionic instead of acetic acid as suggested by johansen (1940). 14ie quality of the fixation is im))ro\('d if the jjerfectly fresh ovaries are cut into thin disks. 1 he most consistent results for all stages have been obtained with the Allen-Bouin modifications, especially 11 and 111 (fable 3.2). A closely graded alcohol-xylene or acetone-xylene series can produce excellent results (Fig. 11.1 d) . l)ut failures are frequent. 1 lie glycerin- evaporation method, the dioxan series or TEA are nuich more re- liable. 1 he (onteiUs of the matinc emhiNo sac are apparent 1\ highly fhiid and parti( iilai ly dillicult to preserve without excessi\e plas- molysis, l)ut Allen-Bouin II usually yields adequate fixation. Staining sections of young ovaries i)rior to meiosis is one ol the easiest tasks. A simj^le hemalum stain witli or wiihoiu cixtlnosin is adecpiate for elementary classwork. iron liematoxvlin and safianin- fast green yield brilliant preparations. I'he meiotic aiul gamelophylic division figures and nuclei should l)e stained with iron luinatoxylin, safranin last greiii or safranin-geniian \ iolet. Ihe last (ombinaiion and the triple slain show the s|)in(!le libers exceptionally will. Reproductive Structures of Vascular Plants 773 5. 'J «, 3f " A.*^'- V^-''"'^#* '■•^'^^':« A' 1 (>•» ♦'*5 ^-. ^i^ « ^ .f^- Fig. 15.2— rt, Transverse section of anther of LUium regale; b. somatic divisions in developing archesporinm; r, archesporium, surrounded by differentiating tapetum; d, sporocytes in pre-leptotene pfiase. The manufacture of lily ovary slides showing the seven-to-eight- nucleate stage is unproductive and expensive. Most of the slides obtained from a ribbon show incomplete embryo sacs. Cutting an ovule longitudinally through the center and having all the nuclei in one section is a matter of chance. Commercial manufacturers have a sales outlet for slides having incomplete sacs and can therefore sell the few choice slides having complete sacs at reasonable cost. For routine teaching, with its attendant breakage of slides, it may be more satisfactory to purchase slides of the seven-to-eight-nucleate stage than to make them. Good fixation has been obtained with fair regularity with Allen-Bouin II and n-butyl alcohol dehydration. 174 Botanical Microtechnique Fig. 15.3— «. Lilltun regale, sectioned in paialiin, fust dixision of nieiosis in micro- sporocytes; b, smeared mlcrosporocytes of Tradescantia hrtuteata. Sax-Hiimphrey method, iron hematoxylin. Liliiim represents a type oi embryo-sac history that differs troni the type found in many of our important crop plants. Slides of lily ovary are relati\ely easy and inexpensive to prepare. This plant should be used to show the transverse floral diagram of the floAver bud (Fig. 17.4 a) ; the carpellary organization of the ovary (Fig. ■' • •• • '-'.y.:: .. ■ ■ '**- I'K.. \'i.\- ((. I rans\erse section ol llowcr Inid ol Llliiiin regale: h. o\ai\ of same. Reproductive Structures of Vascular Plants 175 15.4 /;) ; the origin and development of the ovule and integuments (Fig. 11.3); the origin and enlargement ol the megasporocyte (Fig. 11.3); meiosis, (Fig. 11.4) and the four megaspores in the unparti- tioned embryo sac (Fig. 15.5) . It is of interest that the embryo sac of Liliiim pardalinum is narrow and the megaspores are in linear order, whereas L. iimbellatum has a broad embryo sac and a cruciate quartet (Fig. 15.5) . The so-called normal type, which might better be named the common type, involves the formation of a cjuartet of megaspores, three of which degenerate, the fourth giving rise to the female gametophyte. This type occurs in maize, the legumes, tomato, and many other economic plants. The preparation of each of these is virtually a research task, and the reader who wishes to work on any of these plants should survey the literature on the desired plant. Lilium is a good subject for making preparations showing fertili- zation (Fig. 15.6). Begin collecting 48 hr. after pollination and make collections e\ery 12 hr. Use the killing fluids and methods recommended for the embryo sac. A series of collections will show stages from unfertilized mature embryo sac to young embryos (Fig. 15.6 fl). ^JV^4i3 ^^^ SI. Fig. 15.5-rt, First binucleate stage in embryo sac of Lilium tigrinum. Note the lag- ging cliromosomes; b, linear megaspores of L. pardalinum; c, cruciate arrangement of megaspores of L. umbcllatum. 776 Botanical Microtechnique Fig. 15.6— Fei til i/,atioii in Liliitin regale; a, sperm and egg in conlaci: h. spcini appressed to egg, zygote wall evident. THE EMBRYO, SEED, AND FRUIT Embryology is usuall) neglected in elementary courses, in part because of the high cost ol an adecjuate series of slides. Slides of early stages of embryo development are comparatively expensive to make, whereas the manufacture of slides of nearly mature embryos is more jMoductive. The embryo of lily is large and not diliicidt to j)repare. Use species that produce seed, such as Lilluiii regale or /.. lemiifoliin)). Cut the ovaries into disks not over L! mm. thick and dixide loneitu- dinally iiuo three sectors, each sector containing one locule. Good lixation ol the cnibr)o can be obtained consistently xviih Allen- Boiiin II. liaxing the formaldehyde solution reduced to 5',. Section transversely at 15 to IS ii. Ilu flat developing seeds are in long tiers, and a block yields many gootl sections, all cut longitudinalh ^vith the axis of the embryo (Fig. 15.7) . If six or eight sections are moinited on cadi slide, most ol ilie slides \v'\\\ contain at least one acciiiatelv cut ciiibiNo. Ihe most sal islac loiN sl;iin is iron hemalowlin \vith a ver\' li<'lu cotinierstain ol last tireeii, which stains ihc cell xvalls ol the embryo. Reproductive Structures of Vascular Plants 177 Slides of the caryopsis and embryo ol niai/e are not difFicult to make if the fundamentals outlined in the earlier chapters are ob- served. Consult the bulletins of agricultiual colleges for the methods of making hand pollinations. Collect the ears at the desired intervals after pollination. Remove the husks carefully and trim away two rows of kernels without damaging the adjacent rows. With a thin, sharp scalpel cut oft" the intact kernels close to the cob and drop them into a Petri dish of water. Lay a kernel, with the germ upward, on a sheet of wet paper, remove chaff from the base and trim a longitu- dinal slice from each side of the kernel (Fig. 15.1 /, A) . Also prepare some kernels for transverse sectioning by removing the basal and stylar portions of the kernel, saving onh' the portion from the tip of the coleoptile to the tip of the radicle. After the embryo is 25 days old, better infiltration of pieces for transverse sections can be obtained by transversely bisecting the embryo at the scutellar node, as well as removing the basal and stylar regions as above. The essential morphological structures of the kernel are well de- veloped in 25 to 30 days, and the pericarp becomes hard and brittle in 30 to 40 days. It is usually unnecessary to section the entire caryopsis after these dates. The embryo can be extracted easily be- tween the 15th and 40th day, or until the kernel becomes so hard that the embryo is fractured if an attempt is made to dissect it out. After the kernel has undergone maximum natural drying, or even if the kernel has been artificially dried for storage, the embryo can •A ) w Fig. 15.7— a, Embryo of Lilium regale; b, embryo of Lotus corniculatus (courtesy of Dr. Harold \\\ Hansen) ; c, embryo of Lycopersicum esciilentum. 178 Botanical Microtechnique be extracted. Soak the kernel in a solution that is based on the steeping liquor of corn processing j^lants. The solution used at present contains 2% sodium sulfite and 2[', lactic acid. It is necessary to test the time and temperature factors with each lot of grain. Try 20° and 35°C., and intervals of 1 to 3 days. When the germ can be loosened easily, trim away unessential parts of the germ, subdivide if desired, and drop into the fixing fluid. Small kernels, up to 10 days after pollination, are well fixed in Craf I or II. Older kernels and extracted embryos are penetrated better by Craf III. Xylene is the poorest solvent for infiltration, and chloroform is satisfactory only to the 15th day. Thereafter, a dioxan- normal or dioxan-tertiary butyl alcohol series makes possible the sectioning of 40-day kernels in paraffin (Sass 1945) . (See frontis- piece) . Embedded kernels must be soaked in warm water before section- ing. Adhesion of sections to the slide requires careful flattening of the ribbon, without overheating. For some research problems, hema- lum alone permits adequate diagnostic observation. A safranin-fast green stain is attractive, and for exhibition purposes it is possible to make gaudy muhiple stain preparations. Ages of kernels are given in the legends of figtnx' 15.8. Capsella bursa-pastoris is a favorite subject for embryology. The siliques are soft and easy to section. Although the seeds lie in the locules at various angles, seeds are so abundant that almost every section has complete embryos. Remove the fruits from the inflores- cence, and classify them roughly into age groiqxs in accordance with their distance from full-blown (lowers. Process each class in a separate bottle. A sequence of stages in embryo development can be built up bv sectioning fruits from the se\eral lots. Trim two sides of the silicpie to piomotc penetration. The long sili(|ue of Mdllhiola also max be used. Divide transversely into 2- to 3-nun. lengths for killing, and cut microtoine sedions longitudinally. Use Craf I for either of these criicifers. Lycojjcr.slf inn <'S( iiJoil iini . tlie tomato, is an cxcelU'iit subject lor dicot embryology. Use the small currant tomato, L. piiiipiiicllifoliiiui, seeds of which are obtainable from seed dealers. Slides of fertiliza- tion and the very young embryo are dilTicult and time-consuming Fio. 15.8— Zrn mays: n, kernel of pop corn 10 days after pollinalion; h. enibrvo of (Itnt (oui, 10 davs; c. cinhiAo. 15 davs: d. transverse section of kernel of dent corn, 25 da\s: r. kernil ot pop iomi. "20 da\s. ISO Botanical Microtechnique to obtain in quantity. Ten days after pollination the growing end of the embryo has developed into a large sphere that can be found in sections with adequate frecjuency (Fig. 15.7 c) . Collect develop- ing fruits of various sizes, slice out a transverse median disk of ap- proximately one-third depth of the fruit, and kill in Craf I. Dehydrate and infiltrate with care. Section transversely, 10 yi for the earlier stages, 15 to 18 |.i for larger embryos. As the seed coat of the maturing seed hardens, sectioning becomes increasingly difficult; however, the most important features of organogeny and the initiation of histogens are evident before the seed coat becomes excessively hard. The legtmies may be tempting stdojects for embryology, but the best known large-seeded species, as well as many small-seeded species yield very few slides for the time expended. Lotus cornkulatus is one of the most promising legumes. The long, straight o^"ar^ con- tains many ovules in straight alignment, and sectioning is fairly pro- ductive (Fig. 15.7 b) . J^'' \:V m >^sr. « -v'-'WKH..' *f* :*^- '■^>-i^ ^'I'^-l "■.'~'" ..: '.-''■''' ' . • .'.V-"^/-" "^ Fif;. 15.9— Transverse section of plumule in kernel of vellow dent corn. 30 (lavs after pollination (ct)inlesy of Dr. Robert S. I'airchild) . Reproductive Structures of Vascular Plants 181 Young fruits are processed in accordance with the methods given above for older ovaries. The curraiit tomato and many siliques are small enough even when nearly mature to have complete sections on a slide. Fruits that are more than 1 cm. in diameter should be subdivided and suitable pieces selected from the regions that are to be studied. The developing drupe of Priimis virginiana is an excel- lent subject. To prepare small cherry fruits for killing, remove a thin vertical slice from each side of some fruits and from the top and bottom of others, thus furnishing material for transverse as well as longitudinal sections. Kill in FAA for vascular study and in Allen- Bouin II or Craf III for better fixation of the embryo. The presence of brown pigmentation in many fruits produces poor contrast with the hematoxylins, but safranin-fast green is usually satisfactory. The great array of fruits available to the technician presents a wide range of size, texture, and other properties, from the juicy berry to the flinty caryopsis. It is, therefore, quite impossible to offer gen- eralized recommendations. The worker who ventures to prepare fruits and seeds has probably gained sufficient experience with easier sub- jects to adapt the fundamental methods given in this manual. /O. Microscope Construction, Use, and Care The microscope is probably the most indispensable ol the instru- ments used in the biological sciences. Intelligent purchase and effec- tive utilization of a microscope require an understanding of at least the elements of its optical and mechanical construction. It is an expensive instrument, built to the highest standards of precision and having possibilities of performance that are not fully utilized by many users. Although ha\ing some structural features that are delicate or even fragile, the microscope has adequate durability to give many years of useful service.* The function of a microscope is to produce an enlarged image of an object. This is accomplished by a system of lenses. A lens may be defined as a transjjarent body having at least one curved surface. A simple lens, consisting of one piece of glass, may be used to illustrate how a lens produces an enlarged image by bending or refracting light. A ray of light coming from the object enters the uj)per j)ortion of the curved face of a lens and is bent downxvard. Simihuly. a ray entering the lower portion of ihe lens is bent upward. The rays which pass through the lens converge and then continue as a diverging cone. If a shed of paper or ground glass is placed to inicKipi the rays which pass through the lens, an enlarged image of the object is pro- duced on the screen. A photographic jilate can l)c |)laccd in ihc (one of light and a pholograj)hi{ image obtained. A hand kiis or the lens on a simple dissecting microscope produces an image on a screen in this manner (Fig. Hi.l ./) . The ol))e(ti\e or loAver lens of a micro- scope consists of two lo nine lenses which act as a unit U) produce an image as described abc:)\e. There are certain limitations on the magnification and quality of image obtainable with the objective alone. Ihe primary image produced by the ol)jccti\e is iiiteicepted * By rourtcsy of llir Bausch X: Idiiih Ojiliral Compaiiv and llir Amciicaii Opiiial Compaiiv, valual)lc siiKUX'slions for Cliaplcis lli and 17 lia\c hrcii made 1)\ nicnd)iis of lluir MafTs. I he aiitlior lias drawn frci'ly on llit- (alalognc-s and sor\ ii c kallcis of the k-adins optical manufacturers. I 182 1 Microscope Construction, Use, and Care 183 and magnified, and improved in quality by an eyepiece. 1 he eyepiece or ocular consists ot two or more lenses working as a unit and having a fixed magnification. It a ground-glass screen, a sheet ol paper, or a photograj:)hic plate is placed at any plane above the eyepoint ot the ocular, an image is produced (Fig. 16.1 B) . Note that the primary image is inverted and the projected image is erect. Objecf Lens Image ^ D Object Objecilve Primary image Ocular Projecfed ■° imaqe Fig. 16.1— Formation of projected images by the microscope: A, simple microscope; B, comjjountl microscope.^ With a given objective and ocular, the size of the image varies with the distance of the screen from the ocular. If the screen is placed approximately 10 in. (254 mm.) from the eyepoint, the size of the image will be approximately equal to the product of the designated magnifications of the objective and ocular. Thus, an objective having a designated magnification of lOX. used with a lOx ocular, gives a total magnification of approximately lOOX- Exact values must be determined by micrometry. The foregoing discussion does not take into account the operation of the human eye working in conjunction with the microscope. However, most microscopic work is done by chrect visual observation with the eye held at the eyepoint of the ocular. Let us turn for a moment to a consideration of the eye as an optical instrument. The lens of the eye operates as a simple lens, and the curved retina is 1 The illustrations in this chapter are highly diagrammatic and simplified and are in- tended only to show the approximate relative positions of the object, the optical elements, and the images. 784 Botanical Microtechnique the receptive surface on which the image is formed. If an object is held at a given distance in front of the e)e an inverted image of definite size is produced on the retina. If a larger object is substittited at the same distance, or if the original object is moved closer to the eye, the visual angle, or the angle of the cone of rays between the object and the eye, is increased, the size of the retinal image is in- creased, and the object appears to be larger. In Fig. 16.2 A compare the two objects shown in solid and dotted lines, respectively, their respective visual angles Va^, Va.,, and the retinal images Ri^, Ri.2- When the eye is held at the eyepoint of the microscope, it inter- cepts the image-forming cone which has a definite angle established by the microscope, and a retinal image of definite size is produced (Fig. 16.2 5). The observer sees a magnified virtual image, which appears to be near the level of the microscope stage, approximately 10 in. from the eye. The retinal image is erect, the virtual image is Object, Objectj r--. J a Object Objective Primary image Ocular Reilnal image B Virtual image Fk;. 16.2— a. Formation of imai^cs hv the eye showinj; relative size of retinal image in relation to \isnal anj;ic: />'. leiiiial and \irtual imai^es ol)taine(i with a (omponnd luinoscopc. Microscope Construction, Use, and Care 185 inverted, and the direction of motion of the object is reversed. I'lic apparent size of the virtual image is the same as if the observer viewed the projected image on a screen 10 in. from the ocular. As a specific case of magnification, let us view an object 0.1 mm. long with a lOOX microscope. This produces a retinal image of the same size and the same impression of magnitude as if we looked at the lO-mm. image projected by the same microscope on a screen 254 mm. from the eyepiece. Properties of Objectives MAGNIFICATION The most obvious property of objectives is magnification, which is a fixed value under the conditions outlined in a preceding para- graph. The objective magnifications used most commonly on standard monobjective microscopes range from 3.2x to lOOX- Magnifications below this range are used on paired-objective stereoscopic prism bi- nocular dissecting microscopes. Objectives above lOOx have rather limited uses. The conventional low-power objective is lOX- The lower powers, from 3.2X to 6X. are not fully appreciated and deserve more serious consideration. WORKING DISTANCE Free working distance is the distance between the objective and the cover glass, using a cover glass 0.18 mm. in thickness. The cata- logues of the manufacturers give the working distances of their objecti\ es. A few selected illustrations show the relation between mag- nification and working distance: lOx, 7.0 mm.; 43x» 0-6 mm.; 45x> 0.3 mm.; 95 X> 0-13 mm. It is obvious that for an elementary class the most desirable high-power objective has a magnification in the forties and the longest available working distance. Objectives of high magnification and short working distance must be used with care to avoid damaging the front lens and the slides. FOCAL LENGTH If a beam of parallel ra)s is projected through a simple lens, the rays converge at a point. The distance from this point to the optical center of the lens is the focal length. In an objective consisting of several components, the situation is somewhat more complex, and a different \alue is used. The manufacttners engrave on the mount- ings and list in their catalogues a value known as the ecjuivalent focus. At the standard projection distance of 250 mm., an objective 786 Botanical Microtechnique that has sexcral components and an E. F. of If) mm., -will give an image ol the same size as a simple lens of 16 mm. focal length. The E. F. should not be confused with working distance. 1 he equiAalent focus decreases as the magnification increases. The experienced worker is in the habit of speaking of an obiecti\e as a 4-nnn. objective, for instance. For class use it is much better to speak in terms of magnification, which is 43x in a certain 1-imn. objective. In the past the manufacturers have paid undue attention to computing their objectives so that the equivalent focus is an even number, and a series of objectives will have the equivalent focus in the orderh progression 16, 8, 4, 2 mm., etc. A magnification may turn out to be some awkward fractional ninnber like 3.2 or 5.1. A more practical series would be a sequence of magnifications such as 3, 5, 10, 20, 40. 60. etc. There is a trend toward the use of the latter system. DEPTH OF FOCUS A minute body or a very thin section has thickness or depth. If a deep cell is being viewed \\ith a lOx objeciixe and the lens is focused on the upper wall of the cell, the bottom wall mav also be in focus. If a 45 X objective is focused on the top wall, the bottom wall may be completely out of focus and practically invisible. If the lens is focused on the bottom wall, the top wall becomes obscured. The vertical extent of the zone of sharp focus is known as the depth of focus. Depth of focus decreases as the magnification increases, although magnification is not in itself the determining factor. There are mathematical limits to the dejMh of \iew encompassed by a given objective. Magnification and other factors being equal, objec- tives of the several manufacturers have the same depth of focus. RESOLVING POWER Resolving power is that projjei t\ of a lens ^vhich makes possible the recognition, as distinctly separated bodies, oi objects that are exceedingly close together, oi subtended b\ a small \'isual angle. I he simplest illustration of resolving power is the visibility of double stars. Although ihe two stars may be sejiarated b\ a vast distance, the visual angle reaching the eye is very small, and the stars appear to be close together. Man) indixiduais can see but one star. Othei persons, whose eyes have better resohing power, can see the txvo stars distinctly. Applying this prin(ij)le to the microscope, a lens of poor resolving powei will show a slender chromosome as a single thread, whereas a lens of good resohing power will show the chromo- Microscope Construction, Use, and Care 187 some as two interwound threads. Tlie question to ask concerning an objective is not "how small a thing can you see?" but "what is the minimal separation between two objects that the lens can resolve?" Ihe mathematical derivation of the formula for determining resolving power can be found in textbooks of optics or physics. The formula contains the following factors: // ^ the lowest index of refraction in the path of the rays, i.e., the index of refraction of water, glass, air, cedar oil, balsam, etc. u = half of the angle made by the effective cone of rays entering the objective. This value can be obtained from a table in Gage (1936) or from the manufacturers. N.A. = numerical apertme, a number that is indicative of relative resolving power. The formula is N.A. = u -sin u The value of the numerical aperture is engraved on most modern objectives and is given in the catalogues of the manufacturers. This number is 0.25 in a lOx objective, for example, and increases through progressively higher magnification, attaining the value 1.4 in an expensive 90 X objective. Knowing the nimierical aperture, we can make a simple compu- tation and arrive at a tangible value of resolving power. Assume that we are using an objective of N.A. 1.0 and using light having a wave length, in round numbers, of 0.0005 mm. The formula is I {= wave length) 0.0005 = = 0.00025 mm. 2 N.A. 2 This means that if two bacteria or two chromomeres on a chromo- some are separated by a space of 0.00025 mm., they can be seen as two distinct bodies. As the numerical aperture increases, the resolving power increases, the working distance and depth of focus decrease, and the cost increases. The practicable upper limit of N.A. 0.95 is obtainable with dry lenses, used with an air space between the objective and the cover glass. In accordance with the foregoing formula, the N.A. can be increased by increasing the value of n or of sin u or both. If the angle of the ray that passes from the glass slide to air is greater than 41°, the light is totally reflected back into the glass. This phenomenon limits the angle that determines ??. If a drop of cedar oil or synthetic 788 Botanical Microtechnique silicone immersion fluid is used to connect the immersion objective with the slide, the values of n and sin u are both increased, and con- secjuently the resolving power is increased. An N.A. of 1.10 can be obtained with a water immersion lens, and N.A. 1.40 using cedar oil. OPTICAL CORRECTIONS The foregoing discussion of the properties of objectives does not take into account the quality of the image produced. A simple lens produces a decidedly imperfect image. Rays of white light which enter the lens are broken up to some extent into a band of colors, a spectrum. These colors are not brought to a focus at a common point; blue is converged at a point closer to the lens than is red. Consequently, the colors of the object are not reproduced accurately, and a color fringe or "rainbow" is \isible along the boundaries of objects in the microscopic image. This is known as chromatic aber- ration. Spherical aberration is a defect that produces poor definition in the center of the field. This defect is aggravated by a cover glass that is not within the maximum thickness range of 0.15-0.21 mm. Image quality also is impaired by variation from the standard tube length of 160 or 170 mm. designated by the manufacturer. Certain objectives have an adjustable correction collar on the objective mount, calibrated for variations in cover-glass thickness. Curvature ol ilie field is another structural defect in the micro- scopic image. If the center of tlie field of \ iew is in sharp locus, the margins may be out of focus. With some oi)jecti\es, the image may be distinctly dome-sha})ed. Ihe degree to which object i\es are cor- rected for the above color and structinal defects of the image will be indicated in the discussion of the optical categories in which objectives are classified. PARFOCALIZATION 1 wo or more objectives are parfocal with eadi other when u is [)ossible to locus one obiecti\e on an ()l)je(t, tmn lo the next objective without focusing, and see the objed in more or less sharp focus. This feature is extremely important wiili large classes of elementary students. If the conventional lOX 1<>^\' power and the 10 lo l.")\ high power are not parhxal. the siudtiii must rclociis with the latter lens, \vhi(h has a short woiking distance, small luld. and shallow depth ol focus. Excessive breakage of slides and scraidiing of objec- tives o((ur if the ol)jectives are not parfocal. Adjusimcnt ol the old- /Microscope Construction, Use, and Care 189 style objecti\es should be left to the manul'acturers or to a skilled insiiunient mechanic. The new Baiisch and Lomb objectives have an internal adjustment, with which the student cannot tamper but which can be easily adjusted with a special wrench. Dry objectives between 10 X and 60 X tan be made parlocal in any combination. The older 4 to 5X objectives cannot be so adjusted, but the American Optical Company now makes a 3.5 and a 5.1 X objective, and Bausch and Lomb makes a 3.2 and a 6X objective which can be made parlocal with the lOX. 'I'l^l parfocal with the 43 X within a quarter turn of the fine adjustment. With a combination of 3.2, 10, and 43 X objectives students should be taught to change magnification up or down in that sequence, thereby mini- mizing damage to slides and lenses. Types of Objectives ACHROMATIC OBJECTIVES These are in the lo-^vest price class and are used on classroom microscopes and for routine work in research. In these oJDJectives chromatic aberration is corrected lor two colors and s})hcrical aber- ration for one color. Achromatic objectives have undergone relatively greater improvement in recent years than have other types. FLUORITE OBJECTIVES In these objectives the mineral fluorite is used in conjunction with special optical glasses. The corrections are of a higher order than those of the achromatic series. Fluorite objectives are particularly useful for photomicrography by virtue of excellent color correction. Thev are aAailable only in magnifications over 40 X- APOCHROMATIC OBJECTIVES These objectives ha\e chromatic aberration corrected for three colors and spherical correction for two colors, affording brilliant images, presented in their true colors and without distortion of shape. The highly actinic violet rays are brought to the same focus as the longer visual rays, making these objectives highly desirable for pho- tography. Apochromatic objectives are expensive because of their complex construction and the scarcity of suitable fluorite. Oculars (Eyepieces) Oculars have distinctive optical characteristics that must be un- derstood in order to use the correct ocular, and the correct combina- tion of ocular and objective under specific conditions. An ocular has a 190 Botanical Microtechnique definite cc)ui\alent focal length. This value nia\ l)e obtained from the catalogues, l)ut a more useful designation, \\hich is engraxed on modern oculars, is the magnification value, which ranges from 1 to 30 X- YoY routine work and for classwork lOx is the most satisfactory magnification. The lower magnifications are likely to ha\e marked curvature of the image. Higher magnifications cause increasingh greater eyestrain, which is very pronounced \\ith the 30X- Further- more, there is an upjjcr limit, beyond which the ocular produces only empty magnification, with no gain in the revealing of detail. The maximum effecti\e ocular magnification, which may l)e used with a given objective, can be computed easily. Assume that a 13 X objective of N.A. 0.65 is l^eing used; the formula is 1,000 (X.A. of objective) (1000) (0.65) Magnification of objective 43 ^ 15Xj tl'^e approximate maximum ocular magnification. It is exident that with a microscope on \vhich the 43 X objecti\e is the highest }K)wer used, an ocular magnification of over 15X is of no value with respect to resolving power but of possible value for counting or drawing by projection. 7 his simple calcu- lation will enable a purchaser to specify the most useful lens combinations. Modern ocidars are parfocal, making it j)()ssiijle to interchange ocidars of diiferent magnifications xvithout reciuii- ing nuK h change of focus. OPTICAL CATEGORIES OF OCULARS Huygenian oculars are of comparati\ely simple two lens con- struction. They are designed for use xvilh achromatic objedives and yield inferior images with apochromatic objet ii\es. Comjjensatiiig (xulars aie designed to correct certain residual aberrations inhereni in ajxx hromatic objectives. It is, therefore, im- perati\c' to use compensating oculais with apoc In oniatit objecti\es, and oculars and objectives must be of the same make. These oculars mav be used with .uhroniatic and Ihiorite objedixcs ha\ing magnifi- cation oxer lOX- Flat IrU! (xuhu's are ol the noiuompeiisaling t\pe and \ ield imaues in wiiich cin\aiuie has been considerably reduced. Ihesc oculars have various iiade nanus. H\|)eri)lane and Planascopic being the l)est-kn()\\ n. A serious oljjection to some oculars ol this type is that the eve nuisi be held rigidly at a restricted eye position. The Microscope Construction, Use, and Care 191 slightest lateral motion of the head cuts off part ol the field, and prolonged use produces marked fatigue. AV^ide-field oculars (noncompensating) have an exceptionally wide field and good correction for curvature but may have a restricted eye position as in the Hat-field type. This objection may be raised concerning high-eyepoint oculars, which are designed to permit the use of spectacles by the observer. Workers who must use spectacles with low-eyepoint oculars find that the lenses of the spectacles and oculars become scratched after some use. A simple remedy is to paste a narrow ring of felt over each ocular. This permits the user to press his glasses against the ocular and to utilize the full field, without damage to the glasses or the ocular even after years of use. Illumination The most common method of ilhuiiinaiing a slide or other transparent object is by transmitted light. 1 he light is projected through the hole in the stage and passes through the preparation. The simplest device for projecting light through the specimen is a concave mirror under the stage, designed to focus a converging cone of ravs at the level of the specimen. Regardless of the character of the light source, whether daylight or artificial light, the curried mirror should be used if the microscope has no condensing lenses under the stage. The intensity of the illumination is controlled either by an iris diaphragm, or by a rotating disk ha\'ing a series of holes of different sizes. Microscopes that are used for advanced work are usually ecj[uipped with a condenser. A condenser is a system of two or more lenses under the stage, designed to receive a beam of parallel rays from a flat mirror or a prism and to converge the light at the level of the stage. The simplest type of condenser, known as the Abbe condenser, consists of two lenses. Although Abbe condensers are not corrected for color or curvature, they are adequate for classwork and for much of the routine work in research. The N. A. is 1.20 or 1.25. The upper lens may be unscrewed (not in an elementary laboratory!) ; the lower lens then serves as a long focus condenser of N. A. 0.30, suitable for use with objectives of lOX (N.A. 0.25) or less. On some Leitz models the upper element of the condenser is on a swinging yoke, whereby the upper lens can be swung aside, leaving the lower lens as a long focus condenser that fills the field of the lowest powers. A three-lens con- denser with N.A. 1.40 is available for use with objectives having an 792 Botanical Microtechnique N.A. greater than 1.25. One or both upper lenses are removable, givino; N.A. 0.70 and 0.40 respectively. Ajjlanatic and achromatic condensers, made by several manu- facturers, have excellent corrections lor color and curvature. The elements, usually in 3 units, are separable, affording combinations with N.A. ranging from 0.20 to the fidl 1.30 or 1.40 of the complete condenser. Hie resolving power inherent in an objcctixe can be utilized only if the illuminating system has a numerical aperture equal to that of the objective. A curved mirror has an approximate N.A. of 0.25: therefore, it meets the aperture requirements of a lOx (16 mm.) objective. Microscopes having objectives of over N.A. 0.25 should be cquipjjcd with a condenser, provided that the users are sufficiently skilled to use the condenser properly. An improperly adjusted condenser is worse than having no condenser. Some teachers prefer not to have condensers for large elementary classes in which thorough training in microscopy and close supervision are difficult. The conventional high-power objective on elementary class microscopes is a 4-mm. objective, 43x or 44x. N.A. 0.65 or 0.66. Many thousand instruments of this type are in use, ecjuippcd wiih an Abbe condenser of N.A. 1.20 or 1.25. If this condenser is not focused accurately it is a handicap, furthermore it does not co\er the field of objectives below lOX- Removal of the condenser or of its upper element, a common jjractice among advanced workers when using low jiowers, is a most undesirable practice in large classes of bcginneis. The need for a condenser designed specilicalh for low and inter- mediate powers has been met by the American Optical C^o. (Spencer Lens C^o.) and the Bausch 8: Lomb 0])tical C>o. Ihese condensers have ninncrical apcriures of .(>() and .70 rcspcc ti\ely. and therefore meet the aj)ci tuie rec|uirements of 4 mm.. 43x or 44x objectixes. and aKo illunu'iiaic tiu' (icld of a 3.2x "•' higher power object i\e. A maximum N.A. of 1.00 can l)e obtained wiiU a (ondenser if the condenser lens and the slide are separated 1)\ a hiNcr of air. Obviously, an oil-immersion ol)je(ti\e of N.A. 1.30 does not \iekl maxinuim j^erformance luiless the (ondenser, as well as the object ixc is (onnccicd to the slide Avith (cdar oil. Rcsearcli woi kers Avho \\ish to obtain maxinunn resohing power make a routine practice ol iiiuiici^ing the condenser. There are some practical objections to this practice for classwork. Dark-field illumination is a neglected, but usclul iniihod of observation. In this nuiliod the light that reaches the eye from the Microscope Construction, Use, and Care 793 object does not pass through the object but is rcflecied from ilie surface of the object. None of the light from the illuminant reaches the eye directly. The object thus appears to be self-luminous against a black background. Illumination of the object is obtained by either a standard condenser provided with an adapter or by means of a special dark-field condenser. The simplest form of adapter consists of a wheel-shaped metal disk inserted into the slot below the condenser. The center of the disk cuts off the central rays of light and illuminates the object with the oblique marginal rays. A more effective adapter is a unit that replaces the upper element of the Abbe condenser. The much more expensive dark-field condensers are of two principal types. Refracting condensers provide an oblique cone of lioht by refraction through the marginal regions of the condenser lenses. A disk below the central region of the condenser shuts out light from that portion. Reflecting condensers produce an oblique cone by total reflection from internal surfaces of the condenser lenses. Diagrams and descriptions of the various types of condensers can be found in the catalogues. Dark-field illumination is recommended for the study of filamen- tous or unicellular algae and fungi, as well as for unstained sections of tissues. The cytoplasmic strands and nuclei of Spirogym and cyto- plasmic streaming in leaves of Elodea and filaments of Rhnojms make striking and instructive demonstrations. The discussion of sources of light for the microscope has been deferred to this point, where the source can be discussed in conjunction with the condenser and the other optical components. Illumination is said to be critical when the source of light is super- imposed on the object. This means that if an unfrosted tungsten coil bulb is the source, the coil is sharply defined upon the object. It is true that the portions of the object that coincide with the coil are under critical illumination, but only a very small part of the field may be so illuminated, and it is obvious that a naked coil cannot be used in this manner. A frosted bulb is some improvement, but the granularity of the bulb is visible under critical conditions, as defined above, and the curvature of the bulb is visible under lower powers. If the condenser is lowered to obscure the granularity and cur\ature, the resolving power is decreased. The desirable source is a flat, luminous, grainless surface of sufficient size to cover the field of the lowest power objective. When 194 Botanical Microtechnique such a source is superimposed on the object fickl. uniloi ni illumination is obtained. For elementary class use, and for many roiuine tasks in research, the nearest approach to optimum ilhmiination seems to be an opal glass disk in a suitable lamp housing, with a 50 to 60 watt frosted blue mazda bulb. Place the lamp 8-12 inches from the microscope and manipidate the mirror until the field of view, with an object in focus, is imiformly illuminated. If the microscope has a condenser, place the point of a pencil against the lamp disk and adjust the height of the condenser initil the pencil point is in focus. If finely ground glass, or a plano-convex lens with a ground fiat surface is used instead of opal glass, the condenser must be moved out of ojjtimum position to eliminate the granularity in the field of \ iew. Ihe use of a lamp that has a condensing lens system and a diaj^hragm is discussed in the chapter on photomicrography, and the Avorker who wishes to do critical visual work should consult that chapter. Mechanical Operation A microscope usually has a set of two to foin- objectives per- manently installed on a revolving nosepeice. The objective are centered and parfocalized, each screwed into its designated opening in the nosepiece. The older nosepieces ha\'e adjustable stops for lateral centering of individual objectives. Improvements in manidacturing methods have made possible the quantity jjroduction of nosepieces of such precision that no adjustments for centering are recjuired on the nosepiece. The removal of objectives should be strictly forbidden in the classroom. The bod\ tube of the microscope on \vhi(h ihe objectives and ocidar are mounted is ni()\cc! up and down b\ two mechanisms, a coarse adjustment whidi produces lapid tlisplacemeiu. and a luu' adjustiiuni whidi movis the i)od\ tid)e very slowly. Ihe coarse adjustment is actuated i)y a lack and pinion. This de\ice is j)ractically identical in the se\eral leading makes. The liiihlness ol the action can be adjusted easily by tightening or loosening the sj)lit bearing block against the pinion shaft b\ means of the readily accessil)le scre^vs. In the Zeiss instnunent the action is tightened by grasping the pinicju heads firmly and screwing them lowaid each other. I'he (ine-adjustmc nt mechanism dillers radically in the dilferent makes. One tyj)e employs a gear-and-sec tor device in which onh a few teetii are in contact. This action, liiough \ ci \ smooth and responsive, is rather delicate and easily damaged. The most rugged Microscope Construction, Use, and Care 195 type is actuated by a s])lit mil which has mniicrous threads in permanent contact with a worm gear, llie threads are almost impossible to strip, and this action has excellent responsiveness. Details ot construction of the various makes may be obtained Irom the illustrations and description in the catalogues. The repair ol line- adjustment actions should be entrusted only to a highly skilled mechanic or to the manufacturer. The normal procedure in using the microscope is to locate the object with a low-power objective and then turn to the next higher power. Objectives of lOX or less are the most satisfactory finder lenses because of their large field of view, considerable depth of focus, and long working distance. Microscopes for elementary work should be equipped w'ith a safety stop on the body tube which prevents contact between the slide and the low-power lens. With an objective ot lOX or less in position, it is safe to rack the boch tube down luitil it is stopped by the safety stop. With the body ttU^e in this jjosition look into the ocular and manipulate the mirror until the field of view is uniformly illuminated. Move the body tube upward with the coarse adjustment until the image is visible, then bring the image into sharp focus with the fine adjustment. Search the section by moving the slide, using the fine adjustment freely to bring into sharp focus structural features at different depths in the specimen. \Vhen it is necessary to turn to a higher magnification, center the desired structure in the field of view and bring it into sharp focus with the lowest power. Without chayiging the focus, ttu-n the objective of 7iext higher magnification into position. A properly parfocalized objective has ample clearance. The image should now be visible, and it should recjuire not more than a cjuarter turn of the fine adjustment to bring the image into sharp focus. The safety stop provided on the barrel does not prevent pressing the high-power objective upon the slide. Iherefore, the high-powder objective should never be used for locating the object. If an objective of 3 to 5X is used, do not change from this low magnification to 43 X' btit go progressively up through the range of magnifications. Similarly, go down the range progressively. The manufacturers can furnish safety stops for installation on the tubes of older microscopes. Some teachers prefer to have the objectives adjusted so that when the object is located with the low power, and the high-power objective is swung into position, a slight ujnvard movement brings the object into sharp focus. The objection to this arrangement is that, if the user inadvertently moves the bod\ tube downward, he is mG\'ing it farther 796 Botanical Microtechnique out of focus and may not stop until the slide is smashed. As an alternati\e arrangement the high-power objective may be parfocalized so that, when it is swung into position, the image is visible and a slight downward movement brings it into sharper focus. An accidental movement in the wrong direction, upward, will then do no harm. Students should be told firmly that there is no excuse for ttuning a fine-adjustment knob more than a half revolution in either direction. On the best modern microscopes very little pressure is exerted on the slide when the bod\ tube is lowered upon it with the fine adjustment. The condenser and illuminant are introduced again at this point. Assume that a grainless disk of a lamp serves as the immediate source of light. Adjust the height of the condenser until the surface of the disk is in view simultaneously with the focused specimen. The object is then within a disk of light of uniform linninosity. Ob\ iously, the condenser does not project a point of light, but a disk of light in the plane of the specimen. Although a groinid-glass source approaches the requirements for correct illumination, the condenser must be lowered to move the granularity out of focus. An opal glass disk permits a closer approach to correct illumination. For general class use, the most practical light source is a lamp with a grainless or nearly grainless diffusing disk. It is preferable to have the lamp fastened to the table in constant relation to the position of the microscope. Under such conditions, the condenser, especially the low N.A. condenser described on jiagc 192, can be mounted in sinijile ring mounts that are not adjustable by the students. Ihc position of the microscope in use depends to some extent on the height of the available table and chair in relation to the physical lu.. 1{)..'! — Hiiigc siDj) lor class- room microscope. l)iiil(l ol the user. Hard and last iiilcs ol posliu'e are lidicidoiis iu a classroom Iia\ing tables aud (liaiis ol lixed. uuilonn height, and students of di\cise l)iiil(l. A \(i\ short person shouUI (ertaiuh lili the Microscope Construction, Use, and Care 197 microscope for most work. However, if a fluid mount is used on a tilted stage, disturbing currents are likely to be set up in the litjuid, and the liquid might drain into the diaphragm; therefore, it is advisable to use wet preparations on a horizontal stage. To forestall the progressive trend of weary students toward a reclining position, a hinge stop can be installed on modern microscopes, preventing tilting beyond 30° (Fig. 16.3) . Micrometry The measurement of minute objects by means of the microscope is an interesting and valuable feature of microscopic study. Although the procedure is simple and rapid, the method does not receive adequate attention in teaching. The simplest form of measuring device is an eyepiece micrometer, a disk of glass having an engraved scale, a series of accurately spaced lines. The spaces do not have a standard value, and each disk must be calibrated for each given ocular and set of objectives. Place the disk upon the metal diaphragm in the ocular. If the diaphragm is in the correct position, the lines on the disk will be in sharp focus. Occasionally, these diaphragms become displaced, but they can be pushed back and forth with a softwood stick until the eyepiece micrometer is in foctis. The stage micrometer with which the calibration is made is a slide bearing an engraved scale with known values, usually in tenths and Stage micrometer scale 13.6^ I HI O.lmm. 0.01mm. Eyepiece scale | -- 50 divisions -^ =0.58 mm. Fig. 16.4-Calibration of an eyepiece micrometer disk and measurement of a minute object. hundredths of a millimeter, but scales in hundredths of an inch are obtainable. When the stage micrometer is brought into focus, the scale of the eyepiece will be seen superimposed on the scale of the stage micrometer. Shift the stage micrometer and revolve the ocular until the two scales are in such position that the values may be compared. A specific case using a 43x objective and a lOX ocular is shown in Fig. 16.4. It will be seen that the 50 small divisions of the 798 Botanical Microtechnique ocular scale, only five of which are shown in Fig. 16.4, are equivalent to 6.8 large divisions or 68 small divisions of the stage micrometer scale. The computation is: 50 eyepiece divisions = 0.68 mm. 1 eyepiece division = 0.0136 mm. = 13.6 microns (m) The curved spore in Fig. 16.4, occupies one space on the ocular scale, and is 13.6 \^i long. The loose eyepiece disks described above are easily lost if they are not kept permanently in the ocular. In a large department it is an economy, over a period of years, to buy special micrometer eyepieces instead of disks. These eyepieces have a built-in disk, and the eye lens is adjustable to focus the scale sharply for the eyes of different indi- viduals. Consult the catalogues for descriptions of micrometric devices. Microprojectlon The discussion of image formation showed that an image is produced if an intercepting screen is placed above the eyepoint of the ocular. With a sufficiently darkened room, a brilliant light source such as an arc lamp, and a good screen, an acceptable image can be obtained with the highest powers of the microscope. However, the most satisfactory results are usually at low and moderate magnifi- cations. An image can be projected on drawing paper and a diagram- matic or detailed drawing made with considerable accuracy. Calibrations must be made for each lens combination and projection distance. This is done by projecting the image of a stage micrometer on the screen, measuring this image Avith an accurate ruler, and computing the magnification. The catalogues and service leaflets of the manufacturers furnish detailed descriptions of a -wide range of typos and price classes of microprojectors. Types of Microscopes In the foregoing discussion of the elements of microscopy, the various types and makes of microscopes were not specifically discussed. A simple microscope is one that uses only one lens unit to magnify the object. The lens unit may be a single lens. A pair of lenses in fixed relation to each other comprise a doublet; a triplet consists of three lenses in a mounting. The most useful magnifications range from 6 Microscope Construction, Use, and Care 199 to 12 X- Magnifications up to 20 X are available, but, as the magnification increases, the size of the field and the working distance decrease. A compound microscope is one in which a lens unit, the objective, produces a magnified image, which is in turn magnified by a second lens unit, the ocular. By far the most common type of compound microscope employs one objective and one ocular in working position at one time. This is known as a monocular monobjective microscope. This type is durable, has a wide range of usefulness, and permits full use of the performance capacities of the optical system. The principal objection is that the user employs one eye at a time, and the tendency to use one eye more than the other causes excessive eyestrain and fatigue, A binocular monobjective microscope uses a matched pair of oculars with a single objective. A system of prisms in the binocular body tube splits the beam coming from the objective and produces two images of identical magnification and intensity. The use of both eyes diminishes eyestrain and fatigue, and there is an impression of depth and perspective to the visual image. Ocular tubes of the binocular body are parallel in the majority of the principal makes. The tubes converge at an angle in the standard Spencer binocular, but this firm will furnish parallel tubes. Convergent tubes present the image to the eye as if the image were at ordinary reading distance. When using parallel tubes the eyes are relaxed, as in looking at an object at a considerable distance. Some microscopists are convinced that they can use only one or the other of these two types of binocular with comfort, whereas other workers can use either type effectively. The binocular body has adjustments for separating the ocular tubes for the inter- pupillary distance of the observer. One ocular tube has a vertical adjustment for correcting slight differences of focus of the two eyes. To make this adjustment, select a minute structure in the specimen, close the eye over the adjustable tube and focus on the object with the fixed tube. Now close the eye over the fixed tube and bring the image into sharp focus in the adjustable tube with the focusing device on this tube. The quality of the image obtained with binocular bodies is equal to that obtained with the single tube. Supplementary binocular bodies that are designed to be placed upon older monocular microscopes, have the tube length increased by the superimposed binocular body. A reducing lens system must therefore be used to bring the magnifica- tion back to the standard designated value. The most modern, and 200 Botanical Microtechnique ill mail) ways most desirable, binocular body has the eyepiece tubes inclined. This permits the head to be held in a comfortable position and greatly reduces fatigue. An important category of binocular microscopes utilizes matched pairs of objectives. This type is customarily known as the dissecting binocular or stereoscopic binocular. These instruments show true perspective and depth. The image is erect, thus facilitating dissection, isolation, and other manipulations of the object. The practical range of total magnifications is from 10 to 150X- Two or more pairs of parfocal objectives can be installed on a nosepiece of either the revolving or sliding shuttle type. In one Spencer model a set of objectives may be permanently installed on the objective changer, a desirable arrangement for class use. For research work, each pair of objectives may be obtained in a removable mounting, readily inter- changeable on an objective changer, which, in the several makes is either a rotating drum, a rotating disk, or a sliding shuttle. Several categories of noncompensating oculars are available for twin-objective binoculars. The standard Huygenian type is the least expensive and probably the most satisfactory for classwork. AVide- field ocidars are well worth the greater cost. Two manufacturers produce a good junior-wide-field ocidar, intermediate in cost and performance between Huygenian and wide-field oculars. High eye- point oculars also are available, but they recjuire that the eyes must be held at restricted eye position, making these ocidars objectionable to some workers. This chapter would be incomplete without a few words concern- ing the durability and life span of the microscope. It must be obvious that the period of service obtainable from a well-constructed micro- sco])e depends uj)on the skill and care with which it is used, the amount of use, and certain environmental conditions, such as atmos- pheric conditions, extremes of temperature, and corrosive chemical fumes. An outstanding illustration of durability is afforded by an occasional microscope that seems to be in excellent mechanical and optical condition after .10 xtais of continuous research service. On the other hand, a classroom instrument may be in poor condition after 10 years of use. Serious scratching and corrosion become CNident first on the 4-mm. dry objective, the oil-immersion objective, and on oculars, especially the type having a raised eye lens. The lower power ol)ieclives should show no contac t wear or corrosion, especially if the instrument has a safety stop on the liody tube. Examination of large numbers of class microscopes has shown that the serviceable Microscope Construction, Use, and Core 201 period of a microscope is approximately 20 years. Replacement of the ocular and high-power objectives after 15 years is a good invest- ment which may extend the life of the microscope for another 15 years. Periodic mechanical overhauling and refinishing of metal parts should be done by a competent fine-instrument mechanic. Major repairs and lens work should be entrusted only to the manufacturer. Considering the first investment, the low cost of upkeep, the large trade-in allowances, and the many generations of students served during a normal life span of a microscope, this instrument is the least expensive item of laboratory equipment. The foregoing brief discussion of the principal types of micro- scopes and of the essential optical and mechanical features can be supplemented by a study of the well-illustrated descriptive catalogues of the leading manufacturers. Details of construction of specific models are available in leaflets provided by the manufacturers. The belief in the superiority of the continental European optics may have been well founded 50 years ago, but is no longer a prime factor in purchasing an instrument. A choice among the better-known makes is now largely a matter of personal preference. The prospective purchaser should examine and, if possible, use various models and base his preference on mechanical and optical features and specifica- tions that meet his needs. /7. Photomicrography The use of photomicrographs for illustrations in teaching and research has become a firmly estaljlished practice. A choice between drawings and photomicrographs should be based on an understanding of the limitations and possibilities of these methods and upon the method of reproduction to be used. A drawing may be said to ex- pound and explain the subject, while a good photograph is an accurate, impersonal reproduction of the subject. A drawing may be a routine diagrammatic record of rather gross structures, or it may represent the interpretations of the microscopist, either in full detail or in idealized, semidiagrammatic form. The routine type can be made by an artist; the interpretation drawing can be made only by the investigator sitting at his microscope. Photographs have similar characteristics and range from mere routine recording to the most critical probing of structural details. Instead of arguing the relative merits of dra^vings and jihoto- graphs, the experienced and versatile worker simply decides which method will best serve a specific need and uses such talent as he has or can hire. A few simple examples will illustrate the criteria by which a choice can be made between methods of scientific illustration. A cross section of a corn stem, or the corn kernel in the frontispiece con- tains several thousand cells. To make a drawing which woidd purport to be an accurate cell-for-cell representation would be an almost in- credibly laborious task (for someone else to do) . A photomicrograph of such subjects reproduces with acceptable accuracy the number, distribution, shapes, and sizes of the numerous cells and, furthermore, reproduces texture in a way that can only be remotely approached by the most talented artist. Photomicrograjihs of this type can be made only by a photographer who is familiar with plant materials. Controversial siil))i(ts or luw and siriking discoveries deserve photographic illustrations. The reader has greater confidence in a [ 202 ] Photomicrography 203 description if it is evident that tlic investioator had presentable preparations. In iUnstrating some materials the very act of making an ink drawing on paper exaggerates magnitude, visibility of details, and texture. For instance, protoplasm does not consist of discrete dots and sharp lines. A photomicrograph accompanied by an inter- pretation drawing affords much more convincing illustrations of many subjects than does either method alone. The making of record photomicrographs is often an essential part of diagnostic routine in clinical, chemical, criminological, and many other studies. Under standardized conditions, especially if there is some uniformity in the character of the subjects, such photomicro- graphs can be made by a well-trained technician. In some fields of research it is desirable to make photomicrographs of specialized subjects. The investigator is the only one who can locate and recognize the structure under the microscope. He must determine the proper focal level, the correct magnification, color filters, and other factors. The exposure time of the first trial may be a vague guess. The negative must then be developed at once, and the exj^osure time corrected. It may be necessary to make several negatives at different foci in the same field of view. After a correctly exposed negative is obtained, the investigator must personally decide from a contact print whether the photograph shows the desired structures. Research photomicrography of this type is clearly an in- separable part of the research and must be done by the investigator in person, with his research microscope and frequently without dis- turbing the slide that has been under scrutiny. It is a common fallacy that a photomicrographer must be primarily a photographer, who can easily and quickly "pick up" what he needs to know about the microscope. On the contrary, he must be a skilled and critical microscopist, furthermore he must be familiar with the struc- ture of the material that is to be photographed. He can learn the pro- cessing of negatives much quicker than he can gain a mastery of micro- scopy. Given a good negative and some supervision by the scientist, the commercial photographer can make excellent contact prints and enlargements. This chapter was written for the research worker or teacher who has modest facilities for making photomicrographs and wishes to utilize them to the best advantage. It will be assumed that the ad- vanced worker who has more elaborate facilities has studied both photography and microscopy beyond the elementary scope of this manual. 204 Botanical Microtechnique Attachment Cameras In view oi the iact that most photomicrography is done with a standard microscope used in conjunction with some form of accessory camera, this type of apparatus will be discussed first. A highly satis- factory type of attachment camera consists of a lightweight metal camera which is fastened to the ocular tube and is carried by the microscope tube without additional support. The microscope may be used for visual study in its normal position, and, when a field of view is to be photographed, the camera can be placed into position without disturbing the microscope. These cameras do not have a bellows or an extendable body and the projection distance therefore is fixed. Magnification is varied by changing objectives and oculars. The largest camera of this type takes negatives 9 by 12 cm., or 31/4 by 41/^ in. The projection distance is such that the magnification factor is IX- Therefore, the image magnification is equal to the product of objective and ocular magnifications. Smaller models take negatives 414 by 6 cm., 6.5 by 9 cm., and 35 mm. respectively, and have a lower magnification ratio, in accordance with the shorter projection distance. Focusing is accomplished in some models by an observation lube having either a telescopic ocular or a ground-glass screen. A prism within the camera diverts all or part of the image-forming rays into the observation tube. When the image is in focus in the observation tid)e or screen, it also is in focus in the plane of the negative. The eyepiece observation tid^e may not give precise focus with objectives of less than lOX. in which case the ground-glass screen in the plane of the cnuilsion nuist be used. The camera fastens to the microscope by a clamping collar. A fixed ocular tube gives greater rigidity than a drawlube, unless the diawtiibc lias a positive locking device. A further aid to rigidity con- sists of a biass sleeve pressed around the upper end of the ocular liil)c and tinned on a lathe luitil the slee^•e makes a tight fit into the (lamp (ollar of the camera. The camera can be re\()l\cd to orient the image — thus a revolving stage is not necessary. Exam))les of such cameras are the Zeiss I'liokii. the Leit/ Makani, and the Erb and Gray Visicam (Fig. 17.1) . In the Zeiss and Leitz, the e\pensi\e local plane shutter, range finder and other devices are a total waste if the camera is to be used oidy foi photomicrography. Ihe Visicam and Histoslide use a simj)le 35-nmi. canieia body, the sole f miction ol which is to hold the spool of film. Fig. 17.1-Attachment cameras: upper left, Leitz 9 x 12 cm. with focusing ocular; lower left, Leitz 35mm. with focusing ocular; upper right, Visicam 35mm. camera with focusing screen; lower right, Histoslide 35mm. camera. 206 Botanical Microtechnique The inexpensive Histoslide camera lacks the observation tube. The object is focused through the microscope ocular and the camera is carefully placed upon the ocular. In another model, the camera is held on a bracket attached to the ocular tube, and the camera is swung into position over the ocular (Fig. 17.1) . Pillar-Type Cameras A rigid and substantial type of apparatus carries the camera on a vertical support, which is attached to a heavy base. Thus, the weight of the camera is not carried on the microscope tube. A simple version uses a bellows camera, without a lateral observation tube (Fig. 17.2) . The latter camera may be used with a compound microscope or with Micro Tessar lenses used directly in the shutter of the camera. Excellent work can be done with such apparatus if the component parts are correctly aligned. However, it is obvious that the microscope cannot be used conveniently for visual work when fastened to the base of the camera shown in Fig. 17.2. The camera must be slid vertically and the post swung back to permit use of the ocular. The base is not large enough to hold the lamp, therefore separate pro- .^gc3-^^ir— Fig. 17.2-Pillar-supiK)rlccl cameras: left, Bausch & Lonil) bellows camera on hinged l)ill:n; ritrfil, Rinisdi J^: I.oinh fixcd-lcnglh camera with observation eyepiece. Camera swings on post. Phofomicrography 207 Fig. 17.3-Combination visual and photomicrographic apparatus, permanently as- sembled on commercial (B & L) metal base. The home-made plywood camera swings on a post and can be removed. vision must be made to fasten the lamp rigidly at the proper distance from the microscope. In order to do both visual and photographic work, the user spends much time tearing down and reassembling the components of the apparatus. The swing-out type of camera permits free use of the microscope for visual work, and the camera can be swung into position accurately. The lateral observation tube permits precise focusing (Fig. 17.2) . A compact and rigid unit can be made by bolting a length of channel iron to the metal base of either style shown in Fig. 17.2, and fasten- ing the lamp permanently to the channel iron. Combined Visual-Photographic Apparatus Experienced research workers know that the taking of a photo- micrograph is inseparably associated with critical visual study. For example, let us assume that a wet acetocarmine preparation has been Fk;. 17.1— American UiJlical Clompaii^s base and cameras. Permanent alii;innent of microscope, lamp and camera permit quick change between visual suutv and photo- graphy: top, 4x5 inch model with focusing tube: left. l\5 inch model uitliout focusing tube: right, 35 mm. model with focusing ocular. Photomicrography 209 studied with a fine binocular research microscope, and it becomes desirable to photograph a loose floating cell that is in satisfactory orientation. It is impossible to remove the slide from immersion con- tact with the objective and condenser, transport the slide to another part of the laboratory or another part of the city, set up the slide on another microscope, and locate the specific cell and photograph it in the original condition. Even if a permanent slide is used, the investigator — who is the only one who knows what is wanted — must personally locate the desired field and focus at the desired level. These conditions call for equipment that permits a quick change from critical visual work to photomicrography, right in the research laboratory. A step in this direction is the use of a commercial metal base on which the microscope and the lamp are permanently fastened and aligned (Fig. 17.3) . This arrangement can be used with attach- ment cameras that ride on the microscope, as well as with a more rigid pillar camera. This arrangement permits comfortable visual study and a quick change to photography. The camera can be removed and used by another worker who has a similar private base, microscope and lamp. The versatile Spencer apparatus in Fig. 17.4 permits quick change-over from visual study to a 35-mm. or 4-by 5- FiG. 17.5-Bausch & Lomb reflex camera that permits maximum versatility for visual study and photograpliN, with Micro Tessar lenses as well as with all powers of the compound microscope. 270 Botanical Microtechnique inch camera. The massive, rigid Bausch & Lomb model L has the above desirable features, as well as convertibility to Micro Tessar and gross photography (Fig. 17.5) . In this apparatus, the microscope and lamp are on a base that is slid as a unit from luider the camera for visual study. This apparatus could be improved by providing a stable base on which the microscope-lamp unit could be fastened for visual study while someone else uses the camera, without mutual incon- venience. The Leitz Lumipan goes one step further by incorporating the illuminating system in the base of the microscope, and making the microscope virtually an integral part of the camera and its base. Optical-Bench Cameras The optical-bench type of photomicrographic apparatus has long been considered the ultimate in precision and rigidity. A standard microscope may be used, fastened to an adjustable platform. The three principal components, the camera, the microscope, and the lamp, are mounted on a heavy metal track on which the units may be slid back and forth in accurate alignment. If the microscope is removed for visual study, the replacement and re-alignment are very time-consuming. There is a temptation to keep an expensive micro- scope, possibly a binocular, permanently on the apparatus where it is not available for visual use, and may be used for photomicrography only a fraction of the time (Fig. 17.6 A) . It is preferable to use a special simplified photomicrographic microscope, which is built on as a permanent part of the apparatus. Several workers can therefore use the apparatus without mutual inconvenience, each worker bringing the objectives and oculars from his personal microscope (Fig. 17.6 B) . For low magnifications. Micro Tessars are used in conjunction with special substage condensers. The sequence of operations for setting up and using these elab- orate outfits is identical in principle with the procedures outlined for simpler apparatus. When used in the horizontal position, no substage mirror is used, and the horizontal beam of light is easily centered with the axes of the microscope and camera. Focusing and centering of the illuminant in the plane of the substage diaphragm are thus easily accomplished, and the setting is practically permanent. A lateral observation and focusing tube is available in some makes, or a ground-glass screen may be used for focusing. A limitation of present models is ihat they use large and expensive plates, 5- by 7-in. or 8- by 10-in. sizes. Reducing kits make possible the use of 314- by Photomicrography 27 J B Fig. 17.6— Optical-bench photomicrographic apparatus: A, Bausch &: Lomb appa- ratus with research microscope in place; B, special simplified microscope perma- nently installed. 4l^-in. plates. Recent developments in the production of fine-grain film of high resolving power will undoubtedly lead to the use of much smaller negatives, especially for expensive color work. Light Sources The character of the light source and the method of illuminating the object are important factors in photomicrography. Artificial light is in almost universal use because of its constant intensity and ease 212 Botanical Microtechnique Fig. 17.7— Horizontal apparatus for use with Micro Tessar objectives. Components, from left to right: ribbon filament lamp; filter holder; revolving stage with con- denser holder; bellows camera with removable lens board, which carries the focusing mount and a behind-the-lens shutter. The commercial focusing screen spring-back is renio\al)le. of control. A 6-volt, 108-watt coil filament or ribbon-filament lamp furnishes a steady, fixed source of adequate intensity. A transformer furnishes G-voh current from the llO-volt alternating-current line. A rheostat may be used to control the intensity if color temperature is not critical. The tungsten-arc and zirconium arc also are excellent illuminants. The carbon arc has a brilliant, homc:)geneous crater, but the crater shifts as the carbons bmii away, and it is difficult to keep the crater exactly in the optical axis. The lamp must be provided with an adjustable condenser and an iris diaphragm. A one-lens spherical condenser or the slightly more expensive aspheric condenser will give good results, biu a better corrected condenser with two or more components is preferred. Focusing Aids 1 he focusing jjanel in commercial apparatus is usually mack' ^vill; sufficient precision to place the ground glass in the same plane as ilif |)hoiographic enmlsion. If correct focusing is not obtained, the iua(( urate positioning of groiuid glass is suspected, the easiest remedy is ilie use of a |)Iate holder as a focusing panel. Rtinoxt' the partition that separates ilu plates in a doubli' iioldci. insert a ground glass into I he plate grooves. This j)la(cs the ground stirface in the same jjlane as the emulsion. Take the photographs with a plate or film holder of the same make as the one used as a focusing panel. The groimd-glass surface prox iiles a satisfactory image for orient- Photomicrography 213 ing the subject, but uot for critical focvisiug. For maximuui sharpness, use the clear window method. Make a fine X mark on the ground glass with India ink, on the diagonals ot the glass. Allow the ink to dry, place a drop of balsam or cover glass resin on the mark and lower a cover glass on the resin. This will make a clear window in the ground surface. A focusing glass may be purchased, but an in- expensive one can be made by fitting a 3 to 5 X magnifier into a metal tube of such length that when the tube rests on the clear area of the ground glass, the X mark is in focus. Bring the image into approximate focus on the ground surface, view the image through the magnifier and bring into sharp focus. Exposure Meters An extensive literature has accumulated on the subject of ex- posure control in photomicrography. The most accessible sources are the indexes of Stain Technology and the Journal of the Biological Photographic Association. Only a brief survey of the principal me- thods can be given here. Extinction meters have been used by skilled photomicrographers, who could probably standardize an apparatus and make good nega- tives without a meter. The photoelectric meters used for general photography will register a significant reading with some photo- micrographic apparatus. This makes possible the calibration of the apparatus and fairly satisfactory exposure control. Several highly sensitive, but expensive, electronic meters are available. These meters give good readings in the plane of the emul- sion, in fact permit probing of small areas of the image. Consult the advertisements of scientific journals for the currently available meters. If an exposure meter is not available, an experienced photographer who can judge negative densities can obtain good negatives with a little expenditure of film and time. Assume that previous experience with a certain magnification suggests an exposure of 15 seconds. Draw the dark slide halfway out of the film holder and make a 10-second exposure. Remove the dark slide and expose for another 10 seconds. The two halves of the film have had 10 and 20 seconds respectively. Develop the negative and decide whether the next exposure must be less than 10 seconds, more than 20 seconds, or an intermediate interval. Negative Materials Orthochromatic emulsions can be used for photomicrography. These emulsions are sensitive to green, blue, and ultraviolet. A black object or one that is rich in green or blue may be rendered accurately 274 Botanical Microtechnique in monochrome with such emulsions. Representative emulsions in this category and Eastman's ortho, process ortho and Verichrome films, D. C. Ortho plates, and Agfa Plenachrome film. Noncolor sensitive emulsions such as process plates have not been given adequate attention for photographing such objects as black- stained chromosomes. Historically, the best-known emulsion for photomicrography is the Wratten M plate. This is a panchromatic plate having compara- tively coarse grain and slow speed, producing negatives of high con- trast. The more recent fine-grain panchromatic emulsions may well bring about a radical revision of photomicrographic techniques. These emulsions are fast, they have a wide range of color sensitivity, and, because of the fine grain of the negative, enlargements of many diame- ters can be made. This makes possible the use of rclati^ely low microscope magnifications, with greater depth of focus and a large, comparatively flat field. A negative will yield a contact print or lantern slide of a large field, and selected portions of the negative may be greatly enlarged to exhibit finer details of structure. Films in this category are Eastman Panatomic X and Agfa Finopan. The speed ratings of emulsions can be obtained from the manufacturers or from the frequently revised tables of makers of exposure meters. The choice between plates and films depends on the size of film, the microscope magnification being used, the type of negati^■e holder, and the focusing method. Large sheet film has considerable concavity, w^hereas a glass plate is flat over its entire area. With the lower mag- nification ranges, up to lOOX. the lack of perfect flatness of the enmlsion does not seriously influence focusing, but, if nujch of the area of a large negative is to be utilized with high magnifications, the use of plates may be necessary. Sheet film holders designed to hold the film along all four sides arc superior to separate adapters that fit into platcholders. Some of these adapters do not hold the emulsion of the film in the same })l;me as when a plate is used in the same holder, therefore the focusing screen or observation tube is not in accurate register with the emulsion, resulting in inaccurate focusing. The forcuoiuQ sources of error sliould he tested for the available apparatus and accessories. Roll film is useful only if the conditions are so well standardized that the length of exposure can be estimated accurately. The smaller sizes lie sufficiently flat for moderate magnifications. Pack film has some advantages over roll fihn. Jndi\idual films (an l)e removed from the pack for development, making it possible to establish exposure Photomicrography 215 time with one or more trial exposures, developing the films ai once. Subsequent exposures under similar conditions can then be made in rapid succession. In the larger film-pack sizes the film has con- siderable curvature along the edges, but the central portions are adequately flat. Processing of negatives will not be discussed. The worker who is not proficient in the processing of negatives, or who does not have available the services of an expert, would be rash to undertake photomicrography. The Setting Up and Operation of the Apparatus Before outlining the procedure used in taking photomicrographs, some suggestions are offered concerning the choice of objectives and oculars for any given subject. The ultimate aim of the photographer is a finished print on paper, or a lantern-slide (transparency) image on a screen. The image should convey to the observer the intent of the photographer: a low-power survey of a large area, with little emphasis on cell detail; a rendering of texture and tone in black and white, without much cell detail; an accurate reproduction of details within a cell or within a minute object; or the sharp outlining of an object against a contrasting background, without detail within the object. The worker may have other aims and may combine them, with emphasis placed where needed. When using the standard oculars that are used for visual work, the best results are obtained with oculars of moderate magnification, 8 to 12X- For use with optical-bench outfits, special photographic oculars, the Homals of Zeiss or the Amphiplans of Bausch and Lomb may be used. These oculars produce a flat field on even the largest negative. The objective to use is one that covers the desired area of the object generously, especially when using visual oculars, so that the important area will be in focus simultaneously and the out-of-focus marginal region can be masked out in the finished product. In addi- tion to adequate coverage, the objective should have adequate re- solving power to show the necessary detail. Keep in mind that, as the magnification and resolving power increase, depth of focus decreases. It may be advantageous to obtain a sharp negative covering the necessary area and depth of the object — but having relatively low magnification — and to enlarge a few diameters in making the positive. However, the positive must show the detail that the photographer intended to show. Some workers prefer to keep the negative image 276 Botanical Microtechnique of such size that lantern sHdes may be made by contact, or that contact prints will be ot ihc correct size for publication in a journal. ^Vider use of the fine-grain methods of miniature photography will promote the use of excellent objectives of comparatively low magnifi- cation, large field, and good resolving power. Examples are the Bausch and Lomb oil immersion, 40x> N.A. 1.00, and several makes of oil-immersion objectives, with magnifications of 60 to 65 Xj N.A. from 1.30 to 1.40. The sequence of operations leading uj) to making the exposme will now be described. It will be assumed that the slide, all lenses, the mirror, and the filters are perfectly clean, and that all units are firmly fastened in place. The procedure \aries \vith the type of il- limiination beino used. When using an ordinary mazda bulb and a sheet of groiuid glass or grainless opal glass the operations are as follows: 1. Locate and focus the object as in visual study. 2. Place a thin wedge of black paper against the diffusion glass, and focus the condenser until the pajjer marker is in focus sinud- taneously with the specimen. Remove the marker. If ground glass is used, the grain of the glass will be visible, and the condenser must be displaced slightly to eliminate this grain. 3. Remove the ocidar and adjust the substage diaphragm uniil the back of the objective is just filled with light. 4. Replace the ocular, bring the camera into position, and adjust the angle of the mirror luitil the illumination on the focusing screen is centered. Slight readjustment of the substage condenser may be necessary to obtain uniform intensit) over the illiuninated field. 5. Focus the image sharply on the focusing screen and make the exposure. The use of the foregoing eciuipnuiii ami [jrocedure may be re- garded as amateur photomicrography, Avhich ne\ertheless alfords valuable training and may yield results that meet some needs. For serious and critical work, the lamp sliould lia\e a concen- trated filament bull), a condenser system of one or more components, and an iris diaphragm. Two systems of illumination are j)Ossible with suitable lamps. Critical illiunination is obtained when the condenser system focuses the incandescent light source (filament) u|)()ii the j)lane ol the specimen cjn the stage. Ihis superimposed fdament image nuist be of adequate area to cover the field of the objective and must be of Photomicrography 217 uniform Ijiilliancc. Many laboratories do not have a lanij) suital)lc for this system, and it is not used extensively. The KoJiler system of illumination is the most practical and widely used method. In the Kohler method the image of the filament is focused on the substage condenser diaphragm, and the image of the lamp diaphragm is focused in the plane of the specimen. The operations usually are performed in the following order: 1. Direct the beam of light upon the mirror, with no filters or other screens in the beam. Open the substage diaphragm completely, reduce the lamp diaphragm aperture, and manipulate the mirror until the light reflected back from the lower lens of the substage condenser to the mirror is projected by the mirror as a spot of light, exactly centered on the lamp diaphragm. This position of the mirror must not be altered. In a horizontal apparatus a mirror is not used, and this step is omitted. If the filter holder is adjustable, insert any dense filter and adjust the holder until (a) the beam of light is centered in the filter and (b) the light that is reflected from the back surface of the filter is centered on the lamp diaphragm. 2. Open the lamp diaphragm, close the substage diaphragm and focus the lamp condenser until the filament image is sharply defined on the substage diaphragm. This is the permanent setting of the lamp condenser. 3. Bring the object into focus with the objective that is to be used to take the photograph. Use a neutral filter to reduce the light enough for visual comfort. 4. Open the substage diaphragm completely and partly close the lamp diaphragm. Rack the substage condenser up and down until the lamp diaphragm, with its edges sharply defined, is superimposed on the sharply focused specimen. 5. Replace the ocular with a pinhole ocular, look down into the tube and bring the spot of light in the back lens of the objective into the exact center by manipulating the centering screws of the substage condenser. (Not by moving the mirror.) Open the substage diaphragm fully. If the polygonal disk of illumination — which rep- resents the lamp diaphragm — does not cover the desired area of the object, remove the upper element of the substage condenser and repeat operation 4. It may be necessary to remove the upper two elements of the substage condenser to obtain a large enough illumin- ated field. 6. Replace the ocular. Hold a 3X to 6X magnifier above the 218 Botanical Microtechnique ocular, adjust the magnifier until the back lens of the objective is in focus. Close the substage diaphragm and open it slowly until the rim of the diaphragm coincides with the rim of the back lens of the objective. 1 he full numerical ajjerture of the objective is utilized only imder these conditions. In practice, the aperture may be reduced by means of the substage diaphragm, but not more than one-sixth of the diameter of the back lens of the objective. Up to this point the operations are identical for vistial study and photography, and the foregoing operations can be performed with the binocular body. This is the time to try Wratten filters — usually in pairs — to obtain the desired contrast or detail. A neutral filter may also be needed for visual work. 7. If a binocular body is in place, replace it with a monocular tube and connect with the camera. Remove all filters and arrange the composition of the essential image on the ground-glass screen by means of a revolving stage or revolving camera. 8. Replace the selected Wratten filters (no neutral filter) , and focus critically. Close the shutter, insert a loaded plateholder, wdth- draw^ the slide from the plateholder, and make the exposure. (See the discussion of exposure meters, page 213) . The foregoing procedure is modified with cameras that have an observation tube (Figs. 17.1, 17.2). Composition and focusing can be accomplished after the shutter has been closed, the film holder inserted and the dark slide withdrawn. The prism or mirror that serves the observation tube is then swung aside and the exposme made. The lowest power objectives, such as the 3.2 X 'ii^<-l J^X' <-l^^ ii^t cover a large enough field, and have too much magnification for some sidjjects. The objectives of stereoscopic binocular microscopes have the desired specifications, but such objectives are not adapted for use on a single-tube microscope of correct tube length. The compoinid system is therefore not suitable for photomicrographs in the 5X to 30X I'lnge (See frontispiece) . Such photographs are taken with special objectives that produce a flat, well corrected image and are never used with an ocular. Objectives of this type are the Micro Tessars of Rausch and Lomb and the Micro-Teleplats of Spencer (American Optical Co.) . Leil/ and Zeiss also make excellent objec- tives of this type. Each Micro lY\ssar must be used with a condenser that has the same focal length as the objective. The manufactmers furnish matching condensers lor their objectives. The ilhiminani Photomicrography 279 must have a flat ribbon filament or a homogeneous arc, and a con- denser lens. The mechanical set-up and the operation of a particular com- mercial apparatus should be obtained from the directions supplied by the manufacturer. The principles will be outlined on the basis of the apparatus shown in Fig. 17.7. The usual sequence of opera- tions is as follows: 1. Adjust the lamp condenser to give a beam of parallel rays. This can be done with adequate practical accuracy by focusing the filament upon a wall 8-10 ft. away. Lock the lamp condenser perma- nently into tliis position in relation to the filament. 2. Remove all optical components from the stage and camera and center the filament image on the ground glass of the camera back. The filament will not be in sharp focus, but do not change the setting of the lamp condenser. 3. Insert the Micro Tessar objective and center it into the filament image by shifting the objective, not the light beam. 4. Insert the substage condenser that has the same focal length as the objective. Center the condenser by moving the condenser, not the light beam. 5. Place the slide on the stage and bring the object into sharp focus on the ground glass. 6. Try various Wratten filters and use the combination that gives the desired balance between contrast and detail. 7. Make the exposure. The above optical system transmits enough light, if the Wratten filters are removed, to register adequately on a good exposure meter such as the Weston or General Electric. Exposure factors can be worked out for a given optical system, filter combination and type of negative material. Bibliography Beatty, a. V. 1937. A method for growing and for making permanent slides of pollen tnbes. Stam Tech. 12:13-14. Beck, C. 1938. The microscope. Beck. London. Belling, John 1930. The use of the microscope. McGraw-Hill. New York. Butterfield, John V. 1937. An illuminating system for large transparent sections in photomicro- grapliy. Jour. Bio. Photo. Assoc. 6:155-161. Chamberlain, C. J. 1932. Methods in plant histology. 5th ed. University of Chicago Press. Chicago. 1935. Gymnosperms — Structure and evolution. University of Chicago Press. Chicago. Chowdhury, K. a. 1934. An improved method of softening hard woody tissues in hydrofluoric acid under pressure. A>in. Bot. 48:308-310. Conn, H. J. 1933. History of staining. Commission on Standardization of Biological Stains. Geneva, N.Y. 1936. Biological Stains. 3rd ed. Commission on Standardization of Biological Stains. Geneva, N.Y. Crafts, A. S. 1931. A technic for demonstrating plasmodesma. Stain Tech. 6:127-129. Crowell, Ivan H. 1930. Cutting microscopic sections of wood without previous treatment in hydrofluoric acid. Stain Tech. 5:149-150. Davenport, H. A., and R. L. Swank 1934. Embedding with low viscosity nitrocellulose. Stain Tecfi. 9:137-139. Davis, G. E., and E. L. Stover 1936. A simple apparatus for the steam method of softening woods for micro- scope sections. Trans. 111. Acad. Sri. 28:87. DUFRENOY, J. 1935. A method of imbedding plant tissues without dehydration. Science 82: 335-336. Eames, Arthur J. 1936. Morphology of vascular jaJants. McGraw-Hill. New York. [221] 222 Bibliography Gage. S. H. 193G. The Microscope. 16th ed. Comstock Publishing Company, Inc. Ithaca, N.Y. GOURLEY, J. H. 1930. Basic fuchsin for staining vascular bundles. Stain Tech. 5:99-100. Hance, R. T. 1933. A new paraffin embedding mixture. Science 77:353. Hyland, F. 1941. The Preparation of Stem Sections of W^oody Herbarium Specimens. Stain Tech. 16:49-52. Jeffrey, E. C. 1928. Improved method of softening hard tissues. Bot. Gaz. 456-458. Johansen, D. a. 1940. Plant microtechnique. McGraw-Hill. New York. Journal of the Biological Photographic Association Williams & Wilkins. Baltimore, Md. Kohl, E. J. and C. M. James 1931. A method for ripening hematoxylin solutions rapidly. Science 74:247. 1937. The use of 72-butyl alcohol in the paraffin method. Slain Tech. 12:113- 119. LeE^ A. BOLLES 1937. The microtomists Vade-Mecum. 10th ed. Blakiston. Philadelphia. LiLLiE, Ralph O., Wu.liam F. Windle, and Conway Zirkle 1950. Interim Report of the Cominittee on Histologic Mounting Media. Resin- ous Media. Stain Tech. 25:1-9. Loom IS, W. E. and C. A. Shull 1937. Methods in Plant Physiology. McGraw-Hill. New York. Maneval, W. E. 1936. Lacto-phenol Preparations. Staiii Tech. 11:9-11. McLane, Stanley R. 1951. Higher Polyethylene Glycols as a water soluble matrix for sectioning fresh or fixed plant tissues. Stain Tech. 26:63-64. McClintock, Barbara 1929. A method for making acctocarmin smears permanent. Stai)i Tech. 4:53- 56. McClung, G. E. 1937. Handbook of microscopical technique. 2nd ed. Paul B. Hocbcr. Inc.. Harper. New York. McWhorter, F. P., and E. Weier 19.36. Possible uses of dioxan in botanical microtechnique. Stain TecJi. 11:107- 119. Mettler, F. a., C. C. Mettler, and F. C. Strong 1936. The cellosolve technic. Stain Tech. 11:165. Miller, D. F., and G. \\\ Blavoks 1938. Methods and materials for teaching biological sciences. McGraw-Hill. New York. Newcomer, E. H. 1938. A procedure for growing, staining and ni. iking permanent slides of pollen tubes. Stain Tech. 13:89-91. North I un. II. T. ]<).'?(). Histological a])pli(:U ions of iinuiic a(i(l and ferri( diloridc. Slain Tech. 11:23-21. Bibliography 223 Ph otom icrography Eastman Kodak Comijany. Rochester, N.Y. PoPHAM, Richard A. 1948. Mordanting Plant Tissues. Stain Tech. 23:49-54. Ra\vlins, T. E. 1933. Phytopathological and Botanical Research Methods. John Wiley and Sons, Inc., New York. Sass, John E. 1945. Schedides for sectioning maize kernels in paraffin. Stain Tech. 20:93-98. 1947. A photographic Apparatus for Use With Micro Tessar Olijectives. Jour. Bio. Photo. Assoc. 15:198-200. Schmidt, Louis 1937. Photomacrography. ]onr. Bio. Photo. Assoc. 6:47-61. Shields, Lora Manc.um, and H. L. Dean 1949. Microtome Compression in Plant Tissue. Amer. Jour. Bot. 36:408-416. Smith, Gilbert M. 1933. The freshwater algae of the United States. McGraw-Hill. New York. 1938. Cryptogamic botany. McGraw-Hill. New York. 2 vols. Smith, Luther 1947. The Acetocarmine Smear Technic. Stain Tech. 22:17-31. Steere, W. C. 1931. A new and rapid method for making permanent acetocarmin smears. Stain Tech. 6:107-111. Walls, G. L. 1932. The hot celloidin technic for animal tissues. Stain Tech. 7:135-147. W'ETMore, R. H. 1932. The use of celloidin in botanical technic. Stain Tech. 7:37-62. 1936. A rapid celloidin method for the rotary microtome. Stain Tech. 11:89-93. WiTTLAKE, Eugene B. 1942. An Efficient Vacuum Apparatus for Microtechnic. Ohio Journal of Science. 42:65-69. ZiRKLE, Conway 1947. Some Synthetic Resins in Combined Fixing, Staining and Mounting Media. Stain Tech. 22:87-97. Index Abies, leaf of, 147 Acer stem, mature of, 136 stem, tip of, 128 Acetocarmine, smears, 106 Acetone, for dehydration, 24 Acorus calamus, root of, 140 Adhesives, for sections, 51 Agaricales, 159 Agropyron, rhizome of, 133 Albugo, 154 Alcohol dehydration witli. 22 isopropyl, 24 normal hutyl, 27 tertiary Inityl, 27 Algae collecting of, 6 preparation of, 149 Allium, root tip of, 122 Angios])ermae, reproducti\e organs 169 Anthoceros, 161 Ascomycetes, 155 Asparagus root of, 138. 140 , stem of, 133-34 Aspergillus, 155 Asplenium. sporangia of, 167 Bacteria, 152 Bacterium steiuartii, 152 Balsam, Canada, 64 Basic fixation, formula for, 20 Basidiomycetes, 157 Boti'ycliium gametophytes of, 166 root of, 142 sporangia of, 166 Bouin's fluid, 18 Bromus. stem apex of, 124 Bryophyta collecting of, 6 processing of, 161 Cameras, photomicrographic, 204-11 Cannabis salii'a. stem of. 132, 135 Capsella. emhryo of. 178 Carex, stem of, 133 Carmine, {see Acetocarmine) Carnov"s fluid, 19 Carya, stem of. 136 Catherinia, 161 Celloidin method. 78 Cercospora beticola, 161 Chara. 151 Chenopodiaceae, stems of, 135 Chrome-acetic, formulas, 16 Chrxsauthemum, stem of. 134 Citrus, leaf of, 145 Clavariaceae, 160 Claviceps, 157 Clearing agents, 25, 61 Coleus, stem apex of, 127 Collecting, for processing, 5 of. Condensers, for microscope, 191-94 Conocephalum conicum. 161 Coprinus, 160 Cordyceps, 157 Crocus, root tip of, 122 Cruciferae, roots of, 142 Crystal violet, (see Gentian violet) Cucurhitaceae, stems of, 134 Cyst opus. 154 Dae t\ I is. stem apex of. 124 Dehydration for em])edding, 22 of reagents, 29 Diantlius. leaf of. 145 Dioxan, for dehydration, 28 Diplodia zeae, 160 Embryos, 1 76 Emulsions, for photomicrography. 213 Equisetum gametophytes of, 166 root, stem of, 143 strobilus of, 166 [225] 226 Index Erigeron. stem of, 134 Eiysiphe, 156 Ethanol, (see Alcohol) Euphorbia, leaf of, 146 Exoascus. 157 Exobasidium, 160 Exposure meters, 213 Farmer's Iluid, 19 Ferns gametophytes of. 167 rhizome, root of, 142 sporangia of, 167 Ficiis, leaf of, 146 Filicales, 167 Fixatives, (see Adhesives) Fixing, (see Killing) Flemming's fluid, 16 Flemming's, triple stain, 71 Floral organs, collecting for processing, 6 Fomes, 160 Fraxinus, stem of, 136 Freehand sections, 91 Freezing microtome, 93 Fruits, 176-81 Fucus, 151 Funaria, 161, 163 Fungi collecting of, 6, 7 imperfecti, 160 ])r()cessing of, 151 Gentian violet-iodine, staining process, 75 Cinko, leaf of, 147 Gladiolus, root tip of, 122 Gleditsia, stem of, 136 Gloeocapsa, 150 Glycerin for dehydration, 21 jelly, 102 Gramineae leaves of, 146 root tips of, 139-40 stems of, 133 Gvmnospcrmae rcpr()dncli\c organs of, 167 stems of, 136 Gy mnosporanginni , 159 Hedera helix, leaf of, 146 Hclianthus, stem of, 134 Mcnialum formulas, 57 staining process, 61 Hematin, 58 ITcinaloxylin iioiialinn, 73 self-mordanted, 57 Hxaciiithiis, root ti]) of, 122 Hvdnaceae, 160 Hypoinyces, 157 Illuminators, 211 Iris, leaf of, 147 Isoetes gametophytes of, 166 root, stem of, 143 sporangia of, 166 Killing (Fixing) formulas, 15, 16, 18, 19, 20 process, 14 reagents, 12 Lactophenol, 102 Leaves, collecting for processing, 5 coniferous, 147 dicot, 143-46 monocot, 146 specific methods for, 143 Lichens, 161 Liliiim embryo of, 177 floral organs of, 169-75 ovary of, 114-15 root tip of, 122 Linum, root of, 142 Liriodendron, stem tip of, 127 Lonicera, stem tip of, 127 Lotus corniculatus. embryo of, 177, 180 Lycopersicum esculentuin embryo of, 177, 178 stem of, 131 stem tip of, 127 Lycopodium gametophytes of, 165 root, stem of, 142 s]>()rangia of, 165 Maceration, of tissues, 108 Maize, {see Zea) Marchantia, 161-63 Matlhiola emhrvo of, 178 floral organs of, 169 Medicago stem 'of, 112, 130 tap root of, 142 Mi'hnnpsora, 159 Melilotns, tap root of, 142 Microchemical tests, 96 Micrometr\, 197 Min()])rojeclion, 198 Microscope illumination systems for, 191-94 nicdianical features of. 194-97 optical system of. 182-91 Mi( rosphaera alui, 156 Index 227 Microtome roiaiy, 45 sliding, 85 Milium. 161, 163 Molds, for casting, 34 Morchella, 157 Mucor, 153 Myxomycetes, 153 Narcissus, root tip of, 122 Nawaschin, formulas, 18 Nectria, 157 Nereum oleander, leaf of, 145 Neurospora, 157 Nitella, 151 Nostoc, 150 Oleander, (see Nereum) Onoclea, sporangia of, 167 Oomycetes, 154 Ophioglossales, 166 OsciUatoria, 150 Paraffin embedding compounds, 33 infiltration in, 35 properties of, 31 PeUia, 163 PcniciUium, 155 Peronospora parasitica, 155 Peziza repanda, 157 Piiaseolus apical meristems of, 125 root of, 140 Photomicrography, 202-19 Phvcomycetes, 153 Phytopliithora, 154 Picea, leaf of, 147 Pinus gametophytes of, 168 leaf of, 147 stem of, 136 strobili of, 168 Pisum, apical meristems of, 125 root of, 140 Plasmodiophora brassicae, 154 Podosphaera oxycantliae, 156 Polygonatiim bifloruin, leaf of, 147 Polyporaceae, 160 Polytrichum, 161, 163 Populus, stem of, 136 Porella, 163 Primus fruit of, 181 leaf of. Ill Pseudopeziza medicagiriis, 157 Pseudotsuga, leaf of, 147 Pteridophyta gametophytes of, 167 sporangia of, 167 Pteris, rhizome, root of, 1 12 Pucciuia coronata, 159 graniinis, 158 Pyronema conjluens, 157 Pyrus root of, 140 stem of, 136 Pythiuiu, 154 Quadruple stain, 73 Qumtuple stain, 73 Ranunculus, root of, 142 Reboulia hemisphaerica, 161 Resins, mounting, 65, 104 Rhizopus, 153 Rhodobjyum, 161, 163 Rhoeo, leaf of, 147 Riccia, 161 Rivularia, 150 Roots collecting of, 6 monocot, 138 specific methods for, 138 tips of, 121 Rubber plant, leaf of, 146 Rusts, 159 Safranin as a counterstain, 67 ^vith fast green, 69 properties of, 60 in triple stain, 71 Sambucus, stem tip of, 127 Saprolegnia, 154 Sarcoscxpha coccinea, 157 Schizomycetes, 152 Sclerotinia fructigena, 156 Seeds, 176 Selaginella, root, stem of, 143 Smilacina racemosu, leaf of, 147 Smilax, root of, 140 stem of, 134 Smuts, 158 Soja leaf of , 1 1 1 root of, 140 stem of, 130 stem tip of, 127-28 Solanaceae, stems of, 134—35 Solanum tuberosum, stem tip of, 127 Sphagnum, 161 Stains selectivity of, 60 solubilities of, 59 228 Index Stems collecting and sulxli\ itling of, G gymnosperm, 136 herbaceous dicot, 134-35 nionocot, 133-34 tips of, 123 woody, 135-38 Synchytrium decipiens. 154 Syringa, stem tip of, 127 Tannic acid-Ferric chloride process. 7G TapJnhia. 157 Thallophvta, 149 Tilia stem of, 136-37 stem tip of, 127 Tillclia tritici, 158 Tradescantia leaf of, 147 microsporocvtes of, 174 Tremellales, 159 Tiifoliiim hybridum, stem of, 131 Tsuga. leaf of, 147 Tullfxi floral organs of, 169 root tip of, 122 Typlia, stem of, 133 Uii( inula salicis, Uredinales, 158 Urnula, 157 Ustilago, 158 156 I'ditrlieria. \\hole mounts of. 105 \enelian tur])cnline method, 104 Ve?ittiria iiiactjualis, 157 Vicid fab a root of. 110 root tip of, 123 J'olvox, whole mounts of, 105 Waxes, water-soluble, 95 (see also Paraffin) Whole mounts, 99 acetocarmine, 106 permanent, 103 semi -permanent. 102 temporary, 102 \Vood sectioning in celloidin. 78 .sectioning uncmbedded. 92 softening of, 84 Zea kernel and emljrvo of. 179 leaf of, 146 root of, 123 root tip of, 122 stem (mature) of, 133 stem apex of. 128 Zebrina leaf of, 146 root tip of. 123 stem of, 133 Zygomycetes, 153 /'