MANUAL OF PLANT HISTOLOGY.

THOMSS AND DUDLEY

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LABORATORY MANUAL

PLANT HISTOLOGY

MASON B. THOMAS, B. S.

Professor of Biology in Waba.sh College,

WILLIAM R. DUDLEY, M. 5.

Professor of Botany in Leland Stanford, Jr., University.

CRAWFORDSVILLE, INDIANA.

1894.

COPYRIGHT, 1894.

\1 A-uN li. THOMAS.
ALL KICIITS Ui-:sKuvi:i>.

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Tin: JOURNAL Co.,

I'lil.N-l'KKS,
( HANVKHiDSVILI.K, I Ml.

ERRATA.

p. VIII, last line, for "Standford" read Stanford.

p. XV, line 37, add Roots of Dicots.

p. 45, line 2 from bottom, for "pillar" read arm.

p. 46, line 5, for "F" read N.

p. 54. line 13, for "Magnenta" read Magenta.

p. 54, line 20, for "c. m." read m. m.

p. 64, line 22, for "magnification" read size.

p. 65. line 3, for "1-1000" read 1000.

p. 69, line 16, for ' demonstrated" read demonstrated.

p. 70, line 20. after "p. 362" add 365-68.

p. 74, line 3, before "p. 118" insert p. 100.

p. 75, line 11 and 12, for "4, 5," read (a), (b).

p. 79. line 2, for "later" read latter.

p. 80, line 7, add p. 47-68.

p. 80, line 29. for "p. 11" read p. 61.

p. 90, line 2 from bottom, after "Fig" add p.

p. 91, line 12, add Fig. 27.

p. 95, line 24, for "vacular" read vascular.

p. 96, line 2 from bottom, for "reserviors" read reservoirs.

p. 97, line 21, for "p. 40" read p. 141.

p. 102, lines 15 and 25, for "vacular" read vascular.

p. 103, line 4, for "management" read arrangement.

p. 104, line 8, for "monocot" read Monocots.

p. 104, line 10, for "Cucurbit" read Cucurbita.

p. 108, line 1, for "System" read Systems.

INTRODUCTION.

Almost every thoughtful teacher of botany in the colleges and
universities of our country is confronted by two problems in con-
nection with his laboratory instruction. He is forced to provide a
course which shall give the general student a fair knowledge of
what the teacher deems the most important phases of plant life;
and on the other hand, if a conscientious instructor, he will encour-
age students to advanced work, inaugurating courses which are
intended not only to inform the mind, but to train the powers of
observation, comparison and scientific judgment, and finally pro-
duce the investigator capable of pursuing problems of science
without aid or admonition, if not without suggestions from his'
professor.

Some ten years ago, the present writer, thert in charge of the
Histology and Cryptogamic Botany at Cornell University, attempted
a revision of his laboratory course in plant anatomy, in order to
adapt it to the advanced courses, chiefly in Cryptogamic Botany,
which followed. The result was a small hand-book, privately
printed in 1886, entitled, "Anatomy and Histology of Plants,"
which evidenced the author's desire to impart some special knowl-
edge of tissues as a foundation for more serious work in any
subsequent subject involving the use of the microscope. It soon
appeared, however, that better methods in the preparation of soft
tissues and delicate organisms must be adopted, if any great
advance was to be made toward the solution of problems of
structural development.

It may here be mentioned that imbedding and cutting serial
sections of delicate plant tissues, had not been put in practice in

vi INTRODUCTION.

American botanical laboratories previous to that time, and even in
Germany it was but little in vogue. To study better methods of
microscopic manipulation in this and other directions, was the
object of the writer in spending the year 1887-'88 in the German
laboratories, where he used for the first time the collodion method
of imbedding. It was seen more and more clearly that in the
future, students were to be trained to useful work in biological
investigation chiefly through a mastery of microscopical technique,
and a thorough knowledge of tissues and cell contents with their
behavior under the influence of reagents. The changes in methods
in the histological course brought about during the four years
following 1888, were made to bear upon the work of students taking
the courses on the higher and lower Cryptogams, with most excel-
lent results. Such changes were included in the plans for a revised
manual, carefully drawn up in 1892.

Mr. Mason B. Thomas, an undergraduate, then Fellow in
Botany in the writer's laboratory, 1888-91, and afterward Profes-
of Biology in Wabash College, was invited to assist in this work.
During his university course he had been able to render me invalu-
able assistance, by refining and abridging the process of imbedding
in collodion, and by devising various laboratory appliances con-
nected with it (still remaining in the laboratory at Cornell), some
of which are described in his papers published in 1891 to 93, * and
detailed at some length in Atkinson's " Biology of Ferns" (1894),
particularly in Part II., Chapter I.

The exactions of work since 1892, in an entirely new field have
obliged me to abandon rewriting the Manual. At my request,
Professor Thomas has done this, so far as it seemed necessai'y.
He has also prepared the part on technique (Part I.), as well as
plates, selecting the illustrations from his many beautiful prepara-
tions made while at Cornell University and since that time. The
fact that some of the best laboratories in this country have adopted
the methods formulated by him makes it particularly appropriate
that he should write this part.

In it no attempt has been made at an exhaustive treatise but

* (1) "The Collodion Met hod in l»ot;in v :" Hep. AIM. Society of Micnxcopists,
1891; (Mi "A Dehydrating App.-n-itu-,." A.m. Monthly Microscopical Jol., Jan., 1H91.
(3) "Sectioning Fern Prothaflla," The Microscope, SOT. 1893.

INTRODUCTION. vn

the matter is presented rather in the form of suggestions to those
who may be at the beginning of their work in micro-chemistry or
technique.

The tests for the different vegetable substances and the gen-
eral properties of reagents have been taken from the best authori-
ties on those subjects, and carefully tested.

I am responsible for the plan of the manual of directions
(Part II.), for some of its phrasiology, and for the selections of
most of the subjects used for study; and any imperfections in
this part must be laid at my door. Nevertheless the plan has stood
the test of many years thoughtful use in my own laboratory, and
more recently in that of Wabash College ; and Professor Thomas
shares completely with the writer the belief that such an element-
ary course, most thoroughly taught, should be made the founda-
tion for advanced instruction on the morphology of the higher and
lower plants, and should enter into the education of a student for
any independent work in anatomy, physiology, or biology.

If other teachers should find the work acceptable, we would
remind them that a course of carefully prepared lectures should
supplement the laboratory work, and we urge them to so present the
subject, that the intergradations of tissues may not be overlooked,
and the larger relations of great tissue masses and their beautiful
adaptations to the necessities of the living plant, may be completely
understood by the student. No true teacher will allow a student
to consider these individual studies in an unrelated way. We
have cited freely text books and reference works of unquestioned
value, such as are to be found on the book-shelves of every good
laboratory, but we have not made a practice of referring to original
papers, as it would be for the most part out of place in a work of
this kind. But this does not release the teacher from the duty of
placing the most important papers bearing directly on a subject of
study within the reach of the student and requiring him to look
them over.

The inconvenience of using plates placed at the end of a book
will not be great, and is offset by the fact that they are removed
from the unavoidable scrutiny of the student as he is executing his
own drawings, but any defect in this or any other direction noticed
and communicated by a teacher may be rectified in another edition.

INTRODUCTIOX.

To the student we would say that they, as men fitting them-
selves for professional or semi-professional scientific careers, have
certain duties to themselves, entirely independent of the formal
requirements of the instructor. Their aim should be a complete
familiarity with the methods suggested, a comprehensive and scien-
tific knowledge of as many facts as possible, and an ability not only
to execute but to finally plan their own work, and themselves solve
their scientific problems. To this end they should not, even in this
elementary course, content themselves with the lines laid down,
but should consult all books suggested in the studies given in the
manual, and read carefully the passages to which reference is
made. They should miss no opportunity to learn of a new work
or an original paper in botany, or any fact concerning the mode of
work of any genuine contributor to the literature of the science.
We have an especial sympathy with the ambitious student whose
superior training or skill enables him to accomplish more than the
average students. For him are suggested the additional studies in
the hand-book, and he will always find his instructor ready to advise
him in regard to further reading. Both teacher and pupil should
recognize the fact that in the present day, a sure foundation may
be laid in undergraduate years, for a subsequent successful profes-
sional career, if the pupil thoroughly learns the use of his tools
and pursues his chosen science with the zeal that belongs to his
time of life.

It is with genuine regret that I lay down this work as well as
the particular plans which were the motive of it, for broadening
and deepening the training of American botanical students; but in
doing so, I am sure that in the hands of Professor Thomas it will
arrive at a better development than in my own, and that his efforts
in this field will find nothing but appreciation.

The authors wish to express their indebtedness to Mr. E. W.
Olive, Instructor in Biology in Wabash College, for the many ways
in which his services have lightened the labors in the prepai'ation
of this manual.

WILLIAM RUSSEL DUDLEY,

August, 1894. Leland Standford. Jr., University.

WORKS OF REFERENCE.

In the selection of the list of hooks and periodicals below, it has been the
intention to give only the more general ones and those that should be in every
botanical laboratory. The list is in no sense intended to be a complete one, and it
is expected that the student will have at his disposal, a number at least, from each
of the groups.

For special or advanced work the original papers and monographs, on each
particular subject considered, must be obtained.

General Botanical Works.

Bastin, College Botany; Engelhard & Co., Chicago, 1890.

Bennett & Murray, Cryptogamic Botany; Long-mans & Co., London, 1889.

Bessey, Botany for High Schools and Colleges; Holt & Co., N. Y., 1892.

Campbell, Structural and Systematic Botany; Ginn & Co., Boston, 1890.

DeBary, Comparative Anatomy of Phanerogams and Ferns; Oxford Press, Lon-
don, 1894.

Engler and Prantl, Die Naturlichen Fflanzen Familien; Englemann, Leipzig; issued
in parts and not yet complete.

Frank, Lehrbuch der Botanik; Engelmann, Leipzig, 1893.

Goebel, Outlines of Classification and Special Morphology; Oxford Press, Lon-
don, 1887.

(iray. Struct nral Botany; Am. Rook Co., New York. 1879.

Sachs, <ie>ammelte Abhandlungen ueber Pflanzenphysiologie; Engelmann, Leipzig,
1893.

Sachs. The Physiology of Plants; Oxford Press, London, 1887.

Sachs, History of Botany; Oxford Press, London, 1890.

Vines, Physiology <>t Plants; Cambridge Press, London, 1886.

Vines' Text Book of Botany; Swan, Sonnenschein & Co., London, 1894.

Laboratory Hanuals.

Arthur, Barnes, and Coulter, Plant Dissection : Holt & Co.. N. Y., 1887.

Bower, Practical Botany; MacMillan & Co., N. Y., 1891.

Davis, Text Book of Biology; Chas. Griffin & Co., London, 1893.

Dodge, Elementary Biology; Harper & Brothers, N. Y., 1894.

Dudley, Histology of Plants; Ithaca, N. Y., 1886.

Goodale, Structural Botany; Am. Book Co., N. Y., 1885.

Huxley & Martin, Practical Biology: MacMillan & Co., N. Y., 1889.

Parker, Elementary Biology; MacMillan & Co., N. Y., 1891.

Sedgwick and Wilson, Biology; Holt & Co., N. Y., 1889.

Spalding, Introduction to Botany; Heath & Co., Boston, 1893.

Strasburger, Practical Botany; Swan, Sonnenschein & Co., London, 1893.

x WORKS OF REFERENCE.

Structural and Technique.

Atkinson, Biology of Ferns; MacMillan & Co., N. V., 1894.

Methods for treatment of li-Mies: strnrture.
Bausch, Manipulation of the Microscope: Koclioit-r, N. Y.

Manipulation and care of inst rumeni .
Beale, How to Work With the Microscope; London, 1880.

St ructure and methods.

Beherens, Guide to the Microscope in Botany: Huston, 1885.
Carpenter, The Microscope and Us Revelations: Philadelphia, ls91.

Manipulation and care of instruments: also struct tire.
Clark, Practical Methods in Microscopy: Heath it Co.. Boston, 1893.
Fry, The Microscope and Microscopical Technology; New York, 1892.

Structure, manipulation and methods.
Gage, The Microscope and Histology: Ithaca, N. Y., 1894.

<*are and manipulation of instruments: also methods of mounting.
Goodale, Physiological Botany; Am. Book Co.
Lee, Microtomists Vade Mecum; Philadelphia, )890.

Methods.

Strasburger, Practical Botany.

Van Heurck, The Microscope, Construction and Management; I). Van Nost rand
Co., N. Y., 1893.

Botanical flicro-Chemistry.

Poulsen, Micro-Chemistry, Trans, by Trelease: (Jinn \ c,,.. Kn-ton, 1886.
Zimmcrmann, Botanical Micro-Techni(|iie, Trans, by Humphrey; Holt & Co.,
N. Y., 1893.

Botanical Journals, and Periodical Publications.

Annals of Botany; Oxford, Clarendon Press, London.
Morphological, Systematic, and Physiological.

Am. Monthly Microscopical .lol.: Washington, D. C.
Microscopical methods and histology.

Botanical Gazette; Lake Forest., III.

Morophological, Physiological, and Systematic.

Botanisches Centralblatt, Cassel; contains original work together with Bibli-
ography of Current Botanical Literature, 1880 —

Bulletin of Torrey Botanical Club, N. Y.; largely systematic.

Just's Botanischer Jahreshericht, Berlin; Bibliography of Botanical Literal lire,
1873 .

Annales des Sciences Naturelles (Botaniquc): Ked. par A. Brongiiiart et .1.
Decaisne, Paris, 1854 ; chiefly original papers.

Botanische Zeitung, Leipzig; original papers and Bibliography, 1843

CONTENTS.

PART FIRST.

flicro-Chemistry and Technique.

REAGENTS 2

Alcohol 2

Separation of Inulin 2

Acetic Acid 3

Test for Crystals 3

Alum 3

Ammonia 3

Test for Middle Lamella 3

Anilin Chloride 3

Test for Lignin 3

Argentic Nitrate 3

Test for Living Protoplasm 3

Chloroform 4

Scil vent for Fats, etc 4

Calcic Chloride 4

Clearing Tissue 4

Cupric Sulphate 4

Trsl for Sugars 4

Carbon Disiilphide 4

Solvent for Carotin 4

Carbolic Acid 4

Solvent for Fats, etc 4

Test for Lignin .">

Chromic Acid '. 5

Sol vent for Cell Wall 5

Cnprainmonia 5

Solvent forCellulose 5

Cleaning- Mixture ,"i

Nitro-Sulphuric Acid 5

IMchi-oniate .Mixture 5

Collodion 6

•-' per cent, and 5 percent, solutions... 6

Ether 6

Glycerin 6

Separation of Inulin 7

Hydrochloric Acid 7

Test for Hypochlorin 7

Iodine 7

Test for Starch 7

Test for Cellulose 8

Act ion on Protoplasm 8

Millon's Reagent 8

Detection of Albuminoids 8

Nitric Acid 8

Macerating- Agent 8

Test for Protein Matters 8

Clearing Tissue of Starch 8

Oxalic Acid 8

Hit-aching Tissue 8

Solvent for Pectose 8

Perosmic Acid 9

Fixing- and Hardening Agent 9

I'otassic Dichromate 9

Hardening Resin Masses 9

Test for Tannin 9

1'ot assic Chlorate 9

Macerating Agent 9

Test for Suberin 9

Pa ratline . 9

Melting Points 9

Phosphoric Acid 10

Test for Crystalloids 10

Rosalie Acid 10

Test for Vegetable Jelly 10

Staiti for Sieve Tissue 10

Sugar 10

Test for Protoplasm .....' 10

Pollen and Spore Cultures 10

Sulphuric Acid 10

Test for Cellulose 10

Action on Starch 1O

Action on Fat Bodies 10

Xll

CONTENTS.

Chlor-iodide of Zinc 11

Test for Cellulose 11

Test for Tannin 11

Action on Fungus Cellulose 11

HARDENING AGENTS 12

Alcohol 12

Dehydrating apparatus 13

Picric Acid 15

Chromic Acid 15

Osmic Acid 15

Hardening Fluid - 15

CUTTING AND MOUNTING TISSUES 10

Free-Hand Sectioning 16

Softening Hard Tissues 16

Use of Pith or Cork 16

Use of Parafflne 17

Parafflne Method 17

Construction of Paper Boat 18

Microtomes 19

Fixing Sections to Slide 19

Staining, Mounting, etc 20

Collodion Method 21

Hardening, Sectioning, etc 22

Ether Vapor Bottle 23

Treatment of Delicate Tissues 24

STAINING AGENTS 26

Ammonium Carmine 2<>

Alum Carmine 26

Eosiu 27

Haematoxylin 27

ANILINE COLORS 27

Methyl Violet 27

Methyl Green 27

Aniline Blue 28

Magenta 28

PICRIC ACID 28

SILVER NITRATK 28

CLEARING AGENTS 29

Cedar Oil 20

Clove Oil 29

Solvent for Collodion 29

Use in the Minute Dissections 29

Origanum Oil 30

Sandal wood Oil 30

Carbolic Acid and Turpentine 30

MOUNTING MEDIA 31

Aluminum Acetate 31

Mounting Algae .'51

Biilsam 31

Clearing :il

Preparation of Balsam „.. 31

Treatment for Air Bubbles 31

Sealing Mounts 32

Balsam Bottle ... 32

Carbolic Acid 32

Calcic Chloride 3~:

Glycerin Jelly 32

K Miser's Formula 32

Glycerin 32

Use for Fresh Tissues 33

King's Mounting Medium 33

Water :: i

Action on Tissues 33

CEMENTS 34

Gold Size 34

Shellac ' 34

Ball Cement 34

Asphalt Varnish 34

White Zinc Cement 34

SERIAL SECTIONING 35

Paralliino, and Collodion Sections 35

Arrangement on Slide 35

DOUBLE STAINING 37

Combination of Stains 37

Use of Mordant 37

FLUID MOUNTS 38

Mount ing in Cells 38

Construction of Cells 38

Mounting Fluid 38

Sealing Mounts 3!i

Dry Mounts 39

EQUIPPING OF LABORATORY 40

Case for Reagents 4O

Supplies and Their Location 40

Waste Vessels 4O

Clearer Bottle 41

Collection and Preservation of Mate

rial 42

Treatment of Soft Tissues 42

Preservation in Alcohol 42

Preservation in Collodion »:!

Preservation on Corks or Blocks 43

THE MICROSCOPE 44

Description of Instrument 45 Hi

METHODS OF STUDY 4 7

Care in Observation 47

Directions for Drawing 47-48

I'se of Camera Lucida. 48

Drawing Material 49

Page of Drawing Book 51)

Preservation of Slides 50

Mailing Boxes 50

Construct ion of Cabinet 51

Catalogue of Preparations with Sample

Card 52

APPARATUS NEEDED 53

Microscopes 53

CONTENTS.

arm

Microtomes 53

Reagents 54

Slides, Covers, Brushes, Paper, Dissect-
ing Needles, Razor, etc v54-55

CARE OF APPARATUS 56

Cleaning' Instrument, Lenses, etc. 56

Testing Tissue with Acids 56

Oiling, Changing Objectives and Ocu-
lar 56

Care of Lenses, Focussing 57

CARE OF EYES 58

Bye Shade 59

Sources of Light 59

Artitii-ial Light - 59

MANIPULATION OF APPARATUS 60

Interpreting Appearances 60

Cloudiness on the Lenses 60

Focussing 61

Optical Sections 61

Air Bubbles 62

Oil Globules 62

MAGNIFICATION 63

Determination by use of a Camera Lu-

cida 63

Determination of Ocular Micrometer

Ratio 64

Micron 64

Tube Length 65

PRACTICAL EXERCISES

65

PART SECOND.

Laboratory Directions.

Divisions of the Subject.

A. Living Cells, (with Protoplasm and Chlorophyll.) 66

B. Contents of Cells, (the secondary products.) 66

C. Elementary Tissues 66

D. The Primary Meristem 66

E. The Systems of Tissues 66

F. The Thickening of Stems, etc., (secondary growth) 66

A. Study of Living Cells 67

1. THOSE LIVING SEPARATE FROM ONE ANOTHER 67

a. Protococcus viridia 67

b. Mother-cells of Pollen,— Begonia 68

c. Mature Pollen Grains,— .Malvaceae 70

d. Culture of Pollen Grains,— Tradescantia 71

2. CELLS IN COLONIES, JOINED TEMPORARILY 72

a. Spirogyra,— (showing Protoplasm, Chlorophyll and Progressive Cell Di-
vision) 72

3. CELLS PERMANENTLY JOINED 74

A. NOT FORMING TISSUE 74

Stamen Hairs of Tradescantia 74

B. FORMING TISSUE 74

Illustrated by the Majority of the Subsequent Studies 74

xiv CONTENTS.

B. Cell Contents 75

1. STARCH GRAINS 7.">

a. Potato Tuber,— Solatium tuheroaum 75

b. Garden Pea, I'ixnni satirum 76

c. Wheat Grain,— Triticum vulgare 76

Structure of (Vrral (i rains 77

d. Grain of Indian Corn,— Zea Mays 78

e. Grain of Oat,— Avena saliva 78

2. CRYSTALS 78

Crystal Prisms,— Onion Bulb, Allium Cepa 79

Haphides,— Roots of Many Plants 80

3. PROTEIN GRANULES 80

a. Crystalloids,— Potato Tuber 75

b. Aleurone Grains, Garden Pea, Corn and Wheat Grains 76

CYSTOLITHS 80

Leaf of Ficus elastica 80

INULIN 80

Root of Dahlia 80

C. Elementary Tissues 82

1. PARENCHYMA TISSUE „ 82

a. Isodiametric,— Stem of Geranium «•-'

b. Ellipsoidal,— Root of Hyarinth 83

c. Irregular and Epidermal,— Leaf of Geranium .~. 83

d. Stellate,— Petiole of Pontcderia *•»

e. Suberous,— Bark of Cork Oak 84

MODIFICATION OP CELL WALL '. 8.r>

a. Cell Walls with Mucilage,— Seed Coat of Flax «.'•

b. Cell Walls with Lignin,— Fibrous Tissue 85

c. Cell Walls with Cutin,— Epidermis of Cycas leaf 86

d. Cell Walls with Minerals,— Stem of Equteetum 86

CONTINUITY OF PROTOPLASM 86

2. COLLENCHYMA TISSUE 87

Stem of Begonia or Geranium 87

ENDODERMAL CELLS, 87

3. SCLERENCHYMA TISSUE 88

a. Roots of Dahlia variabilis 88

b. Ivory Nut, — PliytelepJias macrocarpa 88

c. Rhizome of Pteris aquUina 89

4 and 5. PROSENCHYMA TISSUE 89

Prosenchyma Proper 89

Wood and Bast Cells,— Leatherwood,— Dirca palustrte 90

Tracheary Tissue 90

Tracheids,— Stems of Horse Chestnut, Moon Seed and Grape Vines 91

Tracheids of Coniferae,— Pinus Strobus 92

Tracheae if.'

a. Dotted,— Stem of Grape Vine 93

b. Pitted; c. Spiral; d. Reticulated; e. Scalariform 94

Stem of Castor Oil Bean 94

f. Annular,— Stem of Corn _ 94

g. Trabecular, — Leaf of Juniper 94

6. SIEVE TISSUE 94

Stem of Cucumber, or Pumpkin 95

7. LATICIFEROUS TISSUE 95

Latex Cells,— Stem or Petiole of Euphorbia 96

Latex Tubes 01 Yt-s.-l-, Stem or Petiole of Celandine 96

CONl'ENTS. xv

GLANDS AND WATER POKES 96

GLANDS 96

a. Lemon Skin 96

b. Leaf of Eucalyptus 97

c. Resin Ducts of Pinus 97

WATER PORES 97

Leaf Tooth of Fuchsia , 97

D. Heristem Tissue 99

PRIMARY MERISTEM 99

a. Single Apical Cell 101

Tip of Equisetum, or Fern Root ! 101

b. Group of Initial Cells 99

Tip of Hyacinth Root 99

FIBRO- VASCULAR BUNDLES 102

a. Collateral,— Stem of Moon Seed Vine 102

b. Bicollateral,— Stem of Cucurbita 104

c. Radial,— Root of Corn 106

d. Concentric,— Stem of Pterte, Rhizome of Iris 107

E. The System of Tissues 108

l. EPIDERMAL 108

Formation of Stomates, — "Stone Crop," or Sedum ternatum 108

Ti-ichomes,— Shepherdia Candensis, Nettles, etc .....109

Water Pore,— Fuchsia 110

•2 and .'!. FIBRO- VASCULAR AND FUNDAMENTAL 110

Exogenous Stem 110

Herbaceous,— Begonia 110

Woody,— Moon Seed Vine 110

Endogenous Stem 110

Herbaceous,— Corn 110

Woody,— SmilMJC 110

Com ferae,— Pinus 110

Vascular Cryptogams 110

F. Secondary Thickening .112

Stem of Dicots, — Moon Seed Vine 112

Stem of Monocot,— Smilax Hispida 11 3

Stem of Coniferae,— Pinus 113

Roots of Monocots,— Orchidnceae or Cyperaceae 114

VASCULAR SYSTEM OF LEAVES 114

O.rxlix 114

LENTICKLS AND CORK THICKENING 115

Elder, or Moon Seed Vine ...116

LIST OF ILLUSTRATIONS.

Figure 1. Dehydrating Apparatus Page 13

2. Paper Boat 19

3. Ether Vapor Bottle 23

" 4. Microscope 44

" 'i. Ti -insert ion of Anther of Begonia 50

6. Section of Drawer for Glass Slides 51

" 7. Eye Shade 58

8. Culture Slide 71

art CONTEXTS.

Figure 9. Formation of Pollen Grains, Funkiaovata Phite I

" 1O. Stamen Hairs of Tradesc-antia, I

" 11. Section of Potato Tuber II

" IS. Various Forms of Parenchyma Cells II

" 13. Sclerenchyma cells from Dahlia Root 1 1 1

" 14. Resin Duct from Pinus Ill

" 15. Section of Lemon Peel Ill

" 16. Longisection of Hoot, Cypripcdium pubescem IV

" 17. Transection of Hoot, Cuprlpedium pultesccm V

" 18. Collateral Bundle from the Stem of liiyonia VI

" 19. Collateral Bundle from the Stem of Corn VII

" 20. Bicollateral Bundle from the Stem of Cucurtntu.... VIII

" 21. Transection of Root, Spiranthes ccrnua IX

" 22. Radial Bundle, Root of Spiranthcx cernua IX

" 23. Transection of Pterls stem. Concentric bundle X

" 24. Trichomes from Shepherdia Canadenste XI

" 25. Trichomes, Nettle and Geranium XII

" 26. Stomates from the Leaves of Cyca* and Finns X 1 1 1

" 27. Transection of Stem, Moon Seed Vine XIV

" 28. Transection of Stem, Smttax fowpida XV

J.Moore,

Laboratory Manual of Plant Histology.

!att Jfirst

MICRO-CHEMISTRY AND TECHNIQUE.

REAGENTS.

Certain stains and reagents are absolutely indispensable for
laboratory work while others although not indispensable are never-
theless very desirable and become necessary in thorough and
advanced investigations. The following is an alphabetical list of
the more important chemicals, reagents, and stains, together with
tests for the more frequently occurring vegetable substances.

Alcohol.

For all ordinary laboratory manipulations commercial 95 per
cent. Ethyl alcohol is sufficiently strong, and it is only when all
trace of water is to be removed from a specimen, that alcohol of
absolute strength is needed. Absolute alcohol has like osmic acid
the property of rendering protoplasm rigid and can therefore be
employed to advantage in studying the more intricate structures
of protoplasmic bodies ; as for example, nucleus or cell division.
Common alcohol readily removes air from intercellular spaces,
especially if heat is applied. It is also extensively used in harden-
ing tissues, different strengths being employed to prevent its strong
avidity for water causing too great a shrinkage of the protoplasm
from the cell wall. Tissue hardened in this way can be softened
again by soaking it in water. Alcohol is used for dehydrating sec-
tions that are to be mounted in balsam and for dissolving many
fats, resins, and oils from plant tissues.

It is a solvent for chlorophyll, and is used in the preparation
of many stains. If tissue containing inulin is kept in alcohol, the
inulin is precipitated within the cell in the form of sphaero-crys-
tals. Many sphaero-crystal forming substances separate in the
same way, e. g., hesperidin.

Acetic Acid.

Glacial acetic acid is a valuable clearing agent. In 2 per cent,
solutions it is good for clearing up the cell contents and in study-
ing the nucleus or protoplasmic structure. In strong solutions it
dissolves the cell contents and makes the cell wall clear. It is also
used in testing for oxalate crystals which are insoluble in it, but
which dissolve without effervescence in HC1, while carbonate crystals
dissolve with effervescence in both.

Alum.

Alum is employed as a mordant in various staining processes,
as, for example, in Frey's haematoxylin. It is also used to render
more visible cells that have become too transparent by treating
with KOH.

Ammonia.

Strong aqueous ammonia is sometimes used in preference to
KOH where the action of the latter would be too violent. If tissue
with thick walled cells be placed in nitric acid and then in ammonia,
the middle lamella of the cells will be colored yellow. Ammonia is
also used in the preparation of certain stains and in Schweizer's
reagent for dissolving cellulose without essentially changing its
composition. '

Anilin Chloride.

Aniliu chloride is used as a test for lignin, one of the con-
stituents of wood. The sections to be treated are placed in a dilute
solution until they are thoroughly saturated. They then assume a
pale yellow color which is much deepened upon the addition of
HC1. This is Hoehnel's test for lignin.

Argentic Nitrate.

A dilute alkaline solution of silver nitrate when fresh is used
as a test for living protoplasm. The aldehyde which is contained
in the living protoplasm precipitates metallic silver free in the solu-
tion and colors the protoplasm dark. Dead protoplasm is not
affected in any way.

4 HE AGE NTS.

Chloroform.

Chloroform is chiefly used as a solvent for fats, resins, and
oils ; also to some extent in the preparation of chloroform balsam.
According to VanWisselingh it is a solvent for the various suberin
constituents.

Calcic Chloride.

This salt in an aqueous solution is used as a mounting fluid
and not infrequently it is found useful for clearing. The tissue to
be cleared is moistened with a little water and some of the pow-
dered salt is then sprinkled on it. It is heated gently until most of
the water has been evaporated. The whole is again moistened and
mounted in glycerin when it becomes clear.

Cupric Sulphate.

The pure salt in an aqueous solution is largely used in the
detection of sugars. The test employed by Trommer is as follows :
The tissue under examination is allowed to remain in a concen-
trated solution of the salt for about ten minutes when it is rinsed
with distilled water and placed in a boiling mixture of water and
and potassic hydrate. The reaction with cane sugar in the section
is to turn the cells containing it a light blue, white with grape
sugar (glucose), the reaction causes the cells to become clouded by
the deposition of a fine flocculent or granulated orange precipitate
of reduced oxide of copper. Dextrine when not mixed with protein
compounds assumes a vermilion color. Protein compounds in
young cells, with the above tests, turn a violet color. We are thus
enabled to detect the presence of two and often three kinds of
sugars in this reaction.

Carbon Disulphide.

This agent is used chiefly as a solvent for fats, oils, wax, etc.,
also for carotin, a coloring matter with the same composition as
xanthophyll, chlorophyll yellow, etc.

Carbolic Acid (Phenol).

This acid is sometimes used as a solvent for fat and fatty oils.
When mixed with three parts of turpentine, it makes a good clear-

ing agent for most plant tissues. With carbolic and hydrochloric
acids, lignified cells become yellowish green. The test is best made
by adding a few drops of concentrated HC1 to some crystals of car-
bolic acid, warming slightly, and when cold, add HC1 enough to
dissolve any crystals that may have separated out. This gives a
solution of crystals in just enough HC1 to dissolve them, and in
this the tissue is placed. (See Zimmermanu's Microtechnique, p.
145.)

Chromic Acid.

Chromic acid in strong solutions dissolves the cell wall rapidly
except in those cases where it may be cutinized, silicified, or corky.
If the action is allowed to continue, the cutinized wall will finally
dissolve. In dilute solutions the acid causes the cell wall to swell
and often brings out very clearly the markings or stratifications, as
in those of starch grains. Chromic acid is sometimes used in dilute
solutions as a hardening agent.

Cuprammonia.

This is the so-called Schweizer's reagent and is effective only
in fresh solutions. It is prepared by adding to an aqueous solu-
tion of copper sulphate some sodium Hydrate, until a precipitate of
copper hydrate is formed. The precipitate is filtered out and
washed with hot water, after which it is dissolved in as little
ammonia as will take it up. It forms a deep blue solution and will
dissolve quickly cotton fibers. Cell walls of pure cellulose swell
and are readily dissolved by the solution, but, if they contain
lignin or suberin, the reagent will not act until these substances
are removed in some way, e. g., by Schulze's maceration method.

Cleaning Mixture.

A cleaning mixture that works rapidly and removes balsam at
once from the slide is made by adding two parts of strong HNO3
to three parts of concentrated H2SO4. The mixture must be kept
covered as the fumes are very disagreeable. This mixture cleans
glass very quickly and does not injure it. A dichromate cleaning
mixture is made by dissolving 200 grams of potassic dichro-
mate in 1000 c. c. of water and then adding to the mixture 1000

6 REAGENTS.

c. c. of H,S04. Much heat is generated by the action of the acid
and the operation should be performed in a beaker or earthen dish,
since the heat would probably crack a bottle should an attempt be
made to make the mixture in one. This cleaning mixture will after
a time remove balsam and sealing agents from glass, but it is not
so rapid in its action as the nitro-sulphuric acid mixture, nor is it
so disagreeable to handle, and may therefore be better adapted for
the use of the general student.

Collodion.

This is best prepared by dissolving pure gun cotton (pyroxylin)
in a mixture of equal parts of pure ether and 95 per cent, alcohol.
A 2 per cent, and 5 per cent, solution will be needed. These can
be made by dissolving 2 and 5 grams respectively of gun cotton in
mixtures of 100 c. c., equal parts of pure ether and alcohol. The
solutions should be kept in tightly stoppered bottles to prevent
evaporation of ether and consequent thickening of the collodion.
More satisfactory results will be secured by using gun cotton in the
preparation of collodion than by employing ordinary commercial
collodion or celloidin.

Ether.

Ether is used with equal parts of alcohol in the preparation of
collodion. Ether vapor is useful for sealing collodion sections to
the slide and is often a valuable agent as a solvent for oils, fats, and
resins.

Glycerin.

Glycerin is quite an important substance in microscopic man-
ipulation. It is used largely as a mounting medium and preserva-
tive. It evaporates slowly but absorbs water readily from the air.
Mounts in it should therefore be sealed with a water tight cement
shortly after preparation. Sections mounted in glycerin, unless
stained with a very permanent stain, are liable to become transpar-
ent and of but little use. Glycerin with gelatin is used to make
glycerin-jelly, a very convenient mounting medium for a large
variety of plant tissues. The tissue to be preserved in the jelly
can be mounted directly without dehydrating in alcohol, but in
most cases it should be first hardened to prevent shrinking.

7 REAGENTS.

If tissue containing inulin is placed in glycerin, the inulin
separates out in the form of sphaero-crystals. Kraus has used the
same agent as a test for sugar. Iodine glycerin is used largely a,s
a medium for the study of protein granules. The reagent is made
by dissolving a little iodine in some glycerin in which has been
placed a little iodide of potassium. Glycerin is often used as a
clearing fluid and is especially good as applied to Hanstein &
Russow's methods. (See Poulsen, Botanical Micro-Chemistry.)

Hydrochloric Acid.

This is a valuable macerating agent for woody tissues. It is
also used to distinguish between oxalate and carbonate salts. The
former when treated with the acid dissolve without, and the latter
with effervescence. Pringsheim has used the acid as a test for
hypochlorin, a compound of chlorophyll bodies. The tissue to be
treated is sectioned and placed directly in the acid, where it is
allowed to remain 2 or 3 hours. The hypochlorin will separate out
in the form of brown spherical masses which later become needle
shaped crystals. Hydrochloric is also used in connection with
nitric acid as a cleaning mixture.

Iodine.

Iodine is one of the most useful agents in micro chemistry. It
is used in solutions of glycerin, alcohol, or an aqueous solution of
iodide of potassium, the latter being the one usually employed in
most tests. Dilute solutions generally give the best results and
the reaction is not obscured by the intense color of the reagent.
One quite well adapted for most tests is made by dissolving 1 gram
of iodine and 5 grams of potassic iodide in 100 c. c. of water, yet
even for some purposes, a solution of one-half this strength is
desirable. Iodine is sparingly soluble in water and in cases where
the effect of the pure agent is to be observed, it is better to put a
little metallic iodine in water under the cover glass at the side of
the preparation. In a solution of zinc chloride, iodine forms the
so called Schulze's reagent, which is very generally used as a test
for cellulose. Iodine is an infallible test for starch, coloring it in
the presence of water a rich blue. If the reagent is too strong, the

8 REAGENTS.

starch will be colored a dark brown, and in the presence of an
absolute alcoholic solution of iodine, the same color is secured, but
if water is present, the color will be blue. This forms a ready test
for the presence of water in alcoholic solutions. Cellulose mem-
branes are colored by iodine a pale yellow or deep brown. If a
mixture of two parts of sulphuric acid and one of water be added
just before or after the test, the reaction will give a blue color,
while lignified cells if present will turn brown.

Iodine kills protoplasm quickly coloring it a deep brown.
Alcoholic solutions of iodine deteriorate by standing, due to the
formation in them of hydriodic acid. The deterioration is greatly
augmented by the action of light and the solution should therefore
be kept in the dark. Two solutions of different strengths should
be in the laboratory, since it is important that the correct strength
be employed in the different tests.

Millon's Reagent.

This reagent is used for the detection of albuminoid sub-
stances, it readily causing them to turn a strong red color. The
reagent is prepared by pouring over some pure mercury an equal
quantity by weight of strong HNOa. If the solution is not com-
plete, heat the mixture, then pour over it twice its volume of water.
After allowing it to stand a few hours, decant the clear portion for
use. The reagent will act only when used in fresh solutions.

Nitric Acid.

Nitric acid is used with potassic chlorate as a macerating
agent. When added alone to tissues, it causes the protein matters
to turn a bright yellow. The reaction is made more apparent upon
the addition of ammonia. According to Hoehnel, the acid forms a
good test for suberin. It is also used for clearing tissue of starch,
causing the grains to swell and soon dissolving them.

Oxalic Acid.

An alcoholic solution of the acid is very useful in bleaching sec-
tions that have previously been too deeply stained. Dilute aqueous
solutions are employed with some stains for various tissues, and a
concentrated solution dissolves pectose after treatment with potash.

9 REAGEXT*.

Perosmic Acid.

This acid is very volatile and has an extremely disagreeable
and poisonous odor ; it therefore must be kept in a sealed glass
tube. It is usually employed in an aqueous solution of 1 per cent,
strength, and this should be kept tightly corked in a dark place.
The acid is most useful for killing and fixing at once living pro-
toplasm. It is, therefore, very helpful in studying nuclear and cell
division, since it prevents immediately all further change.

Oils and fats are discolored by the acid, due to its reduction
and the deposition of metallic osmium. As a hardening agent it is
used with 9 parts of 25 per cent, chromic acid, and it not only
hardens, but stains simultaneously meristematic tissue.

Potassic Dichromate.

Potassic dichromate is often used as a substitute for chromic
acid in hardening, and seems to give about the same results. It is
•used especially for hardening resin masses and sometimes for the
detection of tannin. After continued action it colors cells contain-
ing the latter substance a reddish brown.

Potassic Chlorate.

This salt with HNO.j forms Schulze's macerating agent, which
is especially useful in destroying the middle lamella and for the
isolation of wood cells. The macerating is aided by the applica-
tion of heat. Since the fumes arising from the mixture readily
corrode metal, it is very important that the operations with the
agent be performed in a room that does not contain any delicate
instruments. Schulze's agent is also used in the detection of
suberiu. The suberized cell walls resist for a long time the action
of the mixture but finally break down, and a part of the suberin
forms eerie acid, which is readily soluble in potassic hydrate,
ether, chloroform, etc.

Paraffine.

Hard and soft paraffine are both useful ; the former with a
melting point of about 45 °C., the latter with 33 °C. Different
melting points can be secured by mixing in different proportions

10 REAGENTS.

the hard and soft kinds. The melting point can be lowered by
the addition of chloroform. It must be remembered, however,
that before any attempt is made to section imbedded tissue, all
of the chloroform must be driven off in the infiltrating oven,
otherwise the paraffine will be too soft for support. Turpentine
can be used in place of the chloroform and is perhaps quite as
good.

Phosphoric Acid.

Phosphoric acid is sometimes used to remove water from
tissues, and when added to any containing crystalloids, it causes
the latter to swell.

Rosalie Acid.

This acid is used in connection with sodic carbonate as a test
for vegetable jelly, staining it red. It is useful in coloring the
callosities of sieve-tubes and in bringing out the general structure
of cribrose tissue.

Sugar.

If tissue containing protoplasm is left for some time in a thick
syrup of cane sugar and then transferred to H2 SO4, it will turn
a red color. The reaction is not always a certain one. A 10 per
cent, solution of sugar is very useful for pollen and for spore cul-
tures.

Sulphuric Acid.

Sulphuric acid is used in connection with many tests
and is also valuable in breaking down cellulose walls, without
shrinking the protoplasm. This makes it especially important in
demonstrating the continuity of protoplasm. It is also used in
connection with iodine to determine the purity of the cellulose that
makes up the cell walls of any tissue. In this test the tissue is
first treated with a tincture of iodine, and sulphuric acid is then
added. The walls will turn blue if they are composed of pure
cellulose. Dilute sulphuric acid causes starch grains to swell,
while with the concentrated acid they are dissolved. Pure cellulose
walls are likewise dissolved by the acid, while cutinized ones resist
its action. Fat bodies are not soluble in it, but they form small
refrative drops.

11 REAGEXTX.

Chlor-iodide of Zinc.

This is known as Schulze's reagent and is very useful in
detecting the presence of cellulose. This reagent is made by pour-
ing over metallic zinc some hydrochloric acid and then evaporating
the solution with an excess of zinc present, until it becomes of a
thin syrupy consistency. Add as much iodide of potassium as
will be taken up, and then iodine until a saturated solution is
obtained. Keep the reagent in the dark to prevent the formation
in it of hydriodic acid.

Pure cellulose gives with this reagent a blue or violet color,
due to the staining by the iodine of the amyloid which is formed
by the action of the agent on cellulose. Wood, cork, and cutinized
walls are colored yellow, while starch colors blue, but the grains
soon become disorganized. Cells containing tannin are colored by
the reagent red or violet. Fungus cellulose, unlike ordinary
cellulose, remains uncolored by the action of this agent.

HARDENING AGENTS.

The subject of hardening agents is one of very great import-
ance and presents a field in which much yet remains to be learned.
The object of hardening is to bring the tissue in a condition to be
either sectioned directly without crushing, or to allow it to be
infiltrated with some substance that will hold it firm for cutting.
The difficulty to overcome is to find an agent that will harden the
tissue without shrinking it. The method employed to overcome
this is to bring the tissue in contact with the dilute hardening
agent, and then gradually increase its strength to prevent a vio-
lent action between it and the tissue. The former must always be
present in a large excess in order that an equilibrium may not be
established too quickly.

The exact strength of the hardening agent in which the tissue
should first be placed is a matter of some uncertainty and can only
be determined after experimenting with each sort of tissue. After
the object is hardened, it should not be left any great length of
time in the full strength of the hardening agent as it is liable to
become brittle.

In arranging the tissue to be hardened it should be carefully
trimmed and only the portions that are needed for examination
placed in the agent. In the case of large pieces, as, for example,
closed pistils, cut the parts open to allow the hardening agent to
penetrate all parts of the object, otherwise, deterioration will result.

Alcohol.

This is one of the most frequently employed hardening agents,
and for many plant tissues is all that could be desired. For most
soft material 40 per cent, alcohol is dilute enough to begin the

13

HAEDE A / M i AG K XT8.

hardening. From this strength it is transferred to 50 per cent., 65
per cent., 75 per cent., 85 per cent, and 1)5 per cent, alcohol
respectively, allowing it to remain in each solution for about 24
hours, varying the time according to the nature of the tissue. If
it is desirable to preserve the tissue for future examination, the
hardening should cease with the 75 per cent, strength, and in this
it should be kept until needed, when the hardening may be com-
pleted with the 85 per cent, and 95 per cent, strengths.

A very convenient apparatus for hardening plant tissues was
invented by Schulze, and a modified form of it is recommended for
laboratory purposes. It can be made as follows:

Fig. 1. Dehydrating apparatus.

Fig. 1. Apparatus complete, showing dehydrating tube It in place. A.
plaster of paris diaphragm. C. Glass rod supporting the
disk.

Fig. 2. Dehydrating tube. D. Chamois skin diaphragm. E. Spring
holding the diaphragm in place.

In a 9x9 Whitall-Tatum museum jar a disk of plaster of paris
is supported about 5 c. m. from the top by means of legs made of
glass rods. The disk is perforated to allow tubes of various sizes,
from 2-4 c. m. in diameter to pass through. These are the B.Q-

14 HABDENING AGENTS.

called dehydrating tubes. The plaster of paris diaphragm can be
made by first constructing a mould of the desired size, with a paper
bottom and a card-board hoop for the outside. This must be
placed on a level surface. The plaster of paris is then mixed with
water and poured into the mould to about the depth of 1£ c. m.
While it is yet soft the three legs can be inserted near the edge and
holes for the dehydrating tubes cut in the disk with a knife or
pressed out with glass tubing of convenient.size. When the plaster
is dry the hoop can be removed and the disk placed in position in
the jar, which is then filled with alcohol to within about 2 c. m. of
the under side of the plaster. The dehydrating tubes should be
about 12 c. m. long and can be made by cutting off the bottom of
large test tubes. In one end is placed a diaphragm of chamois
skin, which can be fastened in position by means of a spring made
of steel wire or ribbon and forced with the chamois skin in the
tube. A rubber band around the tubes prevents them from falling
through the holes in the disk and enables them to be lowered to
any desired depth in the alcohol.

The tissue to be dehydrated is packed closely in the dehyrat-
ing tube and just enough 50 per cent, alcohol added to cover it.
This is then quickly lowered through the hole in the disk, until the
two liquids are at a level. After from 12 to 24 hours, by osmosis, the
two liquids will be of the same strength. The tissue can then be
taken out and placed in the infiltrating bath at once. This method of
hardening has been tried on nearly all kinds of plant tissue, and in
almost every case it was found to be successful. For the most del-
icate tissues where slow hardening is desired, 5 per cent, alcohol
can be placed in the dehydrating tube, and thick chamois skin used
for a diaphragm, while for some of the more delicate algae it has been
found advisable to use as low as 1 per cent, alcohol in the tube.

The strength of the alcohol in the jar can be kept up by add-
ing to it from time to time some calcic chloride, which will in no
way injure the alcohol. The jar should be tall enough to allow
the cover to be kept on while the tubes are in position and thus
prevent evaporation.

An apparatus of such a form with a dozen dehydrating tubes
can be used for months without changing the alcohol. Other har-
dening agents such as picric, chromic, acetic, or osmic acids can be

15 HAKUEM.\(i Ad'EXTS.

used with equal success. Not more than 24 hours is necessary
for dehydrating and hardening nearly all kinds of plant tissue.

The apparatus does away with the transferring of the tissues
from bottles containing alcohol of different strengths, and since no
such sudden transition occurs, the tissue is less liable to shrink.
Many different materials may be used for a diaphragm and almost
any speed of dehydrating can be obtained. The apparatus can be
made in any size to adapt it for private or general laboratory work.

Picric Acid.

This is to be recommended as a hardening agent. It acts
rapidly and energetically and should be used first in dilute solu-
tions. About one-fifth per cent, is the proper strength. Tissue
hardened in picric acid can be kept in 75 per cent, alcohol until
needed. If a little glycerin is added to the hardening agent, the
tissue will be less brittle.

Chromic Acid.

This agent can be used for hardening and with it the tissue
requires the same treatment as with picric acid.

Osmic Acid.

This acid is very useful as a fixing agent and is often used for
hardening. The tissue must not be kept in it long, as it will
become blackened and brittle.

Hardening; Fluid. See p. 17.

Other hardening agents are strongly recommended, but enough
has been said to enable one to properly prepare tissue for such
studies as may follow in this work.

CUTTING flND MOUNTING TISSUES.

The methods of cutting and mounting tissues are indeed
numerous and only the more important ones will be reviewed.

Free-Hand Sectioning.

Many things can be prepared for study in this way. The tis-
sue must be firm in order that it may not be crushed under the
knife and yet not be too hard or brittle to prevent its cutting readily.
The object to be sectioned should be held between the thumb
and fore finger, while the razor should rest on the tip of the finger
with the edge inward. Draw the knife, with the edge pressing
against the tissue, across the finger keeping the thumb well below
the line of cutting. Do not try to cut large sections but make
them small and if need be wedge-shaped in order to get a
piece thin enough for study. It is usually best to keep the tissue
wet with alcohol or water to make the sectioning easy. Transfer
the sections from the razor to the slide with a camel's hair brush .

If the object to be sectioned is quite small it can be placed
between two pieces of elder pith or cork and sections made through
these which shall include in them the sections of tissue. It is
often very convenient to fasten the tissue with the cork, or if large
enough, directly in a microtome, and section with a microtome
knife or razor, keeping the object and knife wet as before directed.

This method is a very useful one for the transections of woody
stems and firm tissues. These can frequently be softened without
shrinking by leaving them for some time in glycerin, and in the
case of the firmest ones, boiling for five minutes or more. Many
small objects can be held for sectioning by placing them at once in

17 CUTTING AND MOI'\T1.\<; TISSUES.

melted hard paraffine and cooling it quickly by immersing in cold
water. The paraffine block can then be placed in a microtome and
the object sectioned. This method will apply only to those objects
that will not be shriveled by the high temperature of the melted
paraffine.

For thorough, systematic study some method should be used
that will enable sections to be made with accuracy, and if need be,
in a series with a known definite thickness. The method should
also provide something that can be infiltrated into the tissue and
hardened to prevent crushing while being cut. The collodion and
paraffine methods are the ones usually used for this purpose.
Many modifications of both have been suggested but each method
will be outlined to apply to the more general kinds of tissues.
Experience will assist in modifying them for special cases.

Paraffine Hethod.

Much discussion has arisen in regard to the relative merits of
the different paraffine methods, but the general differences are
more or less of an unimportant nature and because of the dif-
erent kind of tissues subjected to treatment. The most im-
portant methods are those of Mohl, (Bot. Gazette, Jan., 1888), and
Schoenland (Bot. Gazette, July, 1887), while most of the others are
modifications of these resulting from the experiments of different
workers on the different classes of tissue.

The tissue to be treated is first hardened in chromic or picric
acid or mixtures of these with other agents. As was suggested by
Mohl, the acids act on the tissues in some way and make them
much more penetrable to the infiltrating mass. Paraffine does not
easily penetrate tissue treated with alcohol, yet many recommend
it as a good hardening agent even under these conditions. A good
mixture for hardening is made from equal parts of 1 per cent, chro-
mic acid, osmic acid, 2 per cent., and acetic acid, 1 per cent. The
osmic acid is very useful in many hardening agents for fixing the
protoplasm, especially if it is desired to demonstrate karyokinesis,
or cell division. Tissue should be kept in this mixture from 24 to
48 hours and then washed with running water 6 to 8 hours, after
which it is placed for 12 hours successively in alcohol of 20 per
cent., 40 per cent., 60 per cent., 80 per cent., and 95 per cent.,

18 CUTTING A ND MO UNTING TISS UES.

strengths. A Schulze's apparatus is to be recommended for this
operation. If alcohol has been used for hardening, the disagreeable
process of washing can be omitted. The alcohol is now replaced
by some solvent of paraffine, as chloroform, tui-pentiue, clove or
cedar oil, xylol, or benzol. Chloroform is readily miscible with
paraffine, but is not very penetrating, and therefore requires a
much longer time for clearing than some of the other solvents.
Further it must be entirely driven off before sectioning, otherwise
the paraffine will be too soft to support the tissue. Turpentine is
recommended by Mohl, but this is often harmful to delicate struc-
tures. Cedar oil is perhaps the best of any of the agents for pene-
trating and clearing. Should turpentine be used, as Mohl recom-
mends, the tissue is first placed for a few hours in a mixture of
equal parts of alcohol and turpentine and an equal length of time
in the pure agent, then after a few hours it should be removed,
placed in a cold saturated solution of paraffine in turpentine, after
which it is placed in a bath of equal parts of turpentine and para-
ffine, kept at the temperature of 30° to 40 °C. In a few hours the
tissue is transferred to pure paraffine, with a melting point of
50°C., and in this it is allowed to remain until thoroughly satur-
ated with the imbedding mass. With cedar oil the object is trans-
ferred directly from the clearing agent to pure paraffine and left
there until infiltrated. The time required for infiltrating any object
depends upon the nature of the tissue. For some objects an hour is
sufficient, while with others one or two days is required. After the
object is infiltrated it is placed in a paper box and pure melted
paraffine is poured over it. A convenient form of a paper box can
be made as follows:

Take a piece of stiff paper of the proportions shown in Fig.
2 and fold inward along the lines A A and B B, one third of
each side. Repeat the operation with the ends, folding them along
the lines C C and D D. At each corner fold inward on a line
bisecting the angle formed by the lines A A and B B, etc., allowing
the crease to follow the lines E E, E'E', etc. Tben turn up the
sides and ends until they are at right angles with the bottom, and
bring back around the ends the portions projecting at the corners.
Fold outward the portion projecting above the sides of the box
and press it firmly against the end thus holding all the parts in place.

19

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This box will be found most use-
ful for various purposes in the labor-
atory. The object in the box can be
!D arranged by the use of hot needles,
while the paraffine is yet in a liquid
state. As soon as a film is formed
over the paraffine, it is plunged into
cold water to harden quickly and
thus prevent the mass from becom-
ing filled with air spaces. After the
paraffine has become hard it can be
readily removed from the box and
fastened in a microtome for section-

A B ing-

Fig. 2. Diagram to show the lines of
folding in making a paper boat.

Several different forms of microtomes are highly recommended.
Minot's is certainly one of the best. A very inexpensive well micro-
tome will answer the purpose. In case the latter is used the block
containing the object should be cut down until it will fit in the well
where it is fastened by pouring over it pure melted paraffine. It is
best that a microtome with movable jaws be used in order that the
position of the object can be changed to vary the angle of section-
ing. This is especially important in lougisections of roots or
stems. If longisections of a root are being made or the whole of
an object is to be studied, it is advisable that a series be made and
only those need be mounted which contain the structure desired.
In order to stain and clear the sections it is necessary that they be
in some way glued to the slide. This can be accomplished in sev-
eral ways. Those methods to be especially recommended are as
follows: With a camel's hair brush spread over the slide a very
thin layer of a mixture of equal parts of clove oil and 2 per cent. '
collodion. Place the sections on the slide and press them gently
against it with a brush or any soft object. The preparation is then
put in an oven with the temperature at 50° C. for 20 minutes, or
until the paraffine has melted and the clove oil evaporated. The
sections will then have become fastened to the slide and can be
stained and washed without danger of loosening. The same result
can be more quickly obtained by heating the slide very cautiously

20 CUTTING AND MOUNTING TISSUES.

over a flame until the odor of the clove oil has disappeared. Care
must, however, be taken not to shrivel the tissue by overheating it.

A better method of fastening the sections, is to albumenize the
slide by soaking it a short time in a mixture of egg albumen 1
part, water 200 parts, and then allowing the slide to drain until
dry. A number should be treated at one time and kept on hand
until desired for use. With these slides, by warming the sections
until the paraffine melts, the preparation will flatten out and
adhere with great tenacity to the albumenized surface. The
albumen will not be effected by staining.

After the sections are fastened, the slide is placed in tur-
pentine, or better, in xylol to dissolve away the paraffine. This
will usually be accomplished in 15 to 20 minutes, when the object
is washed with water and is ready for staining. The stain to be
used and the method of applying it depends largely on the nature
of the tissue and the part one desires to bring out. For general
studies haematoxylin works well and with eosin makes a good
double stain. If the haematoxylin is used, the slide need be left in it
but a few minutes, when it is removed and washed thoroughly with
water to take out the surplus stain, dehydrated with 95 per cent,
alcohol, cleared for a few minutes in clearing mixture, and mounted
in balsam. If the tissue is to be stained in toto, before imbedding,
Mohl recommends that it be taken from the 60 per cent, alcohol,
while being hardened, and placed for 24 hours in a solution of
alum carmine, after which the hardening can continue. With this
treatment, when sectioned, it is only necessai'y that the paraffine be
dissolved away, the tissue cleared with a clearing agent, and
mounted in balsam. With haematoxylin or alum carmine the cell
wall stains well and the nuclei show very clearly. In double stain-
ing, if eosin is applied, the sections need be left in it but 20-30
seconds. If glycerin jelly is to be used as a mounting medium, the
sections can be mounted directly after washing off the surplus
stain with water.

During the Sectioning it may happen that some of the parts of
the section will loosen as they are being cut away. In this case
they may be held together with collodion applied in a 1 per cent,
solution with a camel's hair brush. This is painted over the tissue
just before the section is cut, it dries quickly and hold all the parts

21 CUTTING . \ A I) MOUNTING TISSUES.

in place, while it does not in any way interfere with the staining.
It is advisable that all paraffine sections be mounted in l>;ils;un.
The use of the paraffine method is recommended for the firmer
tissues that cannot be held in place by the collodion.

The objections to the paraffiue method are that heat is em-
ployed in infiltrating and this i« injurious in some degree to most
tissues. Further the length of time required to get the tissue in
condition to section is quite extended, and this is often an impor-
tant consideration. The operations requiring paraffine are not as
clean in the hands of most students as would be desired.

The Collodion Method.

This method is now coming into general use for nearly all
kinds of plant tissue. For the use of collodion for infiltrating we
are indebted to Duval, who first published his results in the
Journ. de 1'Anat., 1879, p. 185. A little later Merkel and
Schiefferdecker suggested the use of celloidin, which is merely a
patent collodion. Some discussion then arose regarding the rela-
tive merits of each, but it is generally agreed that one has little or
no advantage over the other.

The method as applied to plant tissues is a comparatively new
one and many modifications of it are at present recommended by
various workers.

The following directions are in the main taken from a report
of the author on the method, made before the Am. Soc. of Micro-
scopists and published in their proceedings in 1890.

The tissue to be treated is first dehydrated and hardened in
alcohol. For this purpose a Schulze's apparatus is of the first im-
portance, in fact it has been found that some tissue can be har-
dened in no other way without shrinking. With its use, from 12-24
hours is sufficient for hardening and dehydrating any plant tissue.
The material is taken from the dehydrating apparatus and placed
in 95 per cent, alcohol for one hour, to insure complete dehydra-
tion. There is then poured over it a 2 per cent, solution of col-
lodion. In this it is allowed to remain from 12-24 hours, depend-
ing on the nature of the tissue, 24 hours being enough for the very
firmest. It is then transferred for the same length of time to a 5
per cent, solution; or the 2 per cent, solution may be allowed to

22 CUTTING AND MO UNTING TISS UES.

evaporate until it is of the consistency of the former. After this,
it is taken out and arranged in position on a cork or block of wood
of convenient size to fit in the jaws of the microtome. By means
of a camel's hair brush the material on the cork is covered with
successive layers of collodion until it is quite enclosed in the mass.
Allow each coat to dry slightly in the air before applying the next.
After the tissue is covered it is placed in about 80 per cent, alcohol
until hard enough to section. Much difference of opinion exists
regarding the proper strength of alcohol to use for hardening the
collodion, but 80 per cent, answers very well, and the tissue can be
kept in it a long time without deteriorating. After a few hours the
collodion will be firm enough for use. Sections of small or deli-
cate objects can be cut by allowing them to harden in a block of
collodion and then carefully clamping it directly in the jaws of the
microtome. For sectioning, any sliding microtome will answer, but
one especially adapted for the purpose will enable the object, which
can be clearly seen through the collodion, to be inclined in any
desired position and sections taken in any plane. It is also neces-
sary that the sections be removed with a long sweeping cut, since a
direct cross-cut would tear them. The sections should be covered
with alcohol while being removed and then floated from the knife to
the slide. (See P. A. Fish's modification, Proceedings Am. Mic.
Soc. Aug, 1893.) The slower the section is cut, the better it will
usually be. Serial sections can be arranged in their proper place on
the slide. For fixing to the slide, blow some dry ether vapor on
the object (Fig. 3), or add a drop of ether to the side of the prepara-
tion. The ether dissolves the collodion and fastens the sections in
place. The preparation is then washed with water, stained, the
surplus stain washed off with water, the sections dehydrated with
95 per cent, alcohol, cleared, and mounted in balsam.

For clearing, the carbolic acid and turpentine mixture is
to be recommended. It clears quickly and does not injure the
most delicate tissues. For staining, one must use that which
seems best adapted for their purpose, but for general study,
haematoxylin seems especially adapted for collodion sections.

Some difficulty may arise in cutting sections that have in them
free parts. It sometimes happens that they become detached from
the collodion and float away. In this case, the section can be

23.

CUTTING AND MOUNTING TISSUES.

CaCl*

Fig. 3. Ether wash-bottle for blowing ether vapor upon collodion or celloidin
sections to fasten them to slide. The tube of calcium chloride
(CaCZj) is for dehydrating the ether vapor.

collodionized as first suggested by Dr. Mark. This is done by coat-
ing the tissue before each section is cut, with a thin coat of one per
cent, collodion, using a camel's hair brush for the purpose ; then
draw the .knife across the tissue very slowly, keeping alcohol
dripping on it while the section is being cut. In this way beauti-
ful sections can be obtained of material with loose parts, where all
will retain their proper position. Care should be taken that none
of the sections be cut before collodionization, for although it may
not always be necessary to keep the parts in place, yet it is a safe-
guard against their displacement.

The method given is found to work admirably on very delicate
meristematic tissue. No heat being required, the most delicate of
tissues will not shrink. The shortness of the method commends it
for general use. Two days, or even less, is sufficient to go through
the whole operation of hardening, infiltrating, and sectioning, near-
ly all kinds of plant tissues.

The sections after being cut can be easily handled with a
camel's hair brush without fear of breaking.

24 CUTTING A^D MOUNT! \(i Tlssi'KS.

In the case of delicate tissues, like fern prothallia, or the
apical cell of Nitella or Chara, some little variation is made from
the regular method and a detailed description of the process may
be of value. (The Microscope, Nov. 1893.)

The material is first placed in 10 per cent, alcohol in a dehy-
drating apparatus and allowed to remain for 24 hours, when it is
taken out and placed for one hour in 95 per cent, alcohol to insure
complete dehydration. After this the tissue is placed in a 1£ per
cent, solution of collodion and allowed to remain in a tightly corked
vial for 12 hours. The cork is then removed, and by slow evapora-
tion of the ether and alcohol, the collodion will thicken. When it
is of the consistency of ordinary glue, the preparation is poured
out of the vial, with the collodion, into a paper boat, of the kind
used in paraffiue imbedding, or an ordinary watch glass will answer
the purpose. The thick collodion is then poured over the tissue
and allowed to harden in the air until a firm film has formed over
the surface. After this the mass is placed in a jar of 85 per cent,
alcohol and allowed to remain from 5-6 hours until the collodion is
quite tough. Then with a thin knife cut out a block of collodion
containing the tissue inside. The block can be placed in any
desired position on the end of a cork and held while thick collodion
is poured over, until it is covered. Each coat as added should be
allowed to slightly harden before applying the next, until the
operation is completed. After the whole has become firm in the
air, it is placed in a jar of 85 per cent, alcohol where it should
remain 6 to 8 hours. The operation of sectioning and mounting is
the same as outlined for the firmer tissues.

In order that the sections may be all arranged the same side
up, the block of collodion, which should always be trimmed at the
top, can be cut with a notch near one corner, and the notches all
arranged with the same relative position on the slide.

It will be found that with this method perfect serial sections
of any desired thickness can- be obtained from objects which are
not more than one layer of cells thick, and thus render the prepara-
tion of delicate tissues but little more difficult than that of the
firmer kinds found in ordinary stems and roots.

Many substances for infiltrating have from time to time been
suggested, and have met with varying success. The more irnpor-

25 CUTTING A ND MO UNTl \(i TISS UES.

tant ones tried are gelatin, gelatin soap, paper, wax, gum arabic,
and paraffine mixtures, but it is not necessary to outline other
methods here as the few described in detail will enable the student
to prepare material for any work suggested in this manual.

STAINING AGENTS.

Only a few of the more important staining agents will be men-
tioned, and directions given for their general use.

Ammonium Carmine.

This stain is best prepared, as suggested by Hartig. Dissolve
a little carmine in water until the mixture has the consistency of
paste. Add to this a little strong ammonia and evaporate the whole
to dryness over a water bath. The resulting powder dissolved in
water is used for staining.

Alum Carmine.

/

Make a concentrated solution of alum and add to it enough
powdered carmine to give it a deep color, (1 gram to 100 c.c. of
alum solution). Boil for 10 minutes and, when cold, filter. This
agent is much used and is often valuable as a selective stain. It
colors pure cellulose cell walls a bright red, but does not effect
those that are lignified or suberized.

Picro-Carmine (Gage).

Twenty grams of picric acid are dissolved in 200 parts of
water, and mixed with 5 grams of carmine in 250 c.c. of strong
ammonia. Stir the whole thoroughly and evaporate to dryness.
Dissolve the residue in 700 c.c. of water. All of the carmine stains
are very useful, easily handled, and quite selective. Picro-carmiue
turns protoplasm a yellowish red.

27 STAINING Ad K -V TS.

Eosin.

This is a valuable general stain, as it has a great coloring
capacity. It stains nucleus and cell wall readily and is much used
in double staining. It can be applied either in an aqueous or alco-
holic solution ; 1 gram of eosin to 100 c.c. of water is a very con-
venient strength.

Haematoxylin.

This coloring agent can not be recommended too highly. It
has a very wide application and gives uniformly good results. The
stain is made by adding a concentrated solution of haematoxylin
crystals in alcohol, cautiously, to a 3 per cent, aqueous solution
of alum, until a medium purple color is obtained. The solution
becomes darker and better by standing a few days, but deteriorates
after a time and will require filtering often. As suggested by Prof.
Gage the addition of chloral hydrate and proper sterilizing of the
constituents of the stain during its manufacture greatly increases
its keeping power. (Proc. Am. Microscopical Society, Jan. 1893).

The old stain, if kept in a cool place, is very valuable in stain-
ing meristematic tissue. Haematoxylin stains the nucleus a deep
blue or purple and is to be recommended for all general work. It
is sometimes used in staining bacteria and with other stains in
double staining.

ANILINE COLORS.

These colors have of late been very useful in furnishing stains
for histological work. To them we owe much for the present valu-
able and convenient methods of staining. Only a few of the more
important agents can be included in this outline.

Hethyl Violet.

An aqueous solution of this is much used in staining bacteria.
It is also valuable as a selective stain for many plant tissues.

riethyl Green.

An aqueous solution with 1 per cent, of acetic acid is used for
staining the nucleus and chlorophyll grains. As a double stain it is
often used on transections of stems in connection with eosin.
Iodine green is, however, preferable for double staining.

28 STAINING AGENTS.

Aniline Blue.

This stain is much used in connection with the staining of
bacillus tuberculosis, but is also very satisfactory as a stain for
sieve plates and sieve tissue. Cellulose takes a blue color while the
sieve plates become azure. Sections treated with this color are
liable to fade after a time. The stain is good in double staining.

flagenta.

Magenta is used both as a general and selective stain. It
should be applied in an aqueous solution, containing a little acetic
acid. The tissue will need to be left in the solution for some time,
before the proper depth of staining is secured.

Picric Acid.

This is a very convenient and useful ground or general stain
and is usually applied in weak alcoholic solutions. It must be borne
in mind, however, that picric acid will wash out to quite an extent
many of the aniline colors, but in use with haematoxylin, borax, or
alum carmine, it makes a most excellent general stain. In connec-
tion with hydrochloric acid it will readily wash out the carmine
colors.

Silver Nitrate.

In a dilute alkaline aqueous solution this is often used as a test
for living protoplasm, since it colors it black, while dead protoplasm
remains unchanged. The reaction is very delicate and positive.
Tannic acid also colors less dilute alkaline solutions black, while
cells containing glucose are colored brown. As a general stain
the action of silver nitrate is too uncertain for positive directions.

CLEARING AGENTS.

The function of a clearing agent is to make the tissue trans-
parent by penetrating into all parts of it.

It must be a liquid of high refractive index and miscible with
balsam, as well as having the power to drive out all of the alcohol.
The agents employed are very numerous, but a successful one
should replace the alcohol quickly and yet not destroy the tissue by
shrinking. The chief clearing agents are the essential oils.

Cedar Oil.

\
This oil is a very good clearing agent but is quite slow in its

action; however, it does not shrink the tissue nor fade aniline
colors.

Clove Oil.

This is one of the best clearing agents. The clove oil on the
market is usually impure and not suitable for use. The pure oil
can be obtained only from reliable dealers. As a clearer, it pene-
trates tissue very readily and clears most thoroughly. It has a
very high refrative index.

Collodion is dissolved by this oil, and it is therefore not safe to
use with collodion sections unless proper precautions are taken to
prevent the displacement of the disconnected parts.

Tissue that remains in clove oil any length of time is liable to
become brittle, and this is sometimes very helpful in minute dis-
sections.

Aniline colors are often faded by the action of this clearer.

30 CLEARING AGENTS.

Oil of Origanum and Oil of Sandal Wood.

These are both to be recommended as clearing agents, but for
many tissues are not wholly satisfactory.

Turpentine.

Turpentine is much used for clearing paraffine sections, as it
dissolves out the paraffine and clears the sections at the same time.
It is very liable, however, to cause sections cut in alcohol or
collodion to shrink.

Carbolic Acid and Turpentine.

This is to be recommended as the cheapest and at the same
time the most satisfactory of all clearing agents.

The mixture is made of 3 parts of turpentine and 2 of pure
carbolic acid. It clears equally well paraffine, collodion, or alco-
holic sections, and needs but a few minutes to thoroughly permeate
the tissue. It is best to filter the mixture through cotton to remove
all particles of dust. The carbolic acid gives the hands an unpleas-
ant feeling and they should therefore be kept free from it.

MOUNTING MEDIA.

Aluminium Acetate.

In a saturated aqueous solution this salt forms a good mount-
ing medium for many delicate organisms. Most algae can be pre-
served in this way without any deterioration, while such objects as
the young prothallia of ferns can be kept without shrinking or
losing much of the brightness of their chlorophyll or protein
granules. Like all liquid mounting media it must be used in a
cell, and the slide should lie for 24 hours before sealing.

Balsam.

Balsam forms a most excellent and substantial mounting
medium. Sections mounted in it should be free from water and
this can be easily brought about by the use of alcohol. It is best
to begin with alcohol of moderate strength (50 per cent.), and
gradually increase it until the tissue is taken from that of 95 per
cent, strength. Before mounting, the sections must be cleared of
alcohol by the use of turpentine, chloroform, oil of cloves, or bet-
ter, a mixture of 3 parts of turpentine and 2 of pure carbolic acid.

Balsam is prepared by evaporating over a gentle heat common
commercial Balsam of fir until the volatile oils have been driven
off and the residue becomes brittle. The portion that remains is
then dissolved in cedar oil, xylol, or chloroform, and filtered
through glass wool in a paper funnel. The medium should, at
the ordinary temperature, be of the consistency of a thick syrup.
Care must be taken to keep air bubbles from balsam. If, however,
they get under the cover they can be driven out if the slide be left
in a warm place for a few hours.

32 MOUNTING MEDIA.

Although not absolutely necessary to seal the cover glass of
balsam mounts, it is, nevertheless, a good precaution, as the cover
may crack away from the medium and the section be displaced.
The balsam should always be kept in a glass-stoppered bottle, and
applied to the slide from a pointed glass rod.

Carbolic Acid.

Carbolic acid in a 1 per cent, aqueous solution is used as a
mounting medium, also carbolic acid crystals in glycerin, for some
of the firmer tissues. These ageiits have a tendency, to make the
sections clear or faded.

Calcic Chloride.

About one part of this salt to two parts of water is a good
mounting medium for many tissues. A little camphor should be
added to the solution to preserve it.

Glycerin Jelly.

This is extensively used as a mounting medium for small and
delicate objects, which might be injured by clearing for balsam.
Glycerin jelly mounts will, after a time, become transparent, unless
they have previously been stained with a permanent stain. Care
should be taken in mounting sections to prevent air bubbles
getting under the cover glass, since they do not disappear as in
balsam mounts. Glycerin jelly mounts should be sealed as soon
as cold.

Many formulae for the preparation of this mounting medium
are in use, several of which seem to be equally good. Kaiser's jelly
is easily made and keeps well. It is prepared by soaking oue pare
by weight of best French gelatine in six parts of distilled water for
two hours ; 7 parts of glycerin are then added, and 1 drop of con-
centrated carbolic acid for every gram of the mixture. Warm 10-15
minutes, with constant stirring, until the mixture becomes clear,
and then filter while warm though wet glass wool in a hot water
filter. The jelly will require warming before use. The mixture
keeps well for years and is a very convenient mounting medium.

Glycerin.

Glycerin is often used as a permanent mounting medium and,
with proper precautions, gives satisfactory results. To prevent

33 MOUNTING MEDIA.

shrinking, sections to be mounted in glycerin should first be placed
in a mixture of equal parts of glycerin and water and allowed to
remain a little time, when stronger glycerin is added at intervals,
until the section is thoroughly permeated with the pure agent. As
this is hygroscopic, the mounts must be sealed at once as they will
readily absorb a quantity of water sufficient to dilute its strength
appreciably. The cover can be sealed first with a little glycerin
jelly, and, after this has hardened, with shellac or asphalt.

Glycerin is a very good mounting medium for studying fresh
tissues, as it evaporates very slowly. Sections mounted in it can
be kept for examination for some time. Glycerin, like gtycerin
jelly, has the property of making sections clear or transparent.
They should therefore be treated with a permanent stain. Only
the purest commercial glycerin should be used, and this must be
kept in a tightly corked bottle, otherwise, it will absorb a sufficient
quantity of water to render it almost useless as a mounting
medium.

Glycerin and Acetic Acid.

A mixture of equal parts of glycerin and acetic acid is a very
convenient mounting medium for many kinds of tissue. Especially
is this true with some of the fungi. In the preparation of this
mixture, pure glycerin and concentrated acetic acid should be used.

King's flounting Medium.

This is good for many fresh- water algae, and like all fluid
mounts it must be used in a cell. It can be secured of dealers in
mici'oscopical supplies.

Water.

Water is not infrequently employed as a mounting fluid. To
preserve the mounts from deterioration a little camphor should be
added. Water is often used as a medium for the studying of fresh
tissue ; but it should be borne in mind, that it may change the
nature of the tissue materially, owing to the osmosis between the
cell contents and medium. This can be prevented by adding some
substance to the water to make its density equal to that of the tis-
sue, or cell contents. Salt is sometimes used, but is by no means
efficient.

CEMENTS.

The number of cements and varnishes is so numerous that one
is at a loss to know just what is best to use, but the general char-
acters of some of the more common ones will be brieflly noted.

Gold Size.

This cement can be secured of dealers in microscopical sup-
plies and is certainly to be well recommended. If used for balsam
mounts, it is best to first ring the cover glass with a coat of shellac.

Shellac.

Shellac is certainly very convenient and seems to be quite dur-
able. It is prepared by dissolving solid shellac in alcohol until the
solution is of a medium oily consistency. A little aniline dye can
be added to color the mixture to one's fancy, also a few di'ops of
oil should be used to prevent the cement from cracking. The mix-
ture is applied, as are all sealing mixtures, with a small brush and,
preferably, by the use of a turn-table.

Ball Cement and Asphalt Varnish.

These are among the very best of cements and can be obtained
from the regular dealers in supplies.

White Zinc Cement.

This cement is much used by microscopists for making cells.
It is very hard and often shows a tendency to crack.

Many very good cements are on sale by reliable dealers, and
one has but to convince himself of the relative merits of a few of
the more important ones, when he will be able to settle upon some
special one well adapted to his purpose.

SERIAL SECTIONING.

It is often very desirable and indeed quite necessary that the
whole of an organ be studied systematically, but to do this requires
that the parts not only be placed in a condition to be observed,
but also, that the arrangement of parts as sectioned be so system-
atic as to show the relation of each section to the whole. This
can be accomplished by making what are known as serial sections
of the object. In this case it is necessary that the sectioning be
done in a microtome in order to preserve a uniform thickness.
The sections as cut, should be arranged on the slide close to each
other and in the same order as they are removed from the object.
They should also occupy the same position with reference to the
relation of each section to the whole.

With the paraffine method when "ribbon sections" are cut, it
is only necessary to break off pieces of ribbon and lay them along-
side of each other in the order in which they are removed.

With the collodion method as the sections are cut, they should
be taken up with a camel's hair brush and transferred to the slide.
After being arranged in position, they should be sealed by blowing
over them a little ether vapor. The slide should be kept constantly
wet with alcohol to prevent the sections from drying or shriveling.
The arrangement of sections on the slide should be uniform at all
times. A very convenient order is to begin in the upper left hand
corner and place the sections under each other in a row along the
longest axis of the slide. After the row has reached to within 2£
c. m. of the opposite end, a new row is begun at the right of the
old one, and at the top of the slide; 5x2^ c. m. cover glasses will
be required.

36 SERIAL SECTIONING.

When the sections are to be designated by number, they should
be numbered in the order in which they are placed on the slide.
Serial sectioning is very important in any thorough investigation,
since it places before the observer all of the object to be studied,
in condition for careful searching, enabling one to trace the course
of any part in every direction through the object. Serial section-
ing certainly saves much time and prevents many misconceptions
that might otherwise arise from the examination of a single prepara-
tion.

DOUBLE STAINING.

It is often desirable, in order, to bring out more clearly some
part of a specimen, to stain different portions of it with separate
colors. This is very easy to bring about if the proper stains are
employed. For example, the xylem of a fibro-vascular bundle can
be stained one color and the phloem another. These results are
very important in research as one is enabled to recognize in this
way similar tissues in different parts of a specimen where other-
wise they might be somewhat difficult to distinguish. Certain
stains will color one part of a tissue or special part of a cell, and
yet not effect in any way other portions.

The combinations of stains that can be used for this purpose
are very varied, and the results in the case of some quite uncertain
to predict, consequently no general rule can be laid down in regard
to the kind of tissue any stain will invariably act upon.

With regard to the treatment, in general the selective stain
should be applied first, and in dilute solutions. The sections
should then be thoroughly washed and the general stain applied,
the tissue washed again and mounted directly in glycerin jelly or
dehydrated, cleared, and mounted in balsam. Glycerin jelly is per-
haps a better mounting medium for double stained tissue, since the
alcohol and clearer are often injurious in their bleaching effect.
Some combinations of stains whose effects can be depended upon
are haematoxylin and eosin or picro-carmine, iodine green and eosin,
carmine or haematoxylin with picric acid, and methyl green and
eosin.

It is often necessary to use a mordant to fix the stain, in which
case a saturated solution of alum can be applied to advantage. A
little experience with double staining will enable one to . use it in a
way that will be of great assistance to a better interpretation of
the character of many tissues.

FLUID MOUNTS.

flounting in Cells, Etc.

It is frequently desirable to mount small objects in such a way
as to preserve them without crushing or mutilation. In this case
the cover glass must be supported in order that it may not come in
contact with the preparation. This is accomplished by mounting
the object in a cell, which can be made in vaiious ways, depending
on the nature of the object to be mounted. If the specimen is very
small and is to be preserved in balsam or glycerin jelly, it may suf-
fice to place around the object a few pieces of broken cover glass to
keep the preparation from being crushed by the cover. Seal as in
other mounts. If the object to be mounted is quite large, a deep
cell must be made.

The nature of the material of which the cell should be con-
structed must depend on the character of the mounting agent,
that is, the material of which the cell is made must not be of a sub-
stance in any way acted upon by the mounting fluid. For
many mounting media, cells made of shellac are very convenient,
and quite durable.

To make the cells, place the slide on a turn table and when
the table is revolving, touch the slide with a small brush dipped in
shellac. A ring is the result. After the shellac has dried, another
ring should be made on the top of the first. Allow this to dry and
repeat the operation until the cell is of the desired depth. Several
of these cells should be prepared and kept on hand until they may
be needed. Before using, paint over the top a very light coat of
thin shellac. The object to be mounted should be placed in the
center of the cell, and the latter filled completely with the mounting

39 FLUID MOUNTS.

medium. To place the cover glass in position, rest one side of it on
the edge of the ring and lower it slowly over the object, allowing
the surplus mounting fluid to be forced out on one side. Press the
cover firmly in position. Before sealing fluid mounts, fasten the
cover in place by applying three or four small drops of sealing mix-
ture at the edge of the cover glass at different points. As soon as
dry, these hold the cover in place and allow the mount to be per-
manently sealed. Cells may be made of glass, zinc, bone, or cellu-
loid rings which can be secured of any regular dealer in micro-
scopical supplies ; or rings can be cut from sheet lead, tin, paper, or
wax, and fastened in position with shellac or marine glue applied to
the base of the cell and then held firm, by the ring, against the
slide until dry. Seal around the outside and when ready for use,
apply a light coat of sealing mixture to the top, to hold the cover-
glass in place. When the object is mounted, seal the whole prepa-
ration as in the case of ordinary mounts.

Objects can be easily mounted dry in cells, in which case they
should be held in position by fastening them to the slide with a
little glycerin jelly, collodion, or gelatine. Fluid mounts should be
examined from time to time to repair any that may not have been
perfectly constructed.

EQUIPPING Of LflBORflTORY.

In the equipping of a laboratory, one must necessarily be con-
trolled largely by the material and funds at their disposal, but a
few suggestions may serve to lighten somewhat the care of over-
seeing so much manipulation.

Each student should be provided with a case made by boring
two rows of holes about 45 m. m. in diameter in a block of wood
30x14 c. m. and 45 m. m. thick. In this should be placed bottles
containing the more general reagents and stains that will be needed
frequently in the work. It is suggested that the set consist of
Iodine, Acetic, Sulphuric, and Hydrochloric acids, Glycerin, Potassic
Hydrate, Eosin, Haematoxylin, and Clearer. Another case contain-
ing reagents and stains of a more special nature can be placed on a
center table easily accessible to all. The desks should all be
equipped with wash bottles of alcohol and water, and a general
supply of the same agents should be located in a convenient place
in the laboratory. These arrangements will prevent the student
from being compelled to walk about searching for some reagent or
supply. Cases should be provided for containing the general store
of chemicals and glassware. The books should also be in a con-
venient place easily accessible to all.

It is much more desirable that the microtomes, hardening and
infiltrating apparatus be on a special table which should also be
supplied with all the stains and reagents necessary for mounting
the sections. A convenient form of a waste vessel over which the
sections can be treated is made by fastening, with sealing wax, to
a tray or dinner platter, glass rods, parallel and about 4 c. m. apart.
The slides with the sections can be laid on these rods and treated

41 EQUIPPING OF LABORATORY.

with the various reagents, which can be washed off directly into
the tray beneath and thus prevent table or hands being soiled by
the stains or clearer. It is very convenient to have a small supply
of alcohol and water in bottles elevated at a little distance on a
shelf, and provided with a siphon having rubber connections below
to enable it to be used in various parts of the table. The orifice of
the glass tip should be small and the rubber tube provided with
a pinch cock to regulate the supply at pleasure. This arrangement
will be found very convenient to keep knife and tissue wet while
sectioning tissue imbedded in collodion, and also for dehydrating
and washing the sections on the slide.

For the clearer, a wash bottle should be pi-ovided with an
enlargement at the inner end of the exit tube filled with cotton,
thus preventing any particles of dirt from getting into the clearer
used on the slide. The enlargement can be made by cutting off
from the end of a glass tube, just small enough to enter the mouth
of the flask, a piece about 5 c. m. long. Close one end with a cork
and pass through a hole in this exit tube. Then fill the piece of
tubing loosely with cotton. A similar bottle will be found very
useful for the haematoxylin. (Fig.3.) Many devices will be suggested
in the laboratory from time to time and materially lighten the burdens
incident to having the charge of so much laboratory instruction.

COLLECTION AND PRESERVATION Of
MATERIAL.

Much of the material for work in histology must be collected
during the summer, or at times when it cannot be used at once,
and is therefore to be kept stored away until needed for study.

Some difficulty must necessarily arise in the proper preserva-
tion of such material, but if special precautions are taken it can
easily be kept in good condition for histological purposes. The
methods of treatment vary with the nature of the material.

All soft tissue collected, as, for example, leaves, herbaceous
stems, etc., should be placed at once in 40-50 per cent, alcohol and
hardened in the usual way by the use of a Schulze's apparatus, or
by transferring successively, for 24 hours in each, to. 50 per cent.,
67 per cent., and 75 per cent, alcohol, in which it can be kept a
long time without deterioration. The softest tissues such as are
found in algae, etc., should be placed first in about 10 per cent,
alcohol and hardened by the use of Schulze's apparatus, or by
transferring, successively, for 24 hours in each, to 20 per cent., 30
per cent., 40 per cent., 50 per cent., etc., to 75 per cent, alcohol,
where they may be kept as in the case of the firmer tissues. The
material thus hardened when ready for use can be dehydrated with
95 per cent, alcohol, infiltrated with 2 per cent, and then 5 per
cent, collodion. In the latter solution it may remain indefinitely
without shrinking. If the tissue to be sectioned is kept on the
cork in alcohol, it will in time become discolored by the tannin of
the cork.

The tissue of each sort should be trimmed until only such
parts remain as are needed for sectioning. These should be care-

43 COLLECTION AND PRESERVATION OF MATERIAL.

fully tied up in pieces of bibulous paper, with the labels written in
India ink or pencil, inside. In this way, many different things can
be kept in the same jar and thus economize room and solutions.
If the material is to be preserved in thick collodion, the stopper of
the vessel in which it is placed should make the bottle air tight and
should also be held in place in some way, otherwise the evapora-
tion of the ether may force it out and ruin the material by the
hardening of the collodion. If it is desired that the tissue be kept
on blocks in alcohol any great length of time, in order that it may
be ready for use at once, hard rubber rods sawed into convenient
lengths of about 2 c.m. may be substituted for the cork. As the
alcohol does not effect the rods, tissue can be kept in this way any
length of time without deterioration. The rubber rods can be
obtained of the Educational Supply Co., Boston.

Blocks of hard wood have also been suggested for the same
purpose.

In collecting material to preserve for class-room work, much
care should be taken to prevent confusion of labels, etc., and all
important data should be included with the notes on each study.

THE MICROSCOPE.

Fijr. 4. Miriosropo, with the parts lettered to correspond with the description.

4:, THE MICROSCOPE.

A. Base of the instrument and part that forms its entire sup-
port. This may be in the form of a horse-shoe or a tripod, support-
ing at three points. This condition is desirable, as greater solidity
is thereby given to the instrument.

B. Pillar. — This is the upright support from the base and
usually has a joint at the top, by means of which the instrument
may be inclined.

C. Arm. — This carries all of the remaining parts of the
instrument.

D. Body. — This is the tube holding the optical parts of the
instrument that are above the stage. The raising and lowering of
these is controlled either by a gearing or by friction of the outer
stationary part with the inner movable one.

E. Nose-Piece. — A small revolving part fastened to the
lower end of the body and forming an attachment for the objectives.

F. Objective — This is the lower of the lens combinations
above the stage and is usually screwed into the nose-piece. Most
microscope makers use a uniform size of thread and this is known
as the society screw. The function of the objective is to form an
image of the object.

G. Ocular — The part holding the upper combination of
lenses, and fitting into the upper end of the body. The function
of the ocular is to magnify the image produced by the objective.

H. Draw-Tube — This forms the inner part of the body and
moves in an outer sheath. The length of the body may be varied
by the adjustment of the tube in its collar.

I. Collar. — A ring fastened to the upper end of the body and
forming a sleeve for the draw-tube.

J. Course Adjustment. — This is used for raising and lowering
the body. It is provided with two large milled heads (K), which
revolve a pinion that acts upon a rack, and controls the working
of the adjustment.

L. Fine Adjustment. — Used to raise or lower the body
slowly through short distances, in this way obtaining the exact
focus. It consists of a milled head with a screw that acts upon the
body of the instrument.

M. Stage — This is firmly attached to the pillar and is for
the support of the object during examination. Sometimes a

46 THE MICROSCOPE.

movable slide carrier is attached to the stage. The more expensive
instruments are often provided with a mechanical movement that
enables the object to be carried in any desired direction by simply
turning two screws located above or at the side of the stage.

F. Clips — These are to hold the glass slide, on which the
object is mounted, firmly against the stage.

O. Mirror. — This is one of the optical parts below the stage
and is for the purpose of illuminating the object, either by throw-
ing light through it, or on it from above. One side of the mirror
is usually plane and the other concave.

P. Mirror Bar. — A bar attached to the arm and carrying the
mirror. It can usually be swung from side to side to vary the
angle of illumination.

Q. Sub-Stage Ring. — Used to support either the diaphragm
or some of the optical parts that are used below the stage. It is
often attached to the latter but in the more perfect instruments is
borne on a separate bar.

S. Diaphragm. — This is a disk provided with numerous
apertures, of various sizes, and can be turned to regulate the
amount of light coming from the mirror to the object.

Field of the Hicroscope. — This is the clear area seen by look
ing into the microscope, and should be perfectly circular, when the
instrument is properly lighted.

METHODS Of STUDY.

It is very important that the student follow from the begin-
ning a system of work that will enable him to utilize the experi-
ence of those who may have had years of training and have learned
the roads to uniformly good results. A few suggestions may not
be out of place.

Care in Observation.

Avoid making hasty conclusions even though the appearances
seem to warrant them. Do not consider as proven a condition that
can be seen but once and then under difficulties. In important
cases, always verify results by trying the study again with other
material so that there can be no doubt as to the exact state of
things. Never substitute an opinion or inclination for a fact even
though it often involves a disappointment, better that than error.

Selecting Material.

Many of the troubles and difficulties in the way of a proper
study can be overcome by taking due precaution in the selection
and preparation of material. Too often a section is carelessly
made with the hopes that it may show something desired, but it is
better always to select the material carefully, then section and
mount with all the proper precautions.

Directions for Drawing.

It is necessary that the student study and thoroughly under-
stand the tissue under examination before any attempt is made to
reproduce it, otherwise, after the drawing is finished errors in it

48 METHODS OF STl'

will probably be found, and it will not represent the true relations
of parts. Do not draw everything that can be seen, but place on
paper enough to represent accurately the general outline and
minute structure of the object that is being studied. Let the
relations of the parts drawn be so clearly indicated that a correct
picture of the object can be perfectly formed in the mind from the
drawings. The first drawing should usually be an outline sketch
of the whole object that is being studied. Then should follow
a more detailed drawing of each particular part as examined, and
lastly, the minute structure of any special tissue or cell. It is true
that such detail will require much time but practice will soon
reduce this and make the work seem very easy. It is not best to
shade any of the drawings since it may obscure the parts that
should be kept prominent. Often in studying the minute structure
of some organ, a single section will not give a complete outline of
the whole part that is to be represented, in which case, it should
be made up from the several sections that will best show the parts
desired. Be certain, however, that, the relations of parts are
thoroughly understood and correctly represented. One is often
inclined to make the drawings too small but this should be guarded
against, and the sketches made of sufficient size to enable the
parts to be clearly seen without close scrutiny. The drawings can
be made free-hand or by the use of a camera lucida. The latter
method is necessarily the more accurate but lacks much of the ele-
ment of good training given by the former. If the drawings are
made with a camera lucida, much of the detail will need to be filled in
free hand, but the general outline can be made with great ease. The
best camera lucidas are of the Abbe pattern which can be used with-
out tilting the tube of the microscope. With it, some difficulty
may be experienced in seeing the object and pencil point at the
same time, but this can be overcome by having the paper equally
illuminated with the object under the microscope. Other camera
lucidas may be used but it is not necessary to outline the working
of each in these directions. With the Abbe camera lucida, be cer-
tain that the drawing paper is at right angles to the axial line as
reflected from the mirror, otherwise the drawing will be distorted.
The variation a from perpendicular with the table can be deter-
mined by the use of a semi-circular protractor, which will give the

49 METHODS OF STl'DY.

angle to which the mirror is tilted. The drawing paper should be
raised at an angle with the table twice as great as the mir-
ror is depressed, below 45°. The drawing board should be
made with a hinged part that will support the paper and yet
allow it to be raised to any desired angle. The magnification
of the drawing should be determined and indicated underneath it ;
(x300) indicates that the drawing is magnified three hundred
diameters. Free-hand drawings should be made with special
reference to exact proportion, in order that every part may have
the same magnification. Pencil drawings should be made on good
drawing paper and with shai'p-pointed drawing pencils, which had
better be of two degrees of hardness, o^e being quite hard and the
other medium soft. The former can be used for detail work and
the latter for general outline. The objection to pencil drawings is
that they blur by rubbing and are not as clear as those
made with ink. It is better to outline ink drawings at first with a
pencil and then retrace and fill in the detail with the pen. The
different parts of the drawing should be named at the side and the
name connected to them with a dotted line. With a little care, this
can be done without trouble, and the drawing book will look neat
and be easily interpreted. Where any part of an object is to be
repeated several times in the same drawing, it is only necessary to
outline the parts and indicate the repetition. Also where there is
to be shown a large mass of cells of uniform nature, the whole
should be outlined and a few cells drawn to indicate their general
character.

Next in importance to the drawing is the description, and
this must not be neglected. It should always include a careful
outline of the nature and intent of the study, together with a full
explanation* of every part of the drawing with the relations of each
part to the whole object. It is always best to adopt some uniform
way of making drawings and keeping notes. The system outlined
below will be found very useful and convenient for examination.

50

METHODS OF STUDY.

Study of the Transection of the Anther of Begonia.

Aiither cavity containing pollen mother

cells.
Mother cells with young pollen grains.

Empty anther cavity.

Parenchyma cells of the interior of the
anther.

Fibro-vascular bundle extending along
the center of the anther.

Epidermis of the anther.

Fig. 5. Sample page of a drawing; book.

The notes and explanations which accompany the drawings
may be kept on separate sheets or pages in the drawing book.

Preservation of Slides.

To the person who makes a collection of microscopic slides, it
becomes a matter of no little importance as to how they should be
arranged in order that they may be best preserved and at the same
time available for instant reference. Many kinds of cabinets may
be purchased of dealers, but the large ones are quite expensive and
beyond the means of many. A vei-y inexpensive way to preserve
the preparations is in mailing boxes, which hold 25 slides. These
can be filed away but should be kept on end to prevent displace-
ment of the mount. After a time, however, the number of boxes
accumulates so as to become burdensome ; and furthermore, it is
better for slides to be supported from below on each side of the

51 METHODS OF

cover glass, rather than at the ends. A servicable and convenient
cabinet can be made by removing the drawers of an empty spool-
case and making a door for the front. The case can then be filled
with drawers suitable for holding the slides. To make these cut a
board, one-half inch thick and as wide as the case is deep, into
lengths one inch shorter than the width of the case. With the aid
of a buzz-saw and chisel, the boards can be made so that a section
of it will appear as the figure :

a ^

Fig. 6. Diagram showing the construction of a drawer for a slide case.

The grooves are to be cut across the grain of the wood. Cut
thin strips and glue them in position in the grooves 1 and 2. The
distance between these points should be 3£ in. The slides rest
on the surfaces 3 and 4, and are separated by little partitions, about
1£ in. apart, glued into a groove made across the board with the
saw before the partitions 5, 5 ' were placed in position. The little
partitions must necessarily be short, about 2£ c.m. long in order
that they may not come in contact with the pieces 5 and 5 ' . To
remove the slide from the drawer, it is only necessary to press
down on one end when the other will be raised and can readily be
grasped.

Such a cabinet can be easily made by a carpenter in a few
days. Much of the work can be done by machinery. The original
cost of the spool case should not be over $3.00. Double spaces can
be left in the drawers for the large sizes of slides and appartments
arranged in any desired way. A piece ^ inch thick fastened to the
end of the drawer will prevent its warping and at the same time
serve for a groove on which the drawer can slide.

The number of the first and last slide in each drawer can be
fastened on a strip, to the front surface.

To catalogue the sections obtain cards 7|xl2£c.m. of good

52

METHODS OF STl'DY.

bristol board. Each slide should have a corresponding card and
on it the following data should be given:
1. Number of preparation.

Name of object, from what taken, and locality.

Name of preparer and date of mounting.

Object of preparation.

Method of mounting, stain, mounting medium, etc.

Reference to figures, books, and papers.

Remarks.

2.
3.
4.
5.
6.
7.

Sample Card.

No. 1.

TRANSECTION OF LEAF.

Oct. 29, '93. J. Smith, Preparer.

From Begonia Sanguinea. Green house plant.

Shows general structure of leaf, stomates, guard cells,
etc.

Hardened with alcohol, infiltrated with collodion,
stained with haematoxylin, and mounted in bal-
sam.

Strasburger's Pract. Bot. p. 162.

This section is quite thin and should be studied with
the high power, etc.

The cards should be arranged alphabetically according to sub-
ject and can be kept on edge in a box of convenient size, with the
topics separated by tin or card board on which the letters of the
alphabet are pasted. This arrangement places at hand for instant
reference the whole collection of slides, and enables one to easily
find any particular section, while it furnishes a record of valuable
data with each preparation.

When any slide is permanently removed or destroyed, the card
can be taken from its place and the set suffers no injury.

flPPflRATUS NEEDED.

Certain apparatus not supplied by the laboratory will be
needed by each student in his work, while many things not actually
needed will be found very useful and even quite indispensable for a
thorough course.

The laboratory should be supplied with good compound and
simple microscopes. The former should have a magnification of
from 75-600 diameters, and the latter about 20.

As regards the most suitable microscope stand, there need be
no discussion. The stands of any reliable maker can be used. It is
important that the working parts be all accurately adjusted, the
pillar provided with a joint for inclination, and the instrument be
firm and substantial. The Continental stands of American manu-
facture are especially to be recommended, as they are quite compact
and can be fitted with the various sub-stage parts with but little
alteration. The instrument should be furnished with a nose-piece for
the objectives and an ocular micrometer for measurements. For
the simple microscope, the ordinary tripod magnifier answers the
purpose very well and is useful in teasing material with needles
under a low magnification. The laboratory should be provided
with some form of a sliding microtome. Well microtomes can be
used for many things, but for cutting serial sections or material
that has been infiltrated with collodion, a sliding microtome is very
desirable. The patterns of several good makers are on the market
and can be secured of regular dealers. The large microtomes of
Bausch and Lomb, or Reichart are especially to be recommended.
They cost forty or fifty dollars but the advantages in their use will
well repay the expense. The small student's hand microtome of

54 A I' I' A It ATI'S XEEDED.

Bausch & Lomb, is very convenient, and, in case the large sliding
one can not be secured, it will be found serviceable for almost all
kinds of sectioning. Its cost does not exceed ten dollars.

Reagents, Etc.

The following is a list of the more general reagents used in
the laboratory :

Hydrochloric, Nitric, Sulphuric, Acetic, Osmic, Chromic, Picric,
and Carbolic acids.

Caustic Potash, Sugar, Potassic Iodide, Iodine, Chlor-iodide of
Zinc, Glycerin, Schweizer's Reagent, Glycerin Jelly, Balsam,
Shellac for sealing cover glasses, Turpentine, Chloroform, Ether,
Gun-cotton, Xylol, Paraffine, Clove Oil, Clearer.

Haematoxylin (cryst.), Eosio, Carmine, Magnenta, and various
Aniline stains.

Alcohol and distilled water should be provided in quantities
for all the general manipulations in which they are required.

The students should provide themselves with glass slips with
ground edges, 3x1 inches, and cover-glasses of assorted sizes. ^
and £ in. circles are most frequently needed, but serial sections
will require 1 inch square and a few 25x50 c. m.

Adhesive labels, 1 inch square, will also be needed for labeling
the slides. For storing the mounted preparations, mailing boxes
holding 25 slips are most convenient for student's use. Two
camel's hairbrushes will be needed one, for handling the sections,
and the other for brushing dust from lenses, covers, etc.

The drawing material required should be a note book of un-
ruled drawing paper or separate sheets cut the proper size can be
used. "Ledger Linen" is to be especially recommended for this
purpose. The sheets with notes and drawings can be fastened in
a cover of maniila paper by boring two holes through the backs
and fastening with a string.

Two kinds of drawing pencils are needed. If ink drawings are
to be made, India ink and a fine pointed pen ("crow quill" or
Mapping pen No. 291) should be provided.

Dissecting needles can be made by grasping a strong fine
pointed needle between a pair of pliers and forcing the head into
the end of a pine stick or a straight twig with a small pith.

55 APPARATUS NEEDED.

It is very desirable that the laboratory be provided with an
Abbe camera lucida and some high power objectives; e. g., a 1-10 or
1-12 oil immersion. A substage condenser is very desirable when
such high magnification is to be used.

It is necessary that each student be provided with a good
section knife or razor. One of the latter ground flat on one side
and concave on the other is preferable. The edge should be kept
very sharp and keen, otherwise the sections will be uneven and torn.
A suitable hone and strap are needed in the laboratory for keeping
the edge in order. Unless the students can be carefully taught
how to sharpen a knife, it should be taken to some one familiar with
honing razors. For cutting sections of lignified or cutinized ma-
terial, an old razor should be used, as the edge will almost invari-
ably be injured. Various pieces of apparatus not mentioned will
be found very useful and materially aid in careful and thorough
work, yet much can be accomplished without any very extensive
expenditure of money for equipments.

CARE Of flPPflRATUS.

It is very desirable that the student should have had some
previous training in microscopical manipulation, but to those who
have had no such opportunity some few explanations and sugges-
tions are necessary before they can work to advantage with so
delicate an instrument as the compound microscope. Access should
be had to some of the excellent books on microscopical technique
mentioned elsewhere in this manual.

With reference to the care of the microscope it is especially
important that the optical parts be kept free from dust or dirt, and
in any case where the lenses come in contact with anything that
would soil them they should be cleaned at once. The Japanese
bibulous paper recommended for this purpose can be secured of
G. S. Woolman, 116 Fulton street, New York. After cleaning a
lens, the soiled paper should be thrown away or it may be used for
removing liquids from any part of the instrument. Since the glass
from which the lenses are made is quite soft, it should never be
subjected to any hard rubbing, as its surface would be injured by
scratching. Never touch any of the glasses with the fingers or
allow the objective to come in contact with reagents or stains that
may be on the table of the microscope. If balsam or shellac should
get on the face of the lens, wet it with alcohol and remove at once
with a linen cloth as the alcohol would soon injure the mounting.
Glycerin and glycerin jelly can be removed with water.

Many of the operations in testing for different vegetable or
mineral substances must be performed on the stage of the micro-
scope, where the action of the agent can be determined. In
this case, much care must be exercised in order that the

57 CARE OF APPARATUS.

instrument may not be injured during the operation. Where a
stain or a liquid of any sort is to be applied, place a small drop of
it on the slide in contact with the cover. Then hold a bit of
blotting paper on the opposite side in contact with the liquid, and
the reagent will be drawn through under the cover glass where its
action can be observed through the microscope. In case of the
action of the acids that may cause effervescence, see that none of
the particles of acid fly against the objective or stage of the instru-
ment. Do not let any liquid come iu contact with the microscope.

Always keep the stand free from dust, and when necessary to
wipe any part of it rub with the grain of the finish to prevent
scratching.

When necessary to lubricate any of the working parts, use a
little soft tallow and wipe the surface slightly to remove any sur-
plus oil.

If any of the parts become loosened by wear, see that the
tightening screws are turned.

In inclining the body of the microscope, grasp the upper por-
tion of the pillar and never allow any strain to come on the adjust-
ments of the instrument.

In screwing the objectives in place, use both hands, holding
the front of the objective between the first and second fingers of
one hand, with the other turn it in place by grasping the milled
head OD the back of the objective. In removing it, reverse the
operation.

To remove the ocular or change the length of the draw tube
always grasp the course adjustment with one hand to prevent the
objective from being forced down upon the stage.

Keep the lenses in a place where they will not be exposed to
sudden changes of temperature, as the expansion or contraction of
glass or metal might crack them.

In getting the object in focus, run the objective down until it
is nearly in contact with the cover glass. Then look into the
microscope and raise the objective by means of the course adjust-
ment, as the image appears in focus, use the fine adjustment for
further study. Ahcays focus ^tp, never down.

CfIRE Of THE EYES.

Some trouble may at first be experienced by those unaccus-
tomed to the use of the microscope, but this will shortly be over-
come if due care is exercised. Always work with both eyes open
and divide the labor between the two. At first, it may be a little
troublesome to see the objects hi the microscope distinctly with
both eyes open, but by a little perseverance this can be overcome
and the objects outside will not interfere.

A convenient form of an eye shade can be made by covering a
piece of card board, 10xl8c.m., with black cloth. Make a hole in
the card midway between the ends and 2c.m. from one side. Put
the shield over the ocular, letting it rest on the collar of the draw
tube. A rubber band fastened to the card will keep it in place.
(Fig. 7.)

If. C.M.

Fig. 7. Diagram of an eye shade.

This shield will cut off the light from the eye not in use, and
be of material assistance.

Do not use the microscope after the eyes become tired. The
fatigue which is troublesome at first will wear away in a short
time, and one will soon be able to work for hours with the instru-
ment without difficulty.

59 CAEE OF THE EYES.

It is the common experience with microscopists that the eyes
improve with use and are permanently benefited, the same as by
judicious exercise of other portions of the body.

The light suitable for microscopical work should be strong
enough to enable the object to be clearly seen, yet not so bright as
to dazzle the eyes. Always avoid direct sunlight. The light from
a north window is to be preferred, especially if the sky is clear
or covered only with white clouds. Microscopic work can be carried
on in the evening by lamp, gas, or electric light. Lamp and gas
light are not very desirable and usually give unsatisfactory results.

The use of electric light is very highly recommended, provided
a ground glass shade be fitted to the incandescent lamp. This gives
a uniform, modified light and proves to be very satisfactory.

MANIPULATION OF APPARATUS.

Much has been written upon general microscopical technique,
and some excellent books are easily procured, but it may not be
amiss to call the attention of those who are beginning the work
with the microscope to a few important precautions and directions
that must be observed, and also to offer some few suggestions
which may aid in manipulation.

Access should be had to Prof. S. H. Gage's " Microscopical
Methods " which has been freely drawn from in the preparation of
the following exercises.

Interpreting Appearances.

DIRT OR CLOUDINESS ON THE LENSES. — It is important that the
lenses be free from dust or dirt, and any cloudiness seen in the
field of the microscope should be removed at once. For removing
particles of dirt or dust, a camel's hair brush can be used, but for
wiping the lenses an old linen cloth or Japanese bibulous paper
is to be recommended. To determine the location of the par-
ticles of dirt, look into the microscope and revolve the ocular in
its place. If the dirt is on the lenses of the ocular, it will revolve
in the field of the microscope. If it does not, it is on the objective.

Should the trouble be with the ocular, remove it from the
microscope and wipe its lenses thoroughly. If desired the front
lens can be unscrewed and cleaned without injury to the instru-
ment. In replacing the ocular, if it fits tightly, observe the pre-
cautions given elsewhere to prevent the objective from being run
down against the stage of the microscope. If the trouble is not
remedied by cleaning the ocular, remove the objective and wipe the
front lens being careful not to scratch it.

61 MAMI'I'LATIOX OF A I' I' A HATU8.

Do not take the objective apart for cleaning. Should any
repairs need to be made on the inside of it, return to the maker for
examination. For removing balsam or glycerin jelly, see directions
elsewhere, (p 56.) Having secured a clear field, i. e., the round
area seen on looking into the microscope, some studies should be
made in focusing. For this purpose prepare a glass slide as
follows: place in the center of a glass slip a piece of paper about
1 m. m. square on which is the letter (a) in " diamond " type. Place
on this a drop of balsam and cover with a piece of glass about 1
c. m. square and 2 m. m. thick. On this piece of thick glass place
the letter (b) very close to the letter (a), but not over it ; add a drop
of balsam and superimpose a piece of thick glass, the size of the
one beneath. Repeat the operation with the letter (c) and place
over this a thin cover-glass, the size of the thick glass below.
After the balsam has hardened the mount can be sealed with shellac
or asphalt. Place the slide under the microscope, using a £ or
1 inch objective. Focus on the letter (b), then turn the fine adjust-
ment either way, and observe the effect. By continuing the experi-
ment, the working of the adjustments of the instrument will soon
be understood. A knowledge of this is absolutely necessary, since
with higher powers, the direction in which the micrometer screw
should be turned is of the utmost importance. It should also be
known whether a certain part seen under the microscope is at the
top or bottom of the object. It is only by much practice in this
way, that the relations of structures are determined. Observe, also,
that only one letter is in focus at once. This is because the depth
of focus is not great. In general, this distance become less as the
magnification is increased.

In order to examine thoroughly an object of much thickness,
which is readily penetrated by the light, it must be studied in
section; that is, focusing on one part and afterward on one above
or below it.

The location of the parts with regard to their vertical
position can be determined by the working of the micrometer
screw. Such sections of the object as are in focus at any one time
are known as "optical sections." Objects with irregular contour
must be studied in this way.

62 MANIPULATION OF APPAEAT I X

Air Bubbles.

Take a clean slide and place in the center a few drops of muci-
lage or glycerin. Beat this with a knife until it has a frothy appear-
ance due to the presence of numerous air bubbles. Cover with a
cover-glass and focus on a small bubble. If the rays of light from
the mirror pass perpendicularly through the slide, the bubble will
have a light center and a uniform dark border. If the mirror is
not central, the light spot will be at one side of the center. After
taking off the sub- stage attachments, move the mirror bar until it
is at an angle with the stage, and observe, when the light passes
through the bubble, the direction from the center which the light
spot has taken. It will be away from the side to which the mirror
bar was moved.

Now beat some cedar oil or oil of cloves, and repeat the experi-
ment. Observe the direction taken by the light spot in the oil
globule. It will be contrary to that taken by the one in the ah*
bubble, or to the side on which the mirror bar was swung.

It is interesting to mix the mucilage with the oil and find an
oil globule and air bubble side by side. Then study the effect of
oblique light to identify the bubble or globule.

It is advisable that the student familiarize himself with the
more common objects that he will meet, perhaps, as foreign
bodies in his studies. Mounts should therefore be made on a
slide dry or in a drop of water of such objects as spores, dust, cot-
ton and woolen fibers, hair, cork, etc., and studies made of them
with the high and low powers.

MAGNIFICATION.

Any student working with a microscope should be able to
determine the magnification of his instrument together with the
ocular micrometer ratio.

Determination of Magnification by the Use of Wollaston's
Camera Lucida.

Arrange the stage micrometer in position and focus on it until
the lines are clearly visible. Place the camera lucida on the ocular
and tilt the body of the microscope until the tube becomes horizon-
tal. Then raise the base of the instrument upon blocks, until the
distance from the ocular to the table is 25 c. m. Change the mirror
until the light is reflected through the tube of the microscope, after
which modify the quantity of light falling on the white paper,
which should be placed on the table beneath the ocular, until, by
looking through the camera lucida toward the table, the lines of
the stage micrometer will be seen on the paper below. Mark off
with a pencil the distance between the lines of the stage micrometer,
as reflected on the paper, and measure with a rule. To determine
the magnification, divide the size of the image by the size of the
object magnified, and the quotient will be the magnification of the
instrument, under the one condition. If the distance between the
lines on the micrometer is 1-10 m. m., and on the image 50 m. m.,
then the magnification of the instrument in that condition is
50 -=-1-1 0=500.

Determine the magnification with the several combinations of
oculars and objectives.

The magnification can also be determined by the eye-piece
and stage micrometers.

64

In the measurement of most objects the former micrometer is
used. This is a scale ruled on glass and placed in a slit in the
ocular, or inside, by unscrewing the upper lens of the combination.

To Determine the Ocular nicrometer Ratio.

Place the ocular micrometer in position in the slit in the eye-
piece, and move the eye lens up or down, until the lines on the
glass are distinct. Now place in position the stage micrometer,
and the lines on it will appear below under those of the eye-piece
micrometer. Move the two scales until the lines of each are parall-
el with the other. Measure with the scale of the eye-piece micro-
meter, the distance between the lines of the stage micrometer. The
ocular micrometer ratio is obtained by dividing the number of
spaces on the eye-piece micrometer, required to cover a space on the
stage micrometer, by the value of the divisions of the latter. For
example, suppose the markings of the stage micrometer were
1-100 of a in. in., and the number of spaces of the eye-piece micro-
meter required to cover one space in the former was 5, then, the
ocular micrometer ratio would be 5-i-l 100— 500, i. e., the ratio is
500. The value of each division of the ocular micrometer is 1-500
with the above conditions in the microscope. The ratio with the
several objectives should be ascertained.

The magnification of an object can be determined, by dividing
the size of the image, as measured by the ej^e-piece micrometer, by
the ocular micrometer ratio. For example, suppose the dimension
of the image of a cell to be 5 divisions of the eye-piece micrometer,
and the ocular micrometer ratio is 300, then the size of the cell is
5-=-300 = .0166-(-m.m. The size of an object can likewise be deter-
mined by measuring the size of the image, under the conditions
described for determining the magnification of the instrument with
the camera lucida, and then dividing the size of the image by the
magnification of the instrument. Suppose the size of the image is
5 rn.in. on the paper, and the magnification of the instrument is 300
diameters, then the real size of the object is 5^300=. 0166-f-ni.m.

The unit of micrometry as universally used is the 1-1000 of a
m.m. This was suggested by Harting in 1859 and (-tilled, in 1869,
by Listing, the micron. It is designated by the Greek letter //.
The magnification of an object is then always to be given in mi-

65 MA G N1F1CA T1ON.

crons, and the reduction is simply made by multiplying the actual
size of the object by 1-1000.

In all measurements with the microscope, the draw tube
should be pulled out, until the whole tube of the microscope is of
a certain length. This distance is known as the "tube length"
and varies in microscopes of different makers, as do also the points
between which the measurement is made. See Prof. S. H. Gage's
Microscopical Methods, p. 10.

In order that the student may become familiar with the work-
ing of the microscope he should carefully go through the following
exercises:

1. Putting the ocular and objective in position, p. 57.

2. Lighting the field of the microscope, with direct and
oblique light, p. 62.

3. Determine the relative position of optical sections, and the
manipulation of the fine and course adjustments, p. 61.

4. Study of ail- bubbles and oil globules with reference to the
identifying of each by their appearance, under direct and oblique
illumination, p. 62.

5. Study of currents in liquids and their direction upon incli-
nation of the stage of the microscope. These currents can be
produced by grinding upon a slide, with a knife, a little carmine in
water, and covering with a cover glass.

6. Determine the magnification of the instrument with the
various combination of lenses, p. 63-64.

7. Dirt or cloudiness on the lenses, p. 60. Smear the objec-
tive with glycerin and study the appearance. Remove the glycerin
with water.

8. Mount various objects (hair, cotton and woolen fabrics,
bits of wood, paper, thread, etc.), under a cover-glass in water, and
study with the high and low power. In this way one will become
familiar with the adjustments of the instrument and the appear-
ance of the more common "foreign bodies" that might be found in
ordinary preparations.

LABORATORY DIRECTIONS.

DIVISIONS OF THE SUBJECT:

A. Living Cells, (with Protoplasm and Chlorophyll.)

B. Contents of Cells, (the secondary products.)

C. Elementary Tissues.

D. The Primary Meristem.

E. The Systems of Tissues.

F. The Thickening of Stems, etc., (secondary growth.

A. THE STUDY Of LIVING CELLS.

For convenience of study, cells may be classified as to manner
of association as follows:

1. Those which live separate from one another. Examples,
Unicellular Fungi and Algae, Pollen-grains, Spores, etc.

II. Those living in Colonies, i. e., joined temporarily, but
which are able to perform all their normal functions if isolated;
example, Spirogyra, (known as "Frog-Spawn," "Water-Carpet,"
"Pond-Scum," etc.)

III. Those which live permanently joined to other cells and
which cannot ordinarily perform their normal functions if isolated;
(a) they may not form Tissue ; example, Nitella ; (b) they may
form Tissue- example, Roots, Stems, Leaves of Flowering l^lants.

CASE I.

Isolated Cells Containing Protoplasm.

Illustration: Protococcus viridis, Ag. (green slime.)
The plant can be found in damp places, growing on the bark
of trees, or in the corner of buildings on the brick, or stones of the
foundation. In fact so general is the plant distributed that no one
need have any difficulty in getting material in good condition for
study.

PREPARATION FIRST : With a knife remove some of the material
from the substratum and mount in water. Place under the high
power of the microscope and OBSERVE: — 1. The unicellular plants,
often associated in group*.

2. Their size, shape, and general appearance.

fi8 THE STUDY OF LIVING CELLS.

3. The thin colorless cell wall surrounding a central granular
mass of protoplasm.

4. Chlorophyll tjnnt"l<'>* distributed through the protoplasm.
These are the centers of the vital processes in the cell, and in sun-
light by the decomposition of plant food form starch, protoplasm,
etc.

5. The nucleus.

Stain the preparation with iodine and observe the effect on the
wall, and protoplasmic contents. (See p. 7.)

Prepare another slide and stain with a fresh solution of
chlor-iodide of zinc, to observe the effect of the thin cellulose, cell
wall. By pressing on the cover glass, the cell contents can be
forced out and the cell wall be made more visible. (P. 11.)

Examine several specimens to observe the various stages in
the division of the plants into groups of individuals.

Many of the small plants will be seen moving about through
the water. This is due to the presence of cilia which, by their
rapid movements, propel the individual. Parker's Biology, p. 23 ;
Strasburger, p. 214 ; Vines' Text Book, p. 236 ; Campbell's Struc-
tural Botany, p. 22 ; Bibliography of the Literature on the
Plant Cell, by Dr. A. Ziramermanu ; Botanisches Centralblatt,
Beihefte, 1894.

Further Illustration: POLLEN-GRAINS and their MOTHER-CELLS,
from Begonia sp .

PREPARATION FIRST: For the MOTHER-CELLS of POLLEN. Lay out
a perfectly clean glass slide and cover-glass, placing a few drops of
distilled water on the former. Select a young staminate flowe? lm<1
(less than half grown). Moisten the razor edge and make thin sec-
tions across the upper part of the flower buds, and the tips of the
contained anthers. From these sections select the thinnest, espe-
cially those of the anthers and remove by means of a camel's hair
brush to the slide. Examine these sections with a tripod lens or a
dissecting microscope, removing the thicker anther sections and
fragments of the perianth. Cover the sections with the cover-glass.
The latter should be taken up with the forceps, one edge placed in
the water, and the glass lowered, not too suddenly, but so as to
allow the water to run along its lower surface without enclosing
any air bubbles. Examine first with the low power objective (^ in.),

69 THE STUDY o/-' I.IVIXG CELLS.

then with the higher (1-6 in.), using the C eye-piece and draw-tube
when necessary.

OBSERVE: 1. — The outline of the sections of the anthers;
oblong or quadrate, usually with rounded angles.

2. The cavities near the angles ; — later, these coalesce in pairs,
thus forming the two "anther-cells," or pollen-cavities.

3. The epidermal layer of cells, the layer just beneath, and
the irregularly placed cells of the interior of the anther.

4. Isolated rounded cells in the cavities or floating free in the
water. These are the mother-cells of the pollens.

(a) — The very thin cell wall, its smooth surface, etc., has it
perceptible thickness ?

(b) — The protoplasm of the interior, its characteristics.

(c) — The nucleiis, its appearance.

(d) — The nucleoliis.

FURTHER PREPARATION : If (c) and (d) are not clearly demon-
straied apply a drop of iodine solution to one side of the cover-
glass, and place a piece of filter paper on the opposite side. This
absorbs the iodine and water, while the former acts on the proto-
plasm as a staining agent. (The iodine may be removed by using
water in the same way).

Observe the relative amount of color given to the cell wall and
the cell contents; especially the effect on the nucleus after the
mother-cells have remained some time in the stain. Also observe
the increased distinctness of parts. From this study determine the
constant effects of iodine on protoplasm and refer to page 7 for
the use of this reagent in plant histology.

Measure the diameter of the mother-cells by the use of the
camera lucida, or ocular micrometer, selecting those of extreme and
also of average breadth.

If practical retain this slide in a moist condition until the next
preparation has been examined. It is useless to keep slides of soft
tissue containing protoplasm more than a few hours when mounted
in water. A temporary moist chamber can be made by placing a
small plate partly filled with water on a level surface and covering
it with a bell jar. The mount may be laid across a small watch
glass inside. Sketch transection of anther from £ objective and a
mother-cell from 1-6 objective. (Fig. 8).

70 THE STUDY OF LIVING CELLS.

PREPARATION SECOND: For young POLLEN-GRAINS. (Fig. 9.) Take a
staminate flower somewhat older than the first. (The exact stage will
have to be obtained possibly after trying several buds.) Section
and prepare as before. Stain with iodine, letting it run under the
cover one-third of its diameter. Focus on that region bordering on
the stained and unstained portions.

OBSERVE: 1. The mother-cells (of pollen grains), floating free
in the water, and the exceeding thinness of the walls.

2. The contents of the mother-cells — four small -'pollen
grains" ; or if only three appear focus carefully to ascertain the
presence of a possible fourth.

3. Form of pollens ; their nucleus and protoplasmic contents.

4. The readiness with which the protoplasm of the pollen
becomes stained, even when the water medium appears scarcely
tinged.

5. Mother cell wall, almost colorless, although the iodine
must have passed through it. Why?

6. Is the place between the mother-cell wall and the contained
pollens more tinged than the water medium?

Figures of developing pollen grains, Goebel, p. 362; Stras-
burger, p. 313 ; Sachs' Physiology, pp. 99, 100.

Draw one or two mother-cells with contained pollens.

PREPARATION THIRD. For MATURE POLLEN GRAINS. From the
mature anther of an open flower jar' out the pollen, letting it fall on
a dry slide. Examine carefully with a microscope, and then apply
water to the side of the cover-glass.

OBSERVE: 1. Appearance of the pollen grains when on the
dry slide.

2. Change of form when the water is applied; the cause?

3. The wall, its relative thickness as compared with that of
the mother cells.

Measure a large and small grain, the longer and shorter axis.
Draw a grain in the dry condition, and one in the moist. Sachs'
Botany, p. 15; Bessey, p. 23; Gray's Structural Boi, pp. 256-257;
Bot. Centralbl., Beih., 1893, pp. 206-17, 321 54, 401-36.

Illustration Second: POLLEN-GRAINS from the Order Mal-
vaceae, (either Hibiscus or Abutilon.)

71 THE STUDY OF LIVING CELLS.

PREPARATION FIRST. This should be similar to that of pre-
paration third under Begonia.

OBSERVE: I. The short processes, sometimes lobed, covering
the face of the grain.

II. If the water causes the grain to swell.

III. The size, — measuring with the micrometer.
PREPARATION SECOND: Culture of POLLEN GRAIN and the study

of germinating POLLEN TUBES, Tradescantia or Begonia.

Sterilize a slide, cover-glass, and ring, for making a cell. This
can be done by heating them for some time in an oven at about
128° C., or quicker, by passing them slowly through the flame of an
alcohol lamp. Make a 10 per cent, sugar solution and boil it ten
minutes. Then with a platinum wire, cooled after passing through
an alcohol flame, place a small drop of sugar solution in the center
of the sterilized cover-glass. Sprinkle on this a very few pollen
grains and invert over the ring, which is placed in the center of the
slide and held in position by a drop of water at its lower edge.
The culture will then have the arrangement shown in Fig. 8. Re-
move the slide to a moist chamber and allow it to remain in a warm
place for a few hours.

Fig. 8. Showing the method of cultivating pollen grains in \\ hanging drop.
A. Glass slide. B. Glass ring. C. Hanging drop containing pollen grains. D.

Cover-glass.

Examine the pollen grains after a few hours with a 1-6 objec-
tive taking care not to break the cover-glass by forcing the lens
against it.

OBSERVE: 1. The outer and inner coat of the grain; the for-
mer is called the extine, the latter the intine.

2. Pollen tubes of varying lengths projecting from the several
grains, usually but one tube from each grain (there may, however,
be several).

3. The thin icall of the pollen tube continuous with the intine,
and the extine ruptured by the germination.

4. The granular contents of the pollen tube consisting of
protoplasm and starch, together with the nucleus of the grain, on

72 T1IK H'/TJtY OF L/17A7,' ('KLL8.

its passage to the end of the tube, from which it goes to perform
the office of fertilization when in contact with the oosphere of the
embryo sac. If the pollen grains of Malvaceae are used, observe _
the relation of the pollen tubes to ike processes or prottiberences on
the grains.

The culture slide can be kept for several days, and the devel-
opment of the pollen tubes observed at intervals. For figures of
developing pollen tubes see: Bot. Gazette, 1886; Goebel, p. 365;
Strasburger, pp. 304, 320; Goodale, p. 429.

CASE II.

Cells in Colonies, Joined Temporarily.

Showing CELLS in COLONIES, also PROTOPLASM, CHLOROPHYLL in
SPIRAL BANDS, and PROGRESSIVE CELL DIVISION.

Illustration : Spirogyra. This genus of filamentous un-
branched aquatic plants belong to the Conjugatae, a group of
Thallophytes (See Sachs' Text Book of Botany, p. 25; Bessey's
Botany, p. 232.) Spirogyras, when in the vegetative state, are
bright green, and have a silky luster when taken from the water.
They vary much in diameter of the filament in different species, but
are seldom over .15 m. m. They frequently occur in fresh pools or
slow flowing streams,* and may be found in winter in pools that do
not freeze. Most species pass into a reproductive stage in early
summer. But few species are known to conjugate in the winter.

PREPARATION FIRST: Place a few of the filaments on a slide
in water.

OBSERVE : 1. Cells placed end to end.

2. The septa, or transverse partitions, plain in some fila-
ments ; possibly in others a box-like area will be observed at the
septum.

3. The spiral band or bands, of bright green color, part
chlorophyll pigment and part protoplasm. Ascertain if possible
the number of bands. To do this, count the number that cross a
thread between the two points where it touches the opposite sides
of the ceH, and this number plus one, will be the number of bauds
in the cell.

73 THE STl'ltY O/-' LH'IXd C

4. The irregular clearl </ <-nt n«tr</in of the bands, and the
protoplasmic ridge traversing the middle of each. If these are not
well defined the cells are not in a healthy condition.

5. Globose masses or corpuscles, in the band at intervals.
These corpuscles are apparently centers of protoplasm, where
starch grains are produced, under proper conditions. The cor-
puscles and the connecting protoplasmic ridge are regarded by
Schmitz as one continuous "chloro-plastid."

6. The transparent roundish area at the middle of each cor-
puscle,— the pyrenoid of Schmitz.

7. The cell nucleus if present ; its form. It may be more
clearly brought out by the application of a little dilute iodine.

8. If protoplasm can be detected in the cell, except in the
chlorophyll bands.

PREPARATION SECOND: — Stain a preparation with iodine, by
placing a drop at the edge of the cover glass.

OBSERVE: 1. The effect on the corpuscles, nucleus and pro-
toplasm in various parts of the cell.

2. That the cell wall remains unstained or slightly colored
although the iodine must have passed through it.

3. The slow action of the iodine on the chlorophyll bands.
The cause of this slowness.

PREPARATION THIRD: — Stain as before, using strong iodine in-
stead of the dilute solution.

ORSERVE: 1. Contraction of the whole cell contents in some
cases. It is a coagulation of the protoplasm, the water being ex-
pelled by the iodine.

2. The color given to the different parts.

For the study of cell division, keep the plants in a very cool
place over night and bring in to the warm laboratory in the morn-
ing. The division will begin in a short time. If the plants are
kept constantly in a warm place, the division will usually take place
at night. In specimens treated as above directed,

OBSERVE: 1. The ridge of cellulose being pushed inward from
the side walls in some cells. This is a *> /itum in active formation,
(by progressive cell division).

2. The chlorophyll, etc., continuing through the aperture of
the partly formed septum. Sketch the above appearance.

74 THE STUDY OF LIVING CELLS.

Describe the plant and all the phenomena observed, carefully
and concisely, including the effects of the staining agents.

Sachs' Botany, p. 16; Vines Text Book, p. 118; Parker's
Biology, p. 192; Goebel, p. 49; Strasburger, p. 246; McAlpine's
Charts, pi. iv; 19th Smithsonian Contributions, pi. 14 and 15.

For method of making permanent mounts of Spirogyra see p. 38.

CASE III.

This includes by far the largest number of plants existing.
They may be considered under two heads.

A. THOSE WHICH DO NOT FORM TISSUE, of which, Chara and
Nitella may be taken as a type. The same condition exists in
stamen hairs, many trichomes, etc.

B. THOSE WHICH FORM TISSUE, as the higher Cryptoyamia and
the Flowering Plants, which will furnish the illustrations under
the subsequent heading of T'issues.

CASE III. (A.)

ILLUSTRATION FIRST: STAMEN HAIRS of Tradescantia showing
PROTOPLASM in MOTION. (Streaming movement.) (Fig. 10.)

PREPARATION. Remove from a perfectly fresh, newly opened
flower a stamen with the attached hairs or trichomes. Place them
in water taking particular care not to break or injure the trichomes.

OBSERVE: 1. The number and/b?v/# of the trichome cells.

2. The faint pink color of the cell sap, due not to colored
granules, but to a red pigment held in solution.

3. The nucleus.

4. Slender streams of moving protoplasm. Trace their course.

5. Estimate their rate of movement.

For figures of the above see Strasburger, p. 29: Sedgwick and
Wilson's Biology, p. 30; Bessey's, p. 12.

The movement of protoplasm can likewise be studied in the
young plant hairs of Cucurbitaceae, in Nitella, or in Vallisneria.
Any of the specimens can be mounted in water and treated the
same as Tradescantia.

B. CELL CONTENTS.

The several illustrations following, represent the secondary
products of the cell as distinguished from active protoplasm and
its immediate derivaties, the chlorophyll body. The secondary
products are found chiefly in the fundamental or cellular tissue, the
characteristics of which will be observed. The solid forms of these
products will be studied as follows:

1. Starch Grains.

2. Crystals.

3. Protein granules.

4. Crystalloids.

5. Aleurone grains.

Illustration First: Showing PARENCHYMA CELLS with STARCH.
The TUBER of the POTATO. (Solatium tuberoswn.) (Fig. 11.)

PREPARATION: Cut a fresh surface on a tuber, make several
thin sections from 2-3 m. m. in breadth and as thin as possible.
Mount in water.

OBSERVE: 1. Form of the parenchyma cells of the tuber.

2. Ovoid or pyriform starch grains, of various sizes, lying in
and about the cells.

3. Structure of grains; some plainly showing a hiltim,
around which appear rings more or less eccentric. The rings are
layers of greater or less density.

4. Measure some of the larger grains.

5. Sun a drop of weak iodine under the cover and note the
blue color resulting in the grain, the characteristic reaction for
starch. Sketch a tuber cell with contained starch grains. Vines'

76 < HLL CONTEXTS.

Text Book, p. 110; Strasburger, p. 11; Goodale, p. 49; Bessey, p.
54; Bot. Centralbl., LV. (1893) p. 157.

Illustration Second : Showing CRYSTALLOIDS. HYPODERMAL
TISSUE of POTATO TUBER.

PREPARATION : Sections should be made below the cuticle, but
very near the surface, and the parenchyma should contain but a
few small starch grains.

OBSERVE: 1. The rectangular cells with cell nuclei if present.

2. Crystalloids, cubical in outline, surface plane, or some-
times eroded.

3. Upon the application of iodine, observe yellowish tint
given to crystalloids indicating their nitrogenous nature.

4. After drawing off a considerable portion of the fluid under
the cover-glass and subsequently applying a saturated solution of
common salt (NaCl), observe the changes in, and final dissolution
of, the crystalloids. True crystals are not thus acted upon.
Strasburger, p. 25; Goodale, p. 47; Vines' Text Book, p. Ill

Illustration Third : ALEURONE AND STARCH. THE GARDEN
PEA (seed of Pisum sativum.)

PREPARATION: Separate the cotyledons of the pea, and cut
away a portion of one with a knife. Then with the heel of the razor
make several small sections from the smooth surface. Mount in
glycerin.

OBSERVE: 1. The grains of starch; their form and concen-
tric lines of structure or stratification.

2. Their lines of fissure.

3. The minute«grains in the cell with the starch, t\iealciiro»c
grains.

4. The cell structure and intercellular spaces.

Draw, showing the above characters; then treat the section
with strong iodine and observe its effect on the aleur<m<-.

Goodale, p. 47; Vines' Text Book of Bot., p. 112; Strasburger,
p. 18.

Illustration Fourth : STARCH, ALEURONE, and the STRUCTURE
OF CEREAL GRAINS. Grain of WHEAT ( Triticntn />ul</<(re.)

PREPARATION : By cross-section cut away one-third of the
grain. Make several thin sections from this surface, which sliull
include only a small portion of the coats of the grain, and of the

77 CELL CONTENTS.

white interior. The coats are better developed near the groove or
sinus of the seed. Mount hi glycerin or water. The sections can
be more easily prepared by soaking the grains for a few hours in
water.

OBSERVE: 1. The form of the cells of the interior of the
grain.

2. The form of the starch-grains in these cells. The stratifi-
cation is not often visible. It can be detected in some upon the
application of dilute Chromic Acid. This is a useful agent in
rendering the stratification of starch-grains, cell-walls, etc., more
distinct.

3. The form of the Aleurone-cells (or " gluten sacs ") in-
closed by broad white walls, whose outlines may be traced even on
their peripheral side. The Aleurone-cells lie next to the starch-
bearing cells.

4. The minute yellowish grains of Ati'm-nne filling these
sacs.

5. The Seed-coats and the Ouary-coatK. '

(a) The Inner seed coat, (or Secundine) made up of two layers
of white homogeneous cellulose, separated by an interrupted gran-
ular line or thin layer. The peripheral of these two layers is
regarded as the true " Inner seed-coat."

(b) The Outer seed-coat (or Primine], usually a narrow brown
band. In this lies the color of "red" and "white" wheat.

(c) The Endocarp, apparently marked at intervals by oblong
transverse pits, pinkish from refracted light. The Endocarp con-
sists of a layer of elongated, thick-walled cells, running transverse
to the long axis of the grain, therefore parallel to this section. The
oblong pits referred to are narrow, nearly-closed passages through
the party walls between these cells.

(d) and (e) The Exocarp. This is made up of several layers
(usually two) of oblong, thick-walled cells running parallel to the
long axis of the grain. The ends of these cells will therefore appear
in the present section as oblong openings. When first mounted they
are not always apparent. These thick-walled cells of the Endoc.-irp,
Exocarp, etc., are Sclerenchyma cells, and characteristic of the pro-
tective coverings of seeds.

78 CELL CONTENTS.

It will be noticed that in the above illustrations the cells inclos-
ing starch, etc., belong to the Cellular Tissue.

FURTHER PREPARATION : The coats above named and their com-
ponent cells will be more clearly understood if two or three thin,
tangential sections be made, i. e., sections parallel to a plane tang-
ent to tbe surface of the grain, and extending only to the starch -
cells. Trace here the Aleurone cells, the Outer seed-coat (1'rimine),
the Endocarp, the Exocarp. If iodine be applied to either the
transection or the tangential section, and be followed by strong
sulphuric acid, the inner seed-coat, the Endocarp and Exocarp turn
a deep blue (the reaction for cellulose), then dissolve, leaving the
brown Outer seed-coat entire, thus indicating the corky nature of
the latter.

The above study should be made with care as all cereals show
the same relative arrangement of seed-coats, aleurone-cells, and
starch-bearing cells. It will make more clearly understood some of
the processes of "high milling," by which the nitrogenous com-
pounds (the Aleurone grains), are separated and saved from the
bran. The ovary and seed-coats form the bran proper; the aleu-
rone-sacs contain most of the nitrogenous compounds or albumin-
oids, and the phosphates; the interior cells contain the starch,
which makes up the bulk of the "white" flour.

For figures illustrating the structure of the coats of the wheat-
grain, see Report of the II. S. Com., at Vienna Ex., 1873, vol. 11,
p. 4, on Art on Vienna Bread ; Goodale, p. 47, 181 ; Strasburger, p.
19; Vine's Text Book of Bot., p. 112; Landois and Stirling,
Human Phys., p. 444.

Illustration Fifth: STARCH. Grain of INDIAN CORN. (Zea
Mays.}

The semi-transparent portion of the dry grain should be used,
and the sections made very thin and necessarily minute. The cells
of the seed should be observed, but particularly the form of the
starch-grains and the peculiar stellate cavities at the center of some
when the section is first mounted in water. For figures, see
Sachs' Botany, p. 61; Goodale, p. 181; Bessey, p. 55; Strasburger,
p. 12; Vines Text Book of Bot., p. 112.

Illustration Sixth: STARCH in COMPOUND GRANULES. GRAIN
of OATS. (Avena sativa.)

79 CELL CONTENT*.

Remove the palets from the grain or seed, and mount the sec-
tions of the later in glycerin to prevent the disorganization of the
compound granules. Observe the reticulated appearance of the
latter, due to the lines of union of simple grains of starch; observe
the form of the compound granules. (For figures see Goodale's
Botany, p. 49, p. 181; Strasburger, pp. 11 and 12.)

With a little care the coats of the above grains may be sec-
tioned for examination at the same time as the starch and compared
with those of the wheat. In Fig. 139 (Goodale) the layer (a) is
that of the palet, and should not be represented in connection with
the true coats of the grain itself. Sections of seeds may be per-
manently mounted by removing the water and adding a drop of
glycerin jelly. Seal when hard. Strasburger, pp. 11 and 12.

1. Raphldes— needle-shaped.

Forms of Plant Crystals-; 2. Crystal Prisms— prismatic.

{ 3. SphiEraphldes— compound, spherical.

Illustration Seventh: CRYSTALS.

Illustration for form 2. The outer dried lamina of an
ONION bulb, (Allium Cepa.)

PREPARATION: Split a thick brown lamina parallel to the sur-
face; this may be done by twisting two parts in different directions
with the hands. The thin margin of the rent including the cells
on the convex surface may be mounted in water.

OBSERVE: 1. The form of the cells in which the crystals lie.

2. The various forms of single crystals, viz: Prisms often
with modified angles, also hexagons and pyramids if present.

3. The number of principal faces to the prisms.

4. The double crystals in the form of an oblique cross; triple
crystals if present. Do the axes of such crystals pass through one
another?

TESTS FOR CRYSTALS:

The greater number of plant-crystals are Calcic Oxalate.
A limited number are Calcic Carbonate.
Crystals of other salts of lime occur but rarely.
Calcic Oxalate is not acted on by Acetic Acid.
Calcic Oxalate is dissolved by Hydrochloric Acid without
effervescence.

Calcic Carbonate is dissolved by both acids with effervescence.
As the composition of the crystals is uncertain, test with

80 CELL CONTENTS.

Acetic Acid first. If it has no effect, draw it off, arid apply Hydro-
chloric Acid. Make sure the acids reach the crystals and particu-
larly observe the effect on any which may have floated out of the
cells.

Tests for Calcic Phosphate and Calcic Sulphate are given in
Poulsen and Trelease's Botan. Micro-Chemistry, p. 96 ; Zimmer-
mann's Microtechnique, pp. 64, 62.

THE STUDY OF GROUP I. Raphides will be reserved until later
in the outline, in connection with an examination of growing roots,
in which these crystals are found in abundance near the tip.

Illustration Eighth : CYSTOLITHS. These are the concre-
tions composed of cellulose and a salt of lime, and are found
chiefly in the epidermal regions of certain Urticaeeae.

Illustration: Leaf of Ficus ELASTICA (one of the " C<i<mt
chouc Trees.)

PREPARATION : Select a piece from the upper part of a leaf
and mount the thin transection in water. See p. 16.

OBSERVE: 1. The upper JZpiderrnis, a layer of small color-
less cells on the upper surface of the leaf.

2. The Hypodermal layer of larger, also colorless cells just
beneath the upper Epidermis.

3. Larger cells at intervals in the hypodermal layer contain-
ing the concretions — the Cystoliths, attached to the peripheral side
of the cell by a stalk.

Apply to this the preceding test for crystals, and demonstrate
what salt of lime is present.

After the test has been completed, and the acid washed out,
apply strong iodine, and observe the skeleton of cellulose re-
maining in the Cystolith. DeBary, p. 44, 104-105; Bessey, p. 11;
Vines' Text Book of Bot., p. 108; Zimmermann's Microtechnique,
p. 61.

Inulin.

Illustration Ninth : A substance of the same chemical com-
position as starch and found dissolved fu the cell sap of many
plants: Dahlia root, or Jerusalem Artichoke.

PREPARATION: Make thin transections of a piece of /><ilil'm
root that has been left for some days in strong alcohol or glycerin.

81 CELL CONTENTS.

OBSERVE : 1. Sphaerocrystals of various sizes, adhering to
the cell walls.

The radial and concentric stratification of the crystal ; seen
more clearly by the application of nitric acid.

Remove the alcohol by water and note that after a little time
all of the crystals have been dissolved. Sulphuric acid also dis-
solves the crystals. Ziinmermann's Bot. Microtechnique, p. 78;
Vines Text Book of Bot., p. 114; Strasburger, 'p. 51; Comptes
Rendus cxvi (1893), pp. 514-17.

C. ELEMENTARY TISSUES.

TABLE OF TISSUE FORMS, OR ELEMENTARY TISSUES.

I. PARENCHYMA TISSUE. IV. FIBROUS TISSUE.

II. / COLLENCHYMA TlSSUE. V. TRACHEARY TlSSUE.

III. ^SCLERENCHYMA TlSSUE. VI. SlEVE TlSSUE.

VII. LATICIFEROUS TISSUE.

1. PARENCHYMA TISSUE. Tissue of the FUNDAMENTAL SYSTEM,
forming the "ground work'1 of plants ; with cell walls of varying
thickness, and although of diverse forms yet never fibrous, as dis-
tinguished from PROSENCHYMA.

The more ordinary forms to be considered are :

1. Isodiametric. 4. Irregular.

2. Stellate. 5. .Epidermal.

3. Ellipsoidal. 6. /Suberox*.
Illustration: ISODIAMETRIC CELLS from GERANIUM STEMS

{Pelargonium inqninans), PITH of most STEMS.

PREPARATION FIRST: A thin transection of the stem can be
made free hand as directed on p. 16 and mounted in water or
glycerin.

ORSERVE: 1. In the center of the section, thin walled isodi-
ametric cells of varying size.

2. Nuclei if present.

3. Contents of parenchyma cells in various parts of the
section.

4. The irregular spaces between the cells, — Inter-cellular
spaces. (Fig. 12.)

5. By careful focusing near the outer edge of the section

83 ELEMENTAE Y TISS VES.

where the cell walls are somewhat thickened, Vbe prtonary partition
between contiguous cells: The 'middle lamella or inter -cellular
substance, as it is sometimes called. This lamella is of much the
same chemical composition as the remaining cell walls of the tissue,
and undergoes modification with it.

PREPARATION SECOND: ELLIPSOIDAL CELLS, from the root of any
herbaceous plant; — those of the JIi/<t<-inth grown in water are easily
obtainable.

For this study a longisectiou of a root should be prepared in
the manner directed for the previous study, or better, after the
directions on p. 21.

OBSERVE: 1. Thin walled ellipsoid cells, beneath the epider-
mal layer, making up the main body of the root — the cortex, — and
extending to the central cylinder.

2. The cells toward the apex of the root are more isodia-
metric.

3. The cell contents, together with the general characters of
the cortical tissue. Bastiirs Bot., p. 152.

PREPARATION THIRD: EPIDERMAL and IRREGULAR PARENCHYMA
cells from the leaf of SCARLET GERANIUM.

Remove a portion of the epidermis of the lower surface of the
leaf. This may be done by raising it at a cut margin and stripping
it back. It will come away as a transparent membrane. Mount in
water with the external surface of the epidermis uppermost.

OBSERVE: 1. The irregular outline of the colorless cj>i<l
cells.

2. tftoniates, with elliptical openings, at intervals.

3. Trichornes in various stages growing from the

The elongated cylindrical cell or cells composing them; the globose
glands at the apex of some. (Fig. 25.) This illustrates the fact that
cell walls, when not acted on from without, will develop with curved
surfaces. Bastin's Bot., p. 15G.

PREPARATON FOURTH: Make thin cross-sections of small por-
tions of leaves, hardened and partly bleached, in 50 per cent, alco-
hol. In these the chlorophyll bodies retain their form and some of
their color. The green tint imparted to the alcohol shows the read,
iness with which the chlorophyll pi</nt< >/f is extracted by its solv-
ents. While sectioning, the razor and the leaf must be kept sup-

84 ELEMENTARY TISSUES.

•

plied with alcohol to prevent access of air. Mount in alcohol or
glycerin.

OBSERVE 1. Epidermis of the upper and lower sides of the
leaf, appearing almost colorless as seen in section.

2. The thickened (cutinized) outside walls of the cells, which
are in very close contact with each other.

3. The palisade cells (ellipsoidal) , and the underlying layers
of irregular parenchyma cells making up the mesophyll of the
leaf. These cells form the main body of nearly all leaves. Note
the large irregular i)itercclh<l<tr .traces in connection with them,
also their contents in the sections of fresh tissue.

4. Stomates in section over small intercellular spaces.
PREPARATION FIFTH: STELLATE PARENCHYMA from STEMS and

PETIOLES of many aquatic plants.

Liliaceve and Pontederiaeeve, furnish excellent material for this
study.

Make several thin transections from the* stem or petiole of
PoMederia and mount in water.

OBSERVE 1. The firm regular epidermal parenchyma with
slightly cutinized walls.

2. The loose isodia metric cells making up the interior of the
section.

3. Large air-passages occupying a considerable portion of the
section.

4. Certain spaces filled with stellate cells, having their project-
ing ends in contact and forming a sieve-like plate across the cavity.
.(Fig 12.)

The function of these plates is probably to form a support for
the cross running fibro- vascular bundles, and at the same time, allow
the free passage of air through the stem (Bot. Jahresbericht; I, I'.Ml.)

5. The large nuclei and granular protoplasmic contents.
PREPARATION SIXTH: Suberous cells, from the "CoRK OAK,"

Quercus Suber, (commercial cork.)

Mount several very thin sections in water; mount others in
alcohol on the same slide. Suberin, the peculiar substance of cork
cells, repels water, and the comparison between the two prepara-
tions should be made as soon as possible.

85 ELEMENTARY TISSUES.

'

OBSERVE: 1. If there is a larger amount of air imprisoned in
one preparation, than in the other.

2. The form, and color of the cells, their thin walls resem-
bling the parenchyma previously studied. Apply concentrated
chromic acid to the section and note the effect. Suberin will enable
the cork to resist for a long time, (often many days and perhaps
continuously) , the action of the acid, while all other modifications
of cellulose are readily dissolved by it.

The probable absence of cellulose from suberized walls, as pre-
sented by Eugene Gilson, is interesting to note in this connection,
(La suberine et les cellules du liege. La Cellule, etc., p. p. Carony,
T. 1890 p. 63.) See Zimmermann's Microtechnique, p. 148.

MODIFICATION OF CELL WALLS.

Those .that produce changes in the chemical composition of the
cell wall may be classified as follows:

1. Mucilaginous Modification.

2. Ligniftcation.

3. Cutinization.

4. Mineralization.

Illustration First: Cell walls containing MUCILAGE. SEED
COATS of FLAX or LINSEED.

Make a thin tangential section of the seed, and place for a few
minutes in water.

OBSERVE: 1. The ready absorption of water, and consequent
thickening of the cell wall.

2. Its gelatinous consistence in seeds that have been left for
some time in water.

In many cases the modification goes so far as to convert the
cell wall into a gum, soluble in water. Examples of this can be
found in the Plum, Cherry, or Peach trees. Vines' Text Book of
Botany, p. 107; Goodale, p. 34; Strasburger, pp. 99, 340.

Illustration Second : Cell walls containing LIGNIN. Fibrous
tissue of the stem of any woody Phanerogams, or Vascular
Cryptogams.

Make a thin section of a woody stem and Amount in a few drops
of cuprammonia. Note the walls are not dissolved, which would
be the case, if they were pure cellulose. (P. 5.)

To a fresh section add iodine and sulphuric acid, or chlor-

8fi ELK MK. \TAIiY TlXS

iodide of zinc, and observe that the cell walls are colored yellow or
In-oirn — the character istic test for Ijiyniti.. What would be the
reaction with pure cellulose? Zimmermann's Microtechique, p.
143; Goodale, p. 36; Vines' Text Book of Bot., p. 107 ; Strasbur-
ger, pj>. 59, 119.

Illustration Third : Cell walls containing CUTIN. Epidermis
of most plants, and in many tissues where cell walls are to be
strengthened, or protection secured.

Make thin transections of the leaf of Pinus Sylvestris or
Cycas, and mount in water.

OBSERVE: 1. The small epidermal cells with outer walls
much thickened, usually in layers by the formation of cutin. The
cutin can be removed by leaving the tissue in strong chromic <i<-'nl
for some time. After this, wash the section with water and add
chlor-iodide of zinc. Note the blue color, the reaction for cellulose,
forming the greater per cent, of the inner part of the wall,
while the outer part consists of nearly pure cutin. Vines' Text
Book of Bot., p. 107 ; Goodale, p. 38 : Strasburger, p. 58 ; DeBary,
p. 78.

Illustration Fourth: Cell walls containing MINERALS, in
CRYSTALLINE or AMORPHOUS forms. (Stems of Cereals, and many
Sedges.) The substance most frequently present is Silica. To
test this, ignite the specimen in a platinum dish, treat with nitric
acid, and again ignite. The silica remains behind, and often
retains the microscopic form of the tissues. This is true with the
stems of Equisetum (horsetails.)

Crystals of Calcium salts can be found in the cell walls of the
bast tissues of the willow and many GynmosperniK. For further
study of the subject, examine Vines' Text Book of Bot., p. 108 ;
Goodale, p. 39 : DeBary, p. 102.

Continuity of Protoplasm.

Illustration: Stem of Aesculus (common horse-chestnut.)
PREPARATION: By the use of a knife remove the outer dark
colored and inner gree;n bark (periderm), from a young growing
stem, about 1 c.m. in diameter. With a sharp razor make thin
tangential sections of the exposed whitisli tissue (cortex), and place
in a solution of iodine in potassic iodide until brown.

87 ELEMENTAL Y 77.s.sr/-;>.

thoroughly with water and add strong sulphuric acid. After a few
minutes repeat the operation of washing.

The acid renders the cellulose cell-wall transparent, and the
protoplasmic strands can be seen connecting the masses of contigu-
ous cells. If this does not show clearly stain the preparation with
aniline blue. The sections can be permanently mounted in a drop
of glycerin jelly.

Strasburger, p. 371 ; Quart. Journ. Micros. Science, 1882, p,
365, 1883, p. 151; Bot. Gazette, 1881), p. 83.

II. Collenchyma Tissue.

This tissue is composed of parenchyma like cells with walls
thickened at the corners, or points of contact, and usually f"j>< /•///</
at the ends. They form cylinders of tissue beneath the epidermis
in many herbaceous stems and petioles. The cells often contain
chlorophyll and are sometimes capable of division.

Illustration: COLLENCHYMA CELLS from a mature stem of the
BEGONIA, or GERANIUM.

PREPARATION FIRST: Transections of a stem 3 to 4 m.m. in
diameter should be cut free hand, stained with haematoxylin, and
mounted in glycerin jelly ; or better, hardened in alcohol, infiltrated
with collodion, sectioned, and mounted as duected on p. 21.

OBSERVE: 1. Thin walled cells forming the epidermal tissue
of the stem.

2. Inside of this a ring of f/lf#h •/////// thick walled cells form
ing a zone about the stem.

3. The latter, the collenchyma tissueis composed of cells with
walls comparatively thin along the lateral surfaces, but thickened
at the angles. This tissue is very strong and contributes greatly
to the strength of the stem.

Make a longitudinal section of the stem and examine the cells
of this zone.

De Bary, p. 119; Strasburger, p. 106; Goodale, p. 65.

Endodermal Cells.

A modified form of thick walled parenchyma is found in the
ENDODERMIS, in most roots a single layer of cells, surrounding the
fibro-vascular bundles of the central cylinder. (Figs. 17, 22.)

88 ELEMENTAL) T1SSTKS.

The walls of the cells are thickened and often folded at the points
of contact between contiguous cells of the sheath. They are
strongly suberized, and have a clear glistening appearance.

Illustration: ENDODEEMIS of the central cylinder in HYACINTH
or CORN root, grown in water.

Make a thin trausection of an old root, and prepare as for the
previous study.

OBSERVE: 1. Near the center of the root surrounding the
Jibro-vascular bundles a single layer of cells thickened only at the
points of contact, but the walls uniformly suberized, thus giving
them a glistening appearance, — the endodermis. De Bary, p. 121 ;
Vine's Text Book of Bot., p. 165; Strasburger, p. 136.

Ill Sclerenchyma Tissue.

The cells under this tissue form vary from the short isodiametric
" Stone cells, " to elongated fibres, with thick walls, passing by
gradations into fibrous tissue on the one hand, and into cellular
tissue on the other, (refer to parenchyma). They will be here con-
sidered as distinct elements.

Illustration: (of "Stone cells), FLESHY ROOTS of Dahlia variab-
ilis.

PREPARATION FIRST: Longitudinal sections should be made
just 'beneath the brown epidermis. The zone containing the cells
will be apparent by cutting or scraping off the epidermis with a
knife, when the sclerenchyma tissue will be found as a hard gritty
layer. Mount in water.

OBSERVE: 1. The thin walled parenchyma, making up the main
body of the fleshy root.

2. Groups of Sclerenchyma cells with very thick walls; the
slender canals, often branched, running through the wall from the
central cavity.

3. The meeting of the canals of adjacent cells.

4. The form of the canal OJH niin/s as seen on the surface of
the cells. (Fig. 13.)

PREPARATION SECOND: SCLERENCHYMA CELLS from the " IVORY
NUT," (Phytelephas macrocurpa} often found made into buttons,
umbrella handles, etc. Soire of the more common nuts may
be used .

89 ELEME ^ 7 '. 1 /,' ) V/.s.s I 'KS.

With an old razor or sharp knife, small sections of sufficient
thinness can be obtained from the surface of the nut. These may
be mounted in water for study, or in balsam for permanent preser-
vation.

OBSERVE: 1. The tissue of the shell composed entirely of
sclerenchyma cells.

2. The narrow cavities of the rectangular cells.

3. Very thick but clear cell walls; cell contents.

4. The tinbranched canals passing through the walls at var-
ious places.

Enlargement of the canals at their place of union between con-
tiguous cells. • In many cases tke septum between the two is not
absorbed.

6. The peculiar branches of the canals at the end walls of
the cells.

PREPARATION THIRD: SCLERENCHYMA CELLS from the underground
stem of Pterls aquilina, (common brake). Place some of the un-
derground stem between two pieces of cork, and fasten in the jaws
of a microtome. With a strong razor kept wet with alcohol cut
thin transections and mount in glycerin jelly for permanent preser-
vation.

OBSERVE : 1. The bands of dark reddixh />/•<>/'•>! tixftue, ex-
tending across the section. These bands are composed of scleren-
chyma cells and assist greatly in strengthening the stem. (Fig. 23.)

2. With the high power, the laminated thick inalls of the
cells.

3. The branching canals through these walls.

4. If the ends of canals of neighboring cells are in contact.

5. By careful focusing, the- ends of these canals at the bottom
of some of the cells.

Vines' Text Book of Bot., p. 133 ; Strasburger, p. 146 ; De-
Bary, 132 ; Goodale, p. 63 ; Sedgwick and Wilson, p. 76.

IV and V Prosenchyma (in its widest sense.)

IV. PROSENCHYMA (proper) or FIBROUS Tissue.

(a) Bast-cells, — in the bark, (derived from Pkhvm..}

(b) Typical Wood-cells — in the wood, (derived from
Xylerri).

90 ELEMENT. \RY 'I 'ISS UES.

V. TRACHEABY TISSUE.

(c) Tracheids — like Wood-cells in form, and like ves-
sels or Trachece in structural markings.

(d) Tracheae—" Vessels " or " Ducts."

Type 1 includes Dotted and Handed Ducts, passing
into Pitted and Scalariform Ducts by gradations.

Type 2 includes Spiral, Annular, and J\<ti<- n/itf«/
Vessels and their gradation. See Goodale, p. 59 ; DeBary IV. *

Illustration: For PROSENCHVMA (in its widest sense.) BARK
and WOOD of LEATHERWOOD (Dirca palustris.)

PREPARATION for (a). Remove the brown cuticle from a branch
of Leathencood, also the loose cells beneath. Make two or more
very thin longitudinal sections of the bark, pulling one end of each
section from the branch instead of cutting it. Many of the silky
bast-fibers will thus be free at the end. Mount in water. Another
mode, is to place bast tissue in nitric acid and potassium chlorate,
and heat for a few minutes. The bast fibres can 'then be separated
under a dissecting microscope. Use a 3-4 and then a 1-5
in. objective.

ORSERVE : 1. The very long bast-fibres; trace out a free one,
and measure its length.

2. What kind of cells, if any, occur with the bast.

3. The clear unmarked walls of bast ; the tapering ends when
not broken.

PREPARATION for (b) and (c). Make (1) very thin, tangential
sections of wood ; (2) very thin transections of the branch includ-
ing wood and bark. Mount in water and stain with haematoxylin.

In the longitudinal section:

OBSERVE: 1. The short unmarked, fusiform wood fibres or
typical wood -cells whose ends over-lap. Measure the longest.

2. The fusiform cells marked by spiral lines, dots, bordered
pits, etc. — Tracheids; see Goodale, Fig. 78, 79; Bessey, p. 81;
Bastin, p. 162 ; Vines' Text Book of Bot., p. 182.

* It will be observed that the classification of 1'rosenchyma tissue is not. the
one usually pre>ented. It is believed that by adherini: Hosely to this one in these
studies, a correct idea can lie obtained of the development of the more perfect
forms of ducts from the simple cells. It is desirable that the chissilicat ions of
others be compared and the points of difference noted.

91 ELEMENTARY TISSUES.

3. Occasional Ducts (long tubes marked with bordered pits,
bands, etc.)

4. Rectangular cells of medullary rays, if present. To Avhat
tissue-form do tbey belong?

In the transection:

OBSERVE: 1. Ends of wood-cells in very regular radial rows ;
thickness of their walls, etc.

2. The larger openings (ducts), marking each years growth.

3. Thin plates of medullary tissue.

4. The bark with ends of bast appearing.

5. Pith at center.

Bastin, p. 159 ; Goodale, pp. 88, 89.

Notice the brittleness of wood, and strength of bast, corres-
ponding to the difference in the length of cells in each.

Usually wood-cells are less regularly distributed than in the
above.

PREPARATION for (a), (b), (c), and (d). Make a radial longitudi-
nal section (i. e., a longitudinal section passing the center of the pith).
Around the pith is a sheath of Spiral or Reticulated vessels (see
Type 1). In the woody tissue of the section are Pitted vessels
(see Type 2).

OBSERVE : 1. The Spiral Ducts.

2. That the spiral appearance is due to the thickened •/•/<///<-
deposited on the inner surface of a thin-walled tube.

3. If there are any Reticulated or Annular vessels in the
medullary sheath.

4. That the Pitted vessels are furnished with "bordered pits"
— the outline of the pit or cavity being nearly circular, and the
"lumen" or central aperture oblong.

Vines' Text Book of Bot., p. 134; Goodale, p. 85; Bessey, p.
74; Strasburger, p. 129.

V. Tracheary Tissue (continued).

Illustration : Sections of the stems of CURRANT, HORSE
CHESTNUT, MOON SEED and GRAPE VINES, are to be mounted to serve as
material for the study of both Tracheids and Ti-(t<-li«». Note
carefully the distinction between the two, the former being '• /!/>•<•
wood cells in outline «>nl ///•> I V.W/N <»• Tr<ic/n <i< in xtrnct nr<il
ma rkings.'1'1 Read carefully DeBary, pp. 1G4-1G6.

02 ELEMENTARY T188VE8,

PREPARATION : Suitable sections can be obtained by fastening
small portions of these steins in the jaws of a microtome ;ui<l
sectioning with a stout razor. Several radial-longitudinal sections
of each should be obtained in order to be certain of including
the proper portion (xylem) of the fibro-vascular bundle.

Other material than that suggested above can be used, but
such common plants have been selected as are found to show
the desired structure. These specimens should be examined with
reference to the study of the general forms indicated in the illustra-
tions of Tracheae 'which follow. Vines' Text Book of Bot., p.
135 ; Gopdale, p. 82 ; DeBary, p. 165.

The Tracheids of Coniferae.

Illustration: Wood of WHITE PINE (/V////.S .s?/W>//x). The
seasoned sap-wood of a young Pine answers the best for the study.

PREPARATION FIRST: Make several thin longitudinal sections
at right angles to the "grain" (the annual layers.) Mount in
water.

OBSERVE : 1. The Tracheids, oblong and fusiform like wood
fibres, but showing " bordered pit* " at intervals. This form of
Tracheid is found throughout the Gymnosperins, fossil and living.

2. The outer and inner ring of the bordered pit. ^By focus-
ing, the inner ring or outline of the lumen on the oppwHle side of
the pit may be seen.

3. The rows of rectangular cells occasionally crossing the
Tracheids at right angles. These are portions of the Medullary
Rays.

PREPARATION SECOND: (1.) Make several thin longitudinal
sections parallel to the "grain." (2.) Make several thin cross-
sections. Mount (1) and (2) in water under the same cover.

OBSERVE in (1.) 1. The small cavities, occurring along the
common-wall of two Tracheids. These are single and lens-shaped
— the bordered pits seen in section.

2. Look for the middle lamella or " limiting membrane "
which originally separated the two halves of the pit and was con-
tinuous with the common-wall of the two Tracheids.

3. The row of roundish or angular cells — three to six in
number occasionally seen between Tracheids. These are large1"

93 ELEMENT A It Y T1S8 UES.

than the sectioned pits and are the cells of the Medullary Rays-in
cross-section.

OBSERVE in (2): 1. The form of the Tracheids in cross-section.

2. The " middle-lamella " in the cell-wall of each.

3. The Bordered-pits and Medullary Mays in the section.

4. The Resin-passages in section. These are large openings
surrounded by irregular thin-walled cells containing resin. These
passages often occur running transversely, following^the larger
medullary rays. (Fig. 14.)

5. The annual layers of tissue (seen best with a low power)
marked by alternating layers of larger and smaller cells, and
crossed at right angles by the Medullary rays. (Strasburger pp.
115, 128a.)

6. The entire absence of true Tracheae, these being found in
Gynmosperins only next the pith. In structure Tracheids seem to
be intermediate between Fibrous tissue and Tracheae. Bessey, pp.
25, 26 ; Goodale, p. 83 ; DeBary, pp. 159, 160 ; Vines' Text Book of
Bot., p. 200 ; Strasburger, p. 56.

V. (d) Tracheae.

Illustration: Sections of steins for the study of TRACHEAE, —
VESSELS or DUCTS. The following general forms will be examined:

(a) Dotted. (c) Spiral. (e) ticalariform.

(b) Pitted. (d) Reticulated. _(f) Annular.

For these studies the woody tissue is to be treated as directed
for the previous illustration, but in the case of herbaceous stems
the tissue should be hardened in alcohol and carried through' the
method for mounting, outlined on p. 21.

PREPARATION FIRST: (a) Longisectioii of GRAPE VINE STEM.

OBSERVE: 1. In the region of the section containing the tibro-
vascular tissue, the large well developed ducts with pitted surfac.es.

2. These dots are true openings allowing free communication
between contiguous cells. Goodale, p. 29 ; Bastin, p. 162.

PREPARATION SECOND: (b) Radial longitudinal section of the
stem of CASTOR OIL BEAN, 1 c.m. in diameter (thin section mounted
in water).

OBSERVE: 1. Rectangular cells of pith and cortex.

2. In the vascular region ; large thick walled ducts, with the
surface covered with bordered pits.

--

94 KLKMK. \TAIl Y

3. The orifice of the pit, consisting of two "acutely divfr</i»<j
lamellae.''1 De Bary, p. 167 ; Sachs' Physiology, p. 137 ; Bessey,
p. 27.

PREPARATION THIRD: (c), (d) and (e) Radial longitudinal sec-
tion of the stem of BEGONIA, BEAN, BANANA, or better the material
used in PREPARATION SECOND. In this preparation,

OBSERVE: 1. Large thin walled ducts with thickened spiral
markings.

2. At various places the spiral bands broken loose from the
wall of the duct.

3. In portions of the preparation some ducts may appear in
longisection, in which case, the ends of the thickened bands may be
clearly seen.

4. The steepness and direction of coils, also the number of
bands.

5. Their branching at various places, often forming in some
vessels a reticulation. This is the origin of the reticulated ducts.
Excellent material for their illustration can also be found in the
Cucurbitaceae and Impatiens.

6. Certain ducts with the broad openings, between the, reti-
culations, arranged one above the other in the form of a ladder —
Scalariform Vessels. Care must be taken to prevent confusing
these with the pitted vessels which they closely resemble. De-
Barj, p. 158.

PREPARATION FOURTH : (f ) Longisection of the stem of CORN.
Zea Mays.

OBSERVE: 1. Lying next the large spiral vessels of the bun-
dles, certain ducts strengthened by transverse thickenings in the
walls in the form of rings, — the Annular Vessels. It is to be
noted that these are but a modification of the spiral ducts. De-
Bary, p. 156; Strasburger, p. 90; Bessey, p. 82; Sachs' Text Book
of Bot., p. 114 ;— Physiology, pp. 91, 136.

A modified form of Annular Tracheae exists in the Trabecular
J)uct, which has its walls strengthened by transverse bars across
the cavity. Good illustrations of this can be found in the fibro-
vascular bundles of the Juniper leaf. Bastin, p. 163 ; De Bary,
p. 156.

<»:. ELEMEM'A

VI. Sieve Tissue.

Illustration-. Stem of Cucurbitaceae (Cucumber or Pumpkin).

PREPARATION: This material should be hardened in alcohol
and treated as directed on p. 21. Both longitudinal and transverse
sections are to be cut, stained with haematoxylin, aHd several of
each permanently mounted. In the longisection, portions must be
secured that contain some of the fibro-vascular tissue. In the lat-
ter section on the outer and inner edge of the bundle,

OBSERVE : 1. The thin walled sieve tubes, recognized by
their sieve like plates, forming septa at the ends, and not infre-
quently on the lateral walls.

2. The protoplasmic contents of the tubes, frequently denser
near the ends.

3. The structure of the sieve plates.

4. The sieve pores, of varying sizes, usually largest at the
center.

5. A clear glistening bluish mass surrounding the sieve
plate, — the callus plate.

6. A mass of slime collected at the end of the tube, with a
portion of it projecting through the pores of the sieve. To a mount
of fresh tissue add qulphuric acid, and observe its effects on the
plates.

In the transectinir.

OBSERVE : 1. The fibro vacnlar bundles arranged regularly
around the peripheral portion of the -ton.

2. Two nearly circular or crescent shaped masses of tissue,
(stained with haeniiitoxyliu a dark purple) one on the axial and one
on the peripheral side of the bundle, — The Phloem.

3. By careful focusing upon some of the larger of the open-
ings in these areas, sieve plates, forming septa.

Goodale, p. 92; Vines' Text Book of Bot., p. 136; DeBary, p.
172 ; Strasburger, p. 121 ; Annales des Sciences Naturelles, (Bot.)
Vol. X., pp. 193-324 ; Prings. Jahrb., 21, 253-292.

7. Laticiferous Tissue.

1. Latex cells.

2. Latex vessels.

Illustration : For simple LATEX CELLS.

96 /•: /. /•; .w /•; .v r. i /,• > vvs.s i "/•;*.

Stems or Petioles of EUPHORBIACEAE or ASCLEPIADACEAE.

PREPARATION FIRST : Make longitudinal sections of the stem
or petiole of a Euphorbia, and mount in water.

OBSERVE: 1. Simple or branched cells or copnocytes, latex
cells — ramifying the various parts of the preparation, and tilled with
a dense milky juice — latex.

2. The very thin walls of the cells and granular nature of the
contents. The latex consists of a watery fluid with various
albuminoids, «»•</<{ /tic «fi<Is. or f///v/A>/V.s-, in solution. The sus-
pended matter may consist of proteid coinp<>i<n<lx, accompanied
sometimes with starch grains.

LATEX TUBES OR VESSELS.

Illustration : Stem or Petiole of (1ln Inlonimn HKIJUH
(Celandine) Stylophornm <f>/>h>///">ti, (Poppy.)

In sections obtained as for the previous study :

OBSERVE : 1. Thin walled profusely branched tubes, often
anastomosing, and extending through the various tissues of the
stem. These latex tubes are formed from rows of cells which be-
come united by the absorption of the partition between, or by its
perforation to allow free communication. The walls of these cells
are usually thin, but frequently become thickened in the form of
striations.

2. The effect of iodine on the latex tissue.

Goodale, p. 94 ; Vines' Text Book of Bot., p. 141 ; DeBary, p.
189 : Bessey, p. 75 ; Strasburger, p. 104 : Sachs' Text Book, p. 8G-7.

Glands and Water Pores.

GLANDS.

Illustration First: SUBEPIDERMAL GLANDS of the "LEMON
SKIN."

PREPARATION FIRST: Harden, section, and mount a piece of
"lemon peel" after, the irethod on p. 21.

OBSERVE: 1. Small cells of the epidermis, often containing
crystals.

2. Near the outer portion of the section large cavities in the
tissue — Tin' (Jluiulx or />.»•» /•/•/'<>/•.< <>f li/xli/cnanx or'nj'in. \. e.,
formed by the breaking down of cells.

97 ELEMKM'MiY TlSSl'ES.

3. Thin-walled cells, with large nuclei, lining the cavities.
In some cases the cells are much disorganized.

4. Crystals in the adjoining tissue.

5. Fragments of fibro-vascular bundles scattered through the
preparation. (Fig. 15.)

PREPARATION SECOND: Make a tangential section and compare
with the previous study.

Illustration Second: GLANDS in the leaf of JKn<'<il,//>tus.

PREPARATION FIRST: Transections of a mature leaf can be pre-
pared as directed for the previous study.

OBSERVE: 1. Large glands located on both upper and lower
sides of the section.

2. The cavity of the gland.

3. The large thin-walled cells, partly disorganized, lining the
gland, and the smaller thicker-walled ones just outside.

4. The mesophyll and palisade cells surrounding the gland.

5. The flattened epidermal cells above.

PREPARATION SECOND: Make transections of the young leaf of
Eucalyptus and trace the formation of the gland, which results
from the breaking down and absorption of the mother cells. Stras-
burger, p. 164, DeBary, p. 201, Vine's Text Book of Botany, p. 40,
Goodale, p. 98.

The Resin Ducts of Pinus were examined in the study of the
Tracheids of Coniferae.

Water Pores.

Illustration: LEAF TOOTH from Fuchsia.
PREPARATION: Several sections should be made serially, and
must include those through the apex of the leaf tooth, (p. 35.)
In the section passing through the center of the tooth :
OBSERVE: 1. The club-like enlargement at the outer edge.

2. The epidermis, consisting of small cells, interrupted at the
apex and forming a circular opening — the pore.

3. Two or three layers of chlorophyll bearing cells beneath
the epidermis.

4. The large region of the center of the section occupied by
the spiral marked tr<idi< nh.

5. The water cavity between the pore and the long pure

98 ELEMENTARY TISSUES.

cells beneath. The section should be preserved for reference in
connection with the study of the leaf structure, of which this is a
type of a very large group.

Bessey, p. 105, DeBary, p. 52, Vine's Plant Phys., p. 91.

D. MERISTEM TISSUE.

MERISTEM, or GENERATING TISSUE, is usually classified under two
heads, viz: —

I. Primary Meristem, giving rise to the PRIMARY STRUCTURE
in plants, such as young cellular tissue, and found at the apices
of young thallomes, stems and roots, and at the apex and base of
young leaves.

II. Secondary Meristem, or CAMBIUM, giving rise to the
SECONDARY STRUCTURE, as the thickening growth of stems, roots of
DICOTYLEDONS, or EXOGENS.

The SECONDARY MERISTEM, as PROCAMBIUM, forms a part of every
fibro-vascular bundle in its earliest stage.

The Primary fleristem.

TYPE A. Of a SINGLE APICAL OR INITIAL CELL. Found in most
of the higher CRYPTOGAMIA.

TYPE B. Of a GROUP OF INITIAL CELLS. This is characteristic
of PHANEROGAMS.

Illustration, Type B: Root from a cultivated Hyacinth grown
in a jar of water.

PREPARATION FIRST: Trans - and longitudinal sections prepared
after the method on p. 21, also for serial sections, p. 35. (Fig. 16.)

In the longitudinal section, observe the general arrangement
of the different layers of cells, viz:

1. That the several series of cells converge into a rounded
cone at a point just back of the apex. This point is the center of
the so-called "Initial Group''' which constitutes the Primary Meris-
tem. It is impossible to say just how far meristematic cells extend
from this point.

100 M Kit 1ST KM T/SsrE.

2. The Cctlyptrogen, a row of cells on the apical skle of the
luitial Group, producing rows of cuboidal cells radiating towards
the apex of the root, the outer ones becoming rounded and loosely
coherent.

3. The DermatOffen, arising from the Initial Group curving
to the right and left and ultimately producing a double layer of
cuboidal or flattish cells bounding the section on each side. In
large roots it consists of more than two rows of cells.

4. The Plerome, axial rows of oblong or cuboidal cells form-
ing a central band immediately back of the Initial Group.

5. The Periblem — thin-walled cells of various forms in several
rows between the Plerome and Dertnatogen.

6. That the differentatiou into the above named layers occurs
at a very early period in the development of the root-tissues. They
retain their original distinctness after passing over into permanent
tissue when they are named as follows:

The Calyptrogen gives rise to the Root-Cap.

The Demiatogen gives rise to the H]>i<l< /•////*.

The Periblem gives rise to the Cortex or Cortical Parenchi/in<(.

The Plerome gives rise to the Axial or Central Cylinder.

1. The first appearance of Spiral Vessels on the right and
left margin of the Plerome. Trace them backwards. They repre-
sent the beginnings of the Fibro- Vascular Bundles which always
arise within the Plerome in both stem and root.

8. The two rows of cells on the peripheral side of the Spiral
Vessels. The inner (the Pericambiuiti) belongs to the Plerome,
the outer (the Endodertnis) belongs to the Periblem or Cortex.
The Endodermis is sometimes called the -'Plerome-sheath" or
"Bundle-sheath/'

9. The peripheral layers of cells of the Plerome itself, extend-
ing back from the Initial Group to the beginning of the Fibro-
Vascular Bundles. These are termed collectively the Procambium,
— as it is from such cells that the tissues of the Bundles themselves
arise by fission and differentiation.

10. Occasional dark lines along the lateral walls of the Cortex
cells. These are due to cylinders of air in narrow intercellular
spaces. (See 2 under observations below on the cross-section.)

0

101 MKBISTEM TISSUE.

11. The Raphides or Needle-shaped Crystals, in black bun-
dles in many cells of the Cortex, and in this tissue only.

To a fi-esh section apply the tests for Crystals given on p. 3.

In the Cross- Section: (Fig. 17.)

OBSERVE: 1. The Epidermis, and outer zone of two or three
layers of cells ; form of latter.

2. The zone of the Cortex cells, with frequent small intercell-
ular spaces. (See obs. 10 above).

3. The Endodermis layer of the Cortex with a dark spot on
the common wall between. adjoining cells, due to a minute fold in
the wall.

4. The Axial Cylinder.

a. The Pericambial layer.

b. The Bundles five to ten in number ; the Xylan mass (that
including the large duct openings) alternating with the thin-walled
Phloem, after the usual plan in roots. See Fig. of Acorns in Sachs'
Botany, p. 115; De Bary, pp. 10,348; Strasburger, p. 184; Good-
ale, pp. 105, 112; Vines' Text Book of Bot., p. 147.

Make a sketch of a cross-section and the longitudinal section,
showing the zones and layers above mentioned. Also an enlarged
drawing of two xylem rays, the intermediate Phloem, Pericambium,
and Endodermal layers. •

A comparison should be made with the roots of a Dicotyledon
(Radish, Pea, etc.), treated after the same manner as directed above.

With reference to the development of the various parts of the
root from distinct initial groups, several types have been described
by Janczewski, ' Flahault, and others.* See Goodale, p. 107; De-
Bary, p. 7.

Type A.

A SINGLE APICAL OR INITIAL CELL.

Illustration : Root of FERN (Pteris) or EQUISETUM.

PREPARATION: The same as for the previous study, except
that serial logitudinal sections mast be made in order to insure tin-
presence of the desired parts. See p. 35. In the median section,
OBSERVE : 1. The cone-shaped root cap covering the tip of the
root, not unlike the one in type B.

2. Just beneath the cap and at the center of the root, the

102 MKIUSTKM TISM'E.

trilateral pyramidal cell with its convex base turned toward
the root-cap, — the apical cell.

3. A transverse partition cutting off the outermost portion,
which becomes the initial cell of the root cap, — Dermatogen.

4. Segments cut from the innermost faces of the cell and, by
longitudinal partition, developing into the tissues of the Pler<>nn>
uinl r< riblem.

Strasburger, p. 188 ; Vines' Text Book of Bot, p. 150 ; De-
Bary, p. 18.

In the transection, OBSERVE : 1. The irregularly thickened
walls of the epidermic.

2. The cortex consisting, on the peripherial portion, of dark
brown parenchyma and merging into sclerenchyma toward the
plerome.

3. The bundle sheath between the cortex 'and fibro-vacular
bundle.

4. The pericambium, a narrow sheath ( just inside the bun-
dle sheath) of parenchyma cells filled with protoplasm.

5. The regular radial bundle of the root.

If time will admit a comparative study should be made of the
apex of the stems of Monocots, Dicots, and higher Cryptogams.

The preparation of the tissues for study would be the same as
that recommended for the roots. DeBary, p. 19 ; Strasburger, p.
177 ; Vines' Text Book of Bot., p. 146.

Fibro=Vacular Bundles.

The following general classes are noted :

A. Collateral.

B. Bicollateral.

C. Radial.

D. Concentric.

Illustration A : Stem of BEGONIA, GERANIUM, MOON SEED
Vine, or SMILAX.

PREPARATION FIRST: The herbaceous stems should be pre-
pared in the usual way, the woody ones may be sectioned by plac-
ing them between pieces of cork in the jaws of a microtome.
Transaction and longisection should be mounted together.

103 MKH1STKM YV.s.srf;.

In the preparation of the Moon Seed Vine, (M<'ni.-<i><
Canadense) a stem of one year's growth should be selected.
(Fig. 27.)

Examine with a hand lens, and observe the general manage-
ment of the tissue systems in a cross-section.

1. Pith, (Fundamental Tissue.)

2. Fibro-vascular bundles, (outline of each is nearly circular).

3. Epidermis.

OBSERVE : 1. The Epidermis; The external walls of its cells
being thickened very greatly, and a light olive in color, the inner
and lateral walls being of ordinary thickness and the cell cavity
small.

2. The Cortical Parenchyma; of two kinds of cells, both
containing chlorophyll.

(a) The outer are regular, closely packed and thick-walled.

(b) The inner are thinner-walled, larger and looser, passing
into the oblong cells of the Medullary Rays.

3. The Fibro - Vascular Bundles :

The Phloem is sharply distinguished from the Xylem, form-
ing a strictly Collateral Bundle.
In the Phloem :

(a) The Crescent -/Shaped band, of thick-walled cells on the
peripheral side of the Phloem ; the lamellate structure of the walls.

(b) The squarish outline of Phloem cells adjoining the thick-
walled Xylem. Some of them somewhat thickened (lignified).

(c) The larger cells lying between (a) and (b). They are
irregular in outline, thin-walled and stain but slightly.

In the Xylem:

(d) The larger duct openings between which the smaller cells
(tracheids, etc.,) appear.

(e) The thick-walled cells of the axial region of the xylem
forming a continuous band, or "bundle sheath," bounding the axial
side of the fibre-vascular bundles.

(f) Between the A'///< //> and Phloem several of narrow, thin-
walled cells — the Cambium. This is the region of growth, in sec-
ondary thickenings.

In the radial-loncjitudinal sections trace out the groups of

104 .)//•;/,'/> 7 '/•:. V TISSl'E.

cells corresponding to those observed under the previous prepara-
tion, and demonstrate the Tissue Forms in each.

Drawings should be made sufficiently in detail to show the
character of the above-mentioned groups of cells in both trans-
and longitudinal section.

DeBary, pp. 319-339, Strasburger, p. 107, Bessey, p. 117,
Vine's Text Book of Bot., pp. 174-182.

Compare with closed collateral bundle of a monocot, (Fig.
19, 28.)

Illustration B: From the stem of Cwm-hit 1'epo.

PREPARATION FIRST: Radial longitudinal and transverse sec-
tions are to be prepared.

ORSERVE: 1. The Fibro- Vascular Bundles: the smaller ones
opposite the external ridges of the stem; the larger alternating with
the smaller, and occupying the ridges projecting into the central
cavity of the stem. To the naked eye the bundles appear as ganglia
of smaller cells.

2. The larger openings in the middle area of the larger bun-
dles, 3 to 8 in number.

3. A group of smaller openings on the axial side of the larger
ones. This middle area is the xylem.

4. Two nearly semi-circular or sometimes crescent-shaped
areas of tissue, one on the axial, and one on the peripheral, side
of the xylem. Both of these are masses of Phloem. Focusing
upon the larger openings of these areas, sieve-like plates may be
observed in some, forming septa. (Sachs' Text Book, p. 113.)

5. Form of cells in other parts of the bundle.

G. The thin-walled parenchyma, forming the greater part of
the stem and surrounding all the bundles. This is the tissue of
the Fundamental system.

7. The Epidermis of the Stem.

8. The "Intra- Corf '/<•<! I A'/////": — a band of several layers of
thick-walled cells, but faintly colored, and separated from the epi-
dermis by slightly stained cells.

9. In the above-named slightly stained band note the sep;i
rated areas of Collenchyma cells. Bessey, p. 30.

Collenchyma cells, as previously noted, are recognized by
the thickening of the walls at the angles.

ior> MI-; i; IST KM TISSUE.

In the middle longitudinal section of one of the bundles :
OBSERVE: 1. The Fin«l<ini<-ntul 7'fsaue (Parenchyma), on
the axial side of the bundle beyond its limits.
In the Axial Mass of Phloem:

2. The Sieve- Tubes; known by the Sieve-like plates forming
the septa or occasionally seen on the lateral walls. See Sachs', p.
113 ; Bessey, p. 78.

3. The Protoplasm enclosed in these tubes.

4. The Fibrous-cells, fusiform in outline, scattered among the
Sieve-tubes or occurring in a band next the Spiral Ducts.

In the Xyletn of the Bundle:

OBSERVE: 5. The Spiral Ducts — one narrow with a loose
spiral; another broader with a close spiral, occasionally becoming
reticulated.

6. A Scalariform duct; see Bessey, p. 27, (Fig. 18 ;) some-
times this approaches (in this plant) a Pitted Vessel in structure.

7. The Pitted Vessels — very broad.

a. The shallow pits on the walls in transverse rows showing
a "border" and "lumen."

b. The ridges on the walls between rows of pits.

c. The frequent occurrence of rings marking the partially
absorbed septa.

8. The thin-walled cainbiform cells, with septa oblique or at
right angles to the lateral walls. This layer sepai'ates the A'////-///
of the Bundle from the Phloem on the peripheral side of the
bundle.

In these bundles it will be noted there exists a pi fnniin //f baud
of cambium, which insures the continued growth of the vascular
tissue. The bundle is therefore said to be an open one.

Iu the closed bundles the procambium tissue becomes differ
entiated into permanent wood and bast, and the bundle undergoes
no further differentiation. The latter condition is the case in the
stems and roots of most Monocots, and Pteridophytes. Vines'
Text Book of Bot., p. 177. Strasburger, p. 83 ; Bessey, p. 121.

In the I'< rifiln nil Muss of Phloem:

9. Another group of S'i, >-, Tubes.

10. A few Jlast Fibres.

Trace in the cross section, the tissues, cells, etc., correspond-

loo

ing to those observed in longitudinal section, particularly noticing
what belongs to the Phloem, and what to the Xylem.

This Bundle is not only a Collateral Hnndle (Bessey, p. 120),
but a Bicollateral I>mt<ll<', (Fig. 20) i. e., one having a layer of
Phloem tissue (Sieve-tubes, etc.), on the axial, as well as on the peri-
pheral part of the Bundle. De Bary, p. 319.

Sketch so much of the cross-section as shall include one Bundle
and the tissue extending from it to the Epidermis ; also the middle
longitudinal section studied. The drawings should be on a scale to
show the characteristics of each tissue-form.

Illustration C: RADIAL BUNDLE in the root of HYACINTH, or
CORN. (Figs. 16, 17, 21, 22.)

In the transection prepared in the usual way,

OBSERVE: 1. The central portion of the root surrounded by
the Endodermis, a single layer of cells easily recognized by the
suberized thickening of the contiguous walls.

2. Just inside the Endoderinis, arranged in a circle, 12-18
groups of thick-walled cells, — vessels in section. These, together
with the wood parenchyma, constitute the xylem of the bundle.

3. Between these xylem areas, groups of thin-walled cells,
with a few large sieve-tubes in section, — The Phloem of the Fibro-
Vascular Bundle.

4. The large isodiametric cells of the Pith.

Vines' Text Book of Bot., pp. 165, 168; Bessey, p. 115; De-
Bary, pp. 348-366; Goodale, p. 109.

Illustration D: CONCENTRIC BUNDLES from the Khizome of
Pteris.

PREPARATION FIRST: Good sections can be obtained by fasten"
ing some of the material, hardened in alcohol, between two pieces
of cork in the jaws of a microtome, and sectioning with a strong
razor. The material must be kept wet during the operation, and
after staining can be mounted directly in glycerin jelly, or after
dehydrating and clearing, in balsam.

OBSERVE: \. Circular or lunar-shaped masses of whitish tis-
sue scattered through the preparation, — The Concentri*- Handles.

2. The well marked bundle sheath, composed of a row of
ellipsoidal cells.

3. With the high power, the xylem of the bundle occupying

107 MEEISTEM TISSUE.

the center, and consisting of thick-walled scalariform vessels and
xylem parenchyma.

4. Outside of the xylem and completely surrounding it, a
band of tissue limited by the bundle sheath, — The Phloem. This
part of the bundle consists of cribose tissue, and phloem paren-
chyma.

Make a longitudinal section of the bundle and identify in it,
all of the tissues seen in the transectiou.

It is to be noted that in this bundle the xylem portion is com-
pletely enveloped by Phloem and both surrounded by the bit //<//>'
sheath. The opposite arrangement of Phloem and Xylem is not
infrequently found.

Illustration Second : CONCENTRIC BUNDLE in the Rhizome of
IRIS. (Fig. 23.)

PREPARATION : Trausections may be cut free-hand or treated
as previously directed for herbaceous stems. By carefully focus-
ing on one of the smaller bundles,

OBSERVE : 1. In the center, the thin- walled cells of the
Phloem with a few sieve plates here and there in the larger open-
ings.

2. The large thick-walled cells outside of the Phloem and
surrounding it, — the Xylem.

Vines' Text Book of Bot., p. 175 ; Strasburger, p. 145 ; De-
Bary; p. 339 ; Bessey, p. 107 ; Sedgwick and Wilson's Biology,
pp. 72, 78.

E. TISSUE SYSTEM (of Sachs).

1. Epidermal Tissue System.

2. Fibro- Vascular Tissue System.

3. Fundamental Tissue System.

i. The Epidermal Tissue System.

Tissues Covering the EXPOSED SURFACES of Plants. Epidermal
Cells and their Modifications.

Illustration First : EPIDERMAL CELLS, STOMATES, and TRICHOMES.

For this study examine the notes and drawings made on p. 83.

Illustration Second : EPIDERMAL CELLS and FORMATION of
STOMATES. A very young leaf of a " STONE CROP," (Echcveria
secunda-glauca), or Sedum ternatum.

PREPARATION: With a razor make a thin section parallel to
lower surface of leaf, extending from the middle to the base, and
including the Epidermis and a little parenchyma. Mount sections
in water.

OBSERVE : 1. The epidermal cells of irregular forms, without
chlorophyll, the outline distinctly sinuous.

2. The stomates, oval or nearly circular when fully formed.

3. The guard cells of a stomate ; lunate in form and contain
ing grains of chlorophyll.

4. The Pore of the Stomate.

5. The several stages of development in the- </<>"»;/ or form
iny stortiMtes; beginning with the initial cell and ending with the
" mother cell " of the stomate just split into the two " guard cells."
The various septa even the primary one may be distinguished
from the original epidermal cell- walls by their straight or curved
(not sinuous), lines.

109 TISSUE S VST KM.

6. The "subsiift'ifri/ cells" surrounding the stoniate. They
are the result of various sub-divisions of the initial cell. Sachs'
Bot., p. 103 ; Bessey, p. 101.

For the study of the more complex forms of stomates an
examination should be made of those in the leaves of Pin us and
Cycas and the stem of Marchantia. (Fig. 26.) Bot. Gazette,
1889, p. 76; DeBary, p. 72; Botauisches Gentralblatt, xxiv, pp. 54,
85, 118, etc.

Illustration Third: (For Trictiomes.) Leaf of Sheplt<-r<ln<
Canadensis. (Fig. 24.)

PREPARATION: With a knife blade remove a mass of scale-like
trichomes, 2 or 3 m. m. square, from the lower surface of the leaf
and mount in alcohol, remove this by water, and mount perma-
nently in glycerin jelly. Seal after a few hours.

Observe the stellate and peltate scale-like trichomes, all grada-
tions of one type. In some cases the stalks of the trichomes may
be seen.

Notice the roots of Indian Corn growing in water. The vel-
vety covering of the roots consists of root hairs, which are tr>n>.
trichomes. (Fig. 12.) Goodale, p. 109; Vines' Text Book of Bot..
p. 158; Sachs' Physiology, p. 260.

Illustration Fourth : STINGING HAIRS from NETTLE, Urtica
dioica. (Fig. 25.)

PREPARATION: Make thin longitudinal sections of the stem
that shall include one of the stinging hairs.

OBSERVE: 1. The firm-walled unicellular hair with a sharp
apex, and broad swelling at the base.

2. The extension of the hair into a cup-like receptacle formed
by the tissue of the stem. In the mature hair, the conical base has
been lifted on a column of tissue, formed from the hypodermal
layer yet covered by the epidermis.

3. The strongly silicious icall of the hair often showing black
striations.

4. The cell contents. The cells also contain a strong acid
poison which enters the wound made by bringing an object into
forcible contact with the trichome.

Further studies of modified hairs may be made with profit if
the material is at hand. Excellent illustrations are found in the

110 TISSUE SYSTBlf.

DIGESTIVE HAIBS of many insectivorous plants, GLANDULAR HAIRS on
the scale of the winter bud of Aesculus Ifippocastanum, and vari-
ous kinds of BARBED HAIRS from Mentzelia ornata.

Strasburger, pp. 72-82; DeBary, pp. 59, 89, 94, 95; Bastin,
pp. 42-47.

It will be noticed that true plant hairs (trichomes) are but a
part of the epidermis which has become differentiated, while thorn's
are modified branches, and are connected with the vascular system
of the plant, e. g., thorns of locust.

For the study f>f Water Pores, in part modifications of the
epidermis, the leaves of Tropaeohnn, Aconitum or Ficus elastica,
furnish excellent material, but are not always readily accessible. It
will be sufficient in this connection to refer to the study made of
the leaf tooth of Fuchsia, p. 97.

Additional studies could profitably be made of many of the
modifications which the epidermis undergoes, but it is believed that
enough has already been given, to familiarize the student with the
more important characters of this interesting tissue system.

2. Fibre Vascular System.

3. Fundamental System.

Illustration: These Systems should be considered in the fol-
lowing groups of plants: (1) EXOGENOUS STEMS, herbaceous (Be-
gonia), and woody (Moon Seed Vine). (2). ENDOGENOUS STEMS,
herbaceous (Corn), and woody (Smilax hispida). (3). CONIFERAE.
(4). VASCULAR CRYPTOGAMS. (Figs. 16, 17, 27, and 28).

Since studies have previously been made of the elements of the
Fibre -vascular bundle, in each of the groups as outlined, it is only
necessary in the brief course here included to examine the distribu-
tion of the tissues in each of the cases mentioned, together with
the relations of the three tissue systems. To this end, careful
series should be made of the tongiseetion and transection of stems
from the plants indicated. De Bary, p. 232; Goodale, pp. 126-
135; Vines' Text Book of Bot., p. 170.

It must be remembered that the VASCULAR SYSTEM of ROOTS,
LEAVES. AND THEIR MODIFICATIONS should be included in the considera-
tion of this subject.

By examination of the material indicated make outline sketches

Ill TISSUE SYSTEM.

that shall represent the arrangement of the three systems in the
types indicated.

The Fundamental Tissiie System includes all tissues not form-
ing a part of the epidermis and its modifications, nor included in
the Fibro-vascular bundles. Examples of this system are found in
the CORTEX, MEDULLARY KAYS, and PITH of stems; CORTEX, PITH, and
ENDODERMIS OF ROOTS, and the MESOPHYLL OF LEAVES. Bessey, pp.
106, 122. (Figs. 14, 16, 27, and 28).

. SECONDARY THICKENING.

This is usually produced by the differentiation and develop-
ment of tissue, by means of OPEN FIBRO-VASCULAR BUNDLES.

I. In Dycotyledonous Stems.

Illustration: STEM of MOON SEED VINE. (Fig. 27.)

PREPARATION FIRST : Refer to the permanent preparation
made from the stem of this plant representing one year's growth.
P. 103.

PREPARATION SECOND: Stem of the Moon Seed Vine of
two or more years' growth. Prepare transections and longitud-
inal sections corresponding to those made for Preparation First.

OBSERVE: 1. The changes in aspect when compared with
Preparation First ; (a) The several annular layers of wood
formed from the xylem ; (b) each layer in any bundle having one
of corresponding thickness in the bundles on the right and left.

THE ANNULAR LAYERS ; (c) The distribution of spiral and
woody vessels ; (d) Of woody fibre ; (e) Tbe slight changes in the
Phloem region of the bundles.

2. THE CAMBIUM RING, of thin-walled cells forming by their
division new tissue. (1) Xylem. (2) Phloem. (3) Medullary.
(4) New Cambium.

This ring of cambium is divided into two portions, (1)
Fascicular, that included in the fibro-vascular bundle. (2) Inter-
fascicular, that between the bundles and included in the JA <//-•/-
lory Rays.

4. The large Medullary JRays, composed of cells whose walls
have not become thickened, (contrary to the general rule in Medul-
lary Bays of several years' growth.)

113 SECONDARY T

5. The isolation of the bundles, which still retain their indi-
viduality, on account of the large Medullary Kays.

6. That the stem has nevertheless been thickened, after the
usual mode in Dicotyledons, i. e., by the addition of a new lajer, in
a uniform manner, to the bundles.

Compare the sections of an Oak or Beech twig where the indi-
viduality of the bundles is not so well preserved. Sachs' Text
Book, p. 129 ; Vines' Text Book of Bot., pp. 1G7, 191 ; Goo.lule.
p. 137. Make sketches of the transection in Preparation Second
to illustrate the changes which have taken place in the thickening
process.

II. In Honocotyledonous Stems.

Illustration: STEMS of Smilax Mspida. Sections from (1)
one year old, (2) several years old. (Fig. 28.)

It is to be noted that there is no cambium /•///// in Endogens,
and the bundles being closed, i. e., without permanent cambium,
are not able to increase, by secondary growth, the size of the stem.
Compare the bundles in the stems of the two preparations and note
the changes, if any, tjjat have occurred during the development of
the plant. In many Monocotyledons there exists a ring of meris-
tematic tissue in the cortex, just beneath the epidermis, where new
closed bundles are formed, thus enabling the stems to increase in
size.

Vines' Text Book, p. 202; Goodale, p. 135; DeBary, p. 618;
Annals of Botany, Vol. VII, p. 21.

Make transectious of the stems of I* inn* which shall include
those from one to several years' growth. Note the changes that
have taken place in the thickening of the stem.

Strasburger, pp. 115, 116; Vines' Text Book, p. 193; DeBary,
pp. 461, 567.

The study of the development of Fern Steins will not be
included in this course. DeBary, p. 623.

Secondary Thickening in Roots.

I. Honocotyledons: As in the case of MONOCOTYLEDONOUS
STEMS, so with most of their roots, no true cambium ::<>n<' is found,
but the roots retain their primary differentiation throughout their
existence, except that the tissue may become firm by age.

114 SECONDARY THICKENING.

Illustration : The ROOTS of ORCHIDACEAE, CYPEBACEAE, or

LlLIACEAE.

PREPARATION : Make transections of the young and old roots
of any of these plants, and note the changes that may have
occurred. DeBary, pp. 360-362, 618; Annals of Botany, Vol. 7, p. 21.

II. Dicotyledons : The changes in the primary structure of
Dicotyledonous roots usually take place soon after the arrangement
of the primary tissues, and continue during the activities of the
plant.

Illustration : Roots of various sizes from the RANUNCULACEAE
and SAPINDACEAE.

PREPARATION : Treat the material as directed for herbaceous
and woody stems.

Compare the sections of different ages, and observe that the
secondary thickening has brought about the following changes: —

1. Formation of a cambium ring, by the longitudinal division
of cells on the axial side of the Phloem areas, and the extension of
the same laterally to their union with the next areas, over the ends
of the xylem rays. VanTieghem, Ann. Sci. Nat., 5 ser., Tom. XIII,
p. 185, pi. 3, 4, 8.

2. Irregular outline of the cambium rimj corresponding to
to the sinuses between the xylem areas.

3. Disappearance of these sinuses in the older roots, and the
nearly circular outline of the cambium rimj.

4. After these changes, the thickening corresponds to that in
the stem.

5. In the mature roots; the wood (xylem), bast (phloem), and
alternating with these plates of parenchyma, — the medullary rays.
DeBary, p. 473.

VASCULAR SYSTEfl OF LEAVES.

The arrangement of the VASCULAR SYSTEM in the leaf, with its
various modifications, or variations in the different groups of
plants, presents a subject too extended to be treated in this brief
course.

Read carefully, Strasburger, pp. 160-169 ; DeBary, pp. 296-
307, 372-373 ; Goodale, p. 155.

In structure the FIBRO- VASCULAR BUNDLES are much the same

115 SECONDARY THICKENING.

as those in the stem, but are often surrounded by a layer of thick
walled fibrous tissue, which adds much to their strength.

Treat a small thin leaf (Oxalis), with KOH and mount. The
preparation may be made permanent by washing thoroughly with
water and mounting in glycerin jelly.

OBSERVE: 1. The anastomosing Fibro-Yascular bundles,
indicating their full development.

2. The free ends of the bundles, consisting of the spiral
tracheids, in close contact with the mesophyll cells of the leaf.

3. The appearance of one of the larger bundles in tran-
section.

LENTICELS.

Illustration: Elliptical wart-like thickenings on the stem of
ELDEB (Sambucus Canadensis}, or Moox SEED VINE (Menispermum
Canadense).

PREPARATION FIRST: Fasten pieces of the stem in the jaws of
a hand microtome, and make several thin transections through a
lenticel. Mount in water and OBSERVE : 1. The ruptured epider-
mis of the stem.

2. Beneath this the true cork cells, not closely united but pro-
vided with inter-cellular spaces, which allow free communication
between the inner tissue and the air outside.

3. Below the loose cork cells, a layer of thin- walled rectangular
ones, constituting the true meristematic region of the cork, — the
Phellogen. The continued development of this tissue produces in
time a ring of cork which breaks away from the stem in the form
of the rough corky bark.

4. Inside of the Phellogen and formed from it is the Phel-
loderm, a layer of cells regular in outline, containing protoplasm,
and in many cases chlorophyll. These cells resemble those of the
primary cortex, and are formed to replace any of the cortical tissue,
that may have been obliterated by the pressure resulting from the
rapid growth of firmer tissue beneath.

Vines' Text Book of Bot., p. 212; Goodale, p. 151: DeBary,
p. 560; Strasburger, p. 153.

Plate L

FIG. 9. Mother cells with developing pollen grains from Funkia oratu The
illustration shows successive figures from A- E and represents, from the tirst divi-
sion of the contents of the mother cell, (A), to near the formation of the four pollen
grains, (E). (.\550). After Sachs.

FiO. 10. Stamen hair of Tradesatntia. Fig. B. Stamen hair showing general
form and relations of cells. A, cell of proximal end: B. nucleus, (x'20.) Kig. A.
Cell much enlarged. A, outer wall of cell; B, nucleus and contained nucleolus; C,
stream of living protoplasm. (x280.)

Plate II.

B

Fio.lt Section of Potato tuber. Solatium tuhcnmim. A. brown epidermis; B,
rectangular cells Jnsl beneath i>piiiermis; (', bypodermal cells containing ertwtal-

loiilx (!•'): l>, starch bearing parenchyma; E, starch grains. (x80). (.Modified after
Landois and Stirling1.)

Fl(3. 12. Various forms of parenchyma cells. Kiir. A. Stellate from the stem of
I'nnttilii in : l-'iir. 15. Ellipsoidal and rectangular from the I'ool of Jlitiirhitli ; Kisr. <;.
Cylindrical from I lie llwtrinlh root, (.1 t richome. or i-oot liair): Fiji. U. and E. Isodi-
ametric and vlohose from I he cortex and pitli of a Geranium stems. A. Cell cavity;
B. Intercellular space. (About xlOO.)

Plate HI.

FIG. 13. "Grit cells," (sclerenchyma), from the Dahlia root. A, Group of cells:
B, thin walled cells surrounding the sclerenchyrmi; C and E. opening of canauln
the cell wall; D, canals In section. Fig. A. (x300.) Fig. B. (xlOO.)

MB.";

Fio. 14. Transect ion of tin- le:if
M"BT. of PIHIW suleextri* A, Cavity of the
' • ' gland; B, thick wslled epidermis; O,
parenchyma of the mesophyll; D,

bundle sheath; E, large thin- walled

cells lining the gland. (.\300.)

•usually more or less disorganized, lining the gland

Plate IV.

FIG. 16. Longisection of root, Cypripcdium pubenctns. A, B, Root-cap, com-
pletely covering the tip of the root; C, cent nil or plerome cylinder, showing- the
thin-walled procambium cells which laier develop into the elements of the fibro-
raacnlar bundle; D, initial group Of meristematic cells from which originates t he
various tissues of the i-(M)t : E, cortex developing from the peribleni of the meris-
tematic region at the tip of the root: F, plerome or bundle sheat h composed of a
single layer of modified parenchyma cells, having the walls uniformly snberi/ed
but folded or thickened at their points of contact; G. epidermis having its origin in
the dermaiogen at the apex just outside of the periblem. (x^O.)

Plate V.

M.B.T,

FIG. 17. Transaction of the root of Cyprlpedlum pubeacens. A. epidermis; II,
cortex; C, bundle sheath; I), xylem ray; E, phloem. S.-e Fig. 16 for parts In lonjji-
sectlon. (x30).

Plate VI.

FIG. 18. Transaction of an open, collateral flbro-vascular bundle from the stem
of BapMria nititla. A, thin-walled cortical parenchyma surroundinj: the bundle; H,
large thick-walled vessels of the xylem; (-. thin-walled cells of the cambium; I),
sieve duct of the phloem; E, wood pareuchyma of the xylem. (.\350.)

Plate VII.

FIG. 19. Tranverse section of a closed, collateral vascular bundle from th» stem
of Indian Corn. Zea Mays. A, thin-walled parenchyma surrounding the bundle; B.
large pitted vessels in section; C, sclerenchymatous cells forming a sheath about
the bundle; D, phloem of the bundle, with sieve-tubes and bast-fibres; E, spiral or
annular vessel in section; F, intercellular space of lysigenous origin. <x200).

Plate V11I.

FIG. 20. Transection of a bicollateral flbro-vascular bundle from tlie stem of
Cucurhita Pepo. A, Parenchyma cells of tlie fundamental system surrounding the
bundle; B, B, larjre thick-walled vessels of the xylem; «.,', t', sieve-tubes of the
phloem; D, I), thin-walled cells of the cambium. (xL>0.)

Plate IX.

FIG. 21. Transeetion of the root of Spirantht* cernua. Showing the general
structure of the root with the relations of the various parts. A, root hair: H,
x>lem of the radial bundle; <', phloem, alternating with the xylem areas; I), epi-
dermis of the root; E, pith; F, bundle sheath or endodermis; G, cortex; II, inter-
cellular space. (x50.)

FIG. 22. The radial bundle in Fig- 21, more highly magnified. The lettering is
the same In both figures. (xlOO.)

Plate X.

M.B.T.

FIG. 23. (Fig. A), Transection of the underground stem of Pterig aquilina. A,
Epidermis; B, concentric flhro-vascular bundle; C, band of thick-walled brown
sclerenchyma; D, thin-walled parenchyma of the fundamental system; E, xylem of
the vascular bundle; F, phloem. (x!7.)

(Fig. B). Enlarged concentric bundle from (Fig. A). A, bundle sheath; B, D,
phloem parenchyma and sieve tissue: C, xylem of the bundle: E, parenchyma cells
of the fundamental system forming the main mass of the stem and the "ground
work" for the other tissues. (xlOO.)

Plate XL

FIG. 24. Trichomcs from the leaf of Sheplicrdia Canadfiisis. Fijr. A, Peltate and
Fig. B, stellate plant hairs. These are hut variations of the same general type. A,
Remains of the stalk. (x250.)

Plah ML

FlG. 25. Plant hairs (tricliomcs).

Kjj;. A. Needle like triehome from the leaf of Geranium. (Pelargonium IIK/UIIKIH.S.)

Fig. B. Stinging hair from the stem of Urtica diotoi. A, epidermis of the stem:
B, cortex; C, column of tissue supporting the hair; D, bull) «>f the hair and con-
tained nucleus: E, slender taper! MSI cell of the tip of the hair; the walls are strong-
ly silicious and usually covered with fine transverse markings. (x">ii).

Fiji- (!. Glandular hair from the leaf of (irranhnn. A, (jland at the apex: B,
epidermis of leaf; C, cells of the stalk; 1), cortical parenchyma. (.\200.)

Plate XI It.

FIG. 26. Fig. A. Transect ion of le:if, Pimm xj/fivxfn'*. A, A, stomates: K. thick-
ened epidermis; C. C, inter-cellular space beneath stoinate; I), mesophyll of tin-
leaf; E, thick-walled cells beneath the epidermis: K, guard cells of the stmnale.
(\250.)

Fig. B. Transect ion through the leaf of Cycdx repoiutd A, opening or pore of
thestomate: B, modified epidermal cells: (', ^ruard cells of the stoinate: l>. inter-
cellular space into which the stomate opens; K. parenchyma cells of the niesophyll
of the leaf; F, epidermal cells with strongly cutini/.ed walls. (.\250).

Plate XIV.

M.B.T

FIG. '27. Transaction of tin- stem of a Dicot, Mcnixprrmuin Cumnli-nxr. (one yr.-n
«>ldi. A, pith: B, epidermis; C, medullary ray: D, xyli-m: E, cambium; F. hast; G,
sieve t ubes; H, cortex. (X^O.) Compare with stem of several years growth.

Plate XV.

FIG. 28. Transection of the stem of a Monocot, Smtlax htepida. A, epidermis;

B, small closed fibro-vascular bundle, formed in the ring of meristematic tissue,
where, by the development of new bundles, the stem is enabled to increase in si/.e;

C, mature vascular bundle; D, cortical parenchyma; E, phloem; F, xylem. (x80).