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1 Introduction

The aim of this book is to present the fundamentals ofplant anatomy in a way which emphasizes their appli-cation and relevance to modero botanical research. It

is intended mainly for students at the intermediatelevel of a first degree course, but provides a readilyunderstandable account of plant structure for the lessadvancedstudent. '

A certain amount of jargon has to be learned inorder to get to grips with any subject, and here 1 makeno excuse for using terms which are specialized intheir meaning. The correct use of technical terms aidsclear thought and helps to make plant anatomy asexact as possible. Rather than define these words asthey arise, and in so doing break the flow of thought, 1have put those which are most used into an ilIustratedglossary which forms Chapter 3.

Examples are chosen from a wide range of plantsfrom temperate to tropical environments - far toomany textbooks neglect the rich tropical flora.Teachers should find plants mentioned which arereadily available to them to illustrate particular cellsor tissues. 1 hope those in tropical countries wilI seizethe opportunity to look at plants growing on theirown doorsteps, instead of having to send to northtemperate lands for microscope slides of unfamiliarplants.

Applied anatomy is the key express ion in this book.Plant anatomy is regarded as a duB subject by manystudents because the'tradition has been to teach it as acatalogue of celI and tissue types with only slightreference to function and development, and no men-tion of the day-to-day use to which this knowledge isput in many laboratories round the world. Textbookshave been written to suit this more usual style ofteaching. These advanced texts are of excellent valuefor the specialist student, but can be daunting to therelative beginner. Complementary to these books arethose consisting largely of illustrations. These are ofmuch benefit to students struggling to recognize whatthey see down the microscope, but again have the

shortcomings that they mainly serve to teach a set ofdescriptive terms.

At Kew, plant anatomy is in everyday use as a tool '

to help in solving baffling problems - many of eco-nomic value and a good number of scientific interest.As such, lhe subject becomes alive and fascinating.We also apply anatomy to help solve rather moreacademic questions of the probable relationshipsbetween families, genera and species. The incorpora-tion of anatomical data with the findings from studieson gross morphology, polIen, cytology, chemistry andsimilar disciplines enables those making revisions ofthe classification of plants to produce more naturalsystems. The economic significance of accurateclassification and hence accurate identification of

plants is frequently overlooked. The plant breeder,the food grower, the ecologist and conservationist alIneed accurate names for the subjects of their study.The chemists and pharmacognosists searching fornew chemical substances must certainly know exactlywhich species or even which varieties yield valuablesubstances. Without an accurate name and descrip-tion, they cannot repeat their experiments, or obtainfurther plant material of the same species, or knowwhich closely related plants might be examined forsimilar properties. In thi~ book, many more reasonsindicating the need for accurate identification ofplants will be given from everyday examples.

Materials and methodsMaterials

Any readily available plant can be used to teach orlearn plant anatomy. Over the years a smalI number ofspecies have become 'approved' as standard plants forstudy. This has produced a stultifying effect. Theplants chosen are thought of as 'typical', but often

(

2 1ntroduction

they are quite atypical. Many botanists go throughtheir lives thinking that maize is a typical mono-cotyledon - but grasses in general are very specializedand represent a very restricted view of monocoty-ledons as a whole. I am sure we frequendy adhere topeas, lettuce, maize and sunflower in our physio-logical work because botanists are often unaware thatother plants which can be grown with equal ease, aremore varied and interesting in their structure.

Material is best if collected fresh. It can be exam-

ined fresh for cell contents, cytoplasmic movementand so forth. Fór ease of sectioning and histologicalstudies it is better to 'fix' the fresh material by chem-ical means. Fixatives, when correcdy formulated, willkill the plant material, preserve its shape and size, andrender the tissues suitable for sectioning.

Fixatives

Seventy per cent alcohol is typically used as a plantfixative in schools, since it has little effect on thestudent should he pour it over himself. This re-agent hardens the plant tissues and can cause changesin shape. The zoologist soon learns to live withformaldehyde, which has to be treated with morerespecto There is no reason why students should notuse more potent liquids for fixing plants if suitablecare is taken. For general histological purposes, thefollowing mixture is found to be excellent:

F ormalin-acetic-alcohol or F AA

850 mI 70 per cent alcohol100 mI 40 per cent formaldehyde50 mI glacial acetic acid

This is a corrosive liquid, and should it comeinto contact with the skin it should be washed offimmediateIy.It is well worth the trouble and careto use material fixed in F AA because it sections well

and can be kept in the reagent indefinitely. Thefumes are harmful and should not be inhaled.

Material to be fixed is normally cut into portions toenable rapid penetration of the fixative. The portionsshould be of such a size that they can be readilyidentified and oriented. Botdes with wide mouths and

plastic screw tops are ideal for storage, and can be ob-tained in a range of sizes. It is best to keep the plantin fixative for at least 72 hours before use. Plantmaterial in the F AA can be stored for as long as re-quired, but the botdes should be inspected regularlyfor evaporation and topped up with 70 per cent alcoholif necessary. This is the most volatile of the consti-tuents.

Specimens to be sectioned are removed with for-ceps and washed in running tapwater for t to I hour.They can then be handled safely.

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Dried herbarium material can often be used for

anatomical studies. Some plants revive easily, butothers are unsatisfactory. If there is no fresh materialavailable, then dried material can be fi~ed after boil-ing in water for 5-15 minutes and cooling. Somepeople add a few drops of detergent to aid wetting.

SectioningThe safety razor blade can be used to produce sec-tions thin enough for study under magnifications of100 to 4°0 x , or sometimes more. Practice is needed.Those with a steady hand will get better sectionswith the thinner, double-sided razor blade, but verythin blades are too flexible. Every anatomist bears thescars of early batdes with tough plant material.

For the production of large quantities of slides ofthe same specimen for class study, a more refinedmethod of sectioning is needed. A rotary or rockingmicrotome is often used, but the method which in-volves embedding the specimen in a block of wax orsimilar material is almost entirely unnecessary forhistological studies. It is only indispensable for sec-tions under 10 Jlm thick, or for subjects such asflower buds where the various parts would separateand become disarranged when not embedded forsectioning.

Normally, sections of between 15-3° Jlm are suit-able for histological study. These can be cut with asledge or sliding microtome. The Reichert OME issuch an instrumento It is highly recommended. It isone of the types in which the specimen to be sec-tioned is firmly held in a universal clamp (to allow forcorrect orientation) and the knife is brought towardsand over the specimen. Models in which the knife isfixed, and the specimen is made to move past theknife are not so universally useful.

The moving knife sledge .microtome can be usedfor the following types of material, using 5° per cent

" alcohol for lubrication (applied to the knife blade witha camel-hair brush).

. l. Tough materials, such as wood, are cut into I cm. cubes, with an orientation as described on p. 8.The cubes are boiled in water until waterlogged, i.e.

I they sink when cold water is added to the container.The cubes are removed, cooled and clamped. Ifthey

. are very hard, they may need to have a jet of steami directed on the surface to be cut (see Fig. l. 1), but\ often they are soft enough to cut direcdy. Some woods!contain silica, a substance so hard that it rapidly\ blunts the microtome knife. Silica can be removed by\ standing the wood for 12or more hours in 10 per cent

\hydrofluoric acid, in plastic containers. Very greatcaution should be taken with this acid. It causes

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fb

safety tube1m+long

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~~ n... wood block~ ) in microtomeclamp

glass beads

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Fig. 1.1 Apparatus for producing a steam jet to soften woodprior to sectioning.

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serious bums even in low concentration, andthe bums heal very slowly. It is not recom-mended for class use, but could be used by trainedtechnicians. After treatment, the wood must bewashed in running water for several hours.2. Twigs may need to be boiled before sectioning.Softer stems are best fixed and washed before

sectioning. Many cylindrical objects need some sup-port in the microtome clamp. Support is normaIlyprovided in the form of cork or pith. Pith tends to be-come soggy when wet, and cork can contain unex-pected sclereids which blunt the knife, but on thewhole I prefer cork. Suitable bottle corks with fewlenticels should be selected (Fig. 1.2A-D). Circularslices about 3-4 mm thick are cut off, using a razorblade. The discs of cork are cut across the diameterand the two halves placed side by side.

F or transverse sections of stems, a notch cut in onehalf of the cork disc will help to keep the specimencorrectly oriented, without aIlowing it to be com-pressed excessively. Alternatively, the cork may becut along its long axis, and the two halves used tomount the specimen to be sectioned.

For making longitudinal sections, the cork slice is

Materials and methods 3

cut across the diameter at an angle (Fig. I.2E-H).When the two parts are placed side by side with thematerial to be sectioned at the bottom of the V, clamp-ing causes the outer parts of the cork to roIl outwards,but the material is retained. If the disc was cut toproduce a fIat surface before clamping, the outwardcurving would release the specimen, as in Fig. 1.3.3. Leaves to be sectioned transversely are rarely justthe right width for the clamp. Wider leaves can befolded once or several times so that they form a sand-wich in the cork, Fig. I.4. With narrow leaves, it isbest to put several leaves between the cork slices -there is more chance of getting some good sections.

Most mesophyte leaves section easily. Some suc-culent or very soft hydrophyte leaves and stems canpose problems. In these plants the cells are thin-waIled and burst easily if compressed when turgid. Asimple and usuaIly effective remedy is to aIlow thespecimen to go limp on the bench for half an hour orso. It can then be firmly clamped, cut, and the sec-tions put in water. If they do not return to theirnatural shape in water, 5° per cent alcohol may beused, or even, for a second or so, immersion in un-diluted bleach such as 'Domestos' or 'Parozone'

(sodium hypochlorite) will cause them to return tothe uncompressed formo

Certain leaves contain silica bodies (particularlythose of grasses and sedges) which blunt the micro-tome knife and tear the section. So if a section appearstorn, first examine it to see if silica bodies are presentoHydrofIuoric acid (IO°k» can be used to remove thesilica bodies, but must be treated with the utmostcaution.

As each section is cut. it will slide onto the knife

blade, lubricated by the 5° per cent alcohol. It shouldbe lightly transported on a paint brush to a petri dishof 5° per cent alcohol. The section can be examinedtemporarily in water, or will keep for several monthsin 5° per cent glycerine solution on a slide (storedfIat). Starch distribution can be studied in suchsections, and chloroplasts and other larger cyto-plasmic inclusions can be seen.

ClearingSo metimes it is an advantage not to have ceIl con-tents obscuring the tissue distribution but; before'clearing' the sections, some should be studied withtheir inclusions. Sections can be cleared by trans-ferring them from the 5° per cent alcohol to a dish ofwater with a brush or fine forceps. Then, using amounted needle or fine forceps they are placed in acavity block containing undiluted Parozone, Domes-tos or other sodium hypochlorite household bleach.

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4 1ntroduction

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tor L.S. clamp

¡-.Q1J ~@] [!!]

~ @]

~ ~~,~ ~~hiS .',' , ) O

,' [Q]. . . torT.S.

Cutting bottle corksto make clamping material ~

[]J~ - [Q] torT.S.

[]J - [EJ tor L.S.

. Z.

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Fig. 1.2 Preparing a cork for holding material to be sectioned. A-D for T.S., E-H for L.S.; note that the oblique cut in cork E helpsto prevent cylindrical stems from being released from the cork on clamping.

The time needed for cell contents to dissolve awayvaries from subject to subject and can be determinedby visual inspection. It usually takes about 5 minutes.The whole section will dissolve if left long enough!After immersion in the bleach, the sections arethoroughly washed in water. Take care not to get thebrush in the bleach - its bristles will dissolve.

StainingAfter thorough washing, the sections are ready forstaining. Two main types of stain can be used, (a)those which are temporary, whose colour fades, orwhich gradually damage the section and (b) thosewhich are regarded as permanent. Even permanentstains may lose their colour if exposed to sunlight, soslides should be stored in the dark.

With care, stains can be selected to give the maxi-mum contrast between the various cell and tissue

types in the planto They might be selected beca usethey colour particular parts of the cell wall structureand indicate its chemical composition. The stainsdescribed below are in daily use in the JodrellLaboratory. Those who want comprehensive listsshould see the books by Gurr, Foster or Peacock - tomention but three of the many guides to micro-technique.

I. Temporary stains(a) 1 per cent aqueous methylene blue All cellwalls turn blue, except cutin or cutinized walls which

remain unstained; cell walls take up a degree of in-tensity of blue depending upon their chemicalcomposition and physical structure; various walllayers frequently stain differently.

The stain may be mixed with 5° per cent glycerine,about 10per cent of I per cent aqueous stain to 9° percent of 5° per cent glycerine, and sections mounteddirectly into this medium. This mixture is also usefulfor staining macerated tissues which are difficult tohandle. A drop of washed macera te in water is mixedwith a drop of the mixture on a slide, and the coverslip put on.

(b) Chlor-zinc-iodine solution (Schulte's solution)This solution consists of: zinc chloride 3° g, potas-sium iodide 5 g, iodine I g and distilled water 14°mI.Cellulose walls turn blue, starch turns blue-black,lignin and suberin turn yellowand moderately ligni-fied walls turn greeny blue.

Sections are placed on the slide,and a drop or twoof CZI added. This can be drawn offand replaced by5° per cent glycerine after 2-4 minutes, but satis-factory results can be obtained by adding 5° per cent .

glycerine directly, and mounting in the mixture.This stain swellsthe wallsand eventuallydissolves

them. Consequently, care must be taken when des-cribing wall thickness.

(e) Chlorazolblack - saturated solutionin 70percentalcohol Stainswallsblackor grey;particularlygoodfor showing pitting.

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. '. i-Clamp

Specimen will come out ifcork edges arecut square

Fig. 1.3 The wrong way to cut cork for making L.S. of material.When clamped, the cork curlsback and the specimen is released.

- (d) Saturated carbolic acid solution Sections aremounted directly in the solution (which should bekept off the hands). Silicabodiesusually turn pink;this helps to distinguish them from crystals whichremain colourless.

(e) Phloroglucin and conc. HCI The phloroglucin isadded to the sectiol\,and then the HCI. Lignin turnsred.

(f) Sudan IV Sections can be mounted directly inthe stain. Fats, cuticle turn orange.

(g) Ruthenium red Mucilage and some gums turnpink. Sections can be mounted directly in the stain.

,

2. Permanent stains

Safranin (1 per cent in So per cent alc~hol) and Dela-field's haematoxylin. CeIlulose turns dark blue, ligninturns red and ceIlulose waIls with some lignin turnpurple.

Freshly mix the safranin with matured Delafield'shaematoxylin in the proportions of I :4; filter. Thestock mixture can be used for up to about one week,but should be filtered before use each day.

Sections should be transferred from water (afterwashing all bleach away) into a cavity block contain-ing the stain, and the block covered with a glass lid.Most sections take up the stain in 2-6 hours; someneed less time. They are then transferred to a petridish of 5° per cent alcohol containing 2-3 drops ofconc. HCI. This solution removes the stain, acting onthe safranin first. The object is to obtain a satisfactorycolour balance, and only experience will tell when thishas been reached.

In general, sections should be removed when theystill appear to be slightly dark or overstained for best

Materials and methods S

~~fold a long leaf and obtain several sections

Fig. 1.4 Long lea ves can be folded several times beforesectioning. Several sections will then be obtained with each cut.

results, the colours look less intense under the micro-scope. The decolorizing action is halted by placingthe sections into a petri dish of 9S per cent alcohol.After about S minutes they can be transferred to abso-lute alcohol in a coyered petri dish. Five ll1inutes laterthey can be transferred either to a SO/50 mixture ofabsolute akohol/xylene in a covered dish, or this stepmay be eliminated and they may be transferreddirectly into~xylene. Xylene fumes should not beinhaled. After ten minutes in the xylene, the sectionscan be mounted in Canada balsam on the microscopeslide. Any milkiness in the section at this stage meansthat water is still present, and the section should betaken back through xylene, then fresh absolutealcohol, fresh absolute alcohol/xylene and freshxylene before re-mounting. Sections which curl up orroIl up should be straightened out in So per centalcohol. As they progressively dehydrate in pureralcohol they become more brittle and cannot be un-roIled without breaking. Curled wood sections can beflattened by drawing them over the edge of a slidepartly immersed in So per cent alcohol (Fig. I.S) - aprocess needing three hands! Alternatively a sectionlifter can be used. Once on the slide they can be 'set'using a few drops of 95 per cent alcohol.

If it is more convenient to sta in overnight, thesafranin/haematoxylin mixture can be used in theproportions of 94: 6. Although fast green can be usedas a counter-stain for safranin, we have found thathaematoxylin produces a colour which photographsbetter on normal panchromatic film.

Fast green can be used on its own as a stain formacerated material. The macerate is dehydrated bydecanting alcohols of 5°,7°,9°,99 per cent and abso-lute in turn from a tube containing the macerate. It isa help if a small hand centrifuge can be used to settlethe cells at each stage. Finally, the cells are transferredto a slide bearing 2-3 drops of euparal containing 2-3drops of fast green per 10 mI. The cover slip is

. applied.

6 lntroduction

Fig. 1.5 Drawing a curled section onto a microscope slide.

Mounting mediaNeutral Canada balsam has the advantage that it isnot likely to remove safranin from the sections. Somemodero substitutes, whilst being less yellow, are tooacid and gradually cause the safranin to leach out, orcontract markedly if over-dried.

Euparal is used either where it is undesirable topass very delicate sections. through xylene after abso-lute alcohol because they may distort, or for macer-ated material, as described above.

Slides are baked fIat, in an oyen at S8°C for 10-14days, to thoroughly dry the mounting media. Thosesections in Canada balsa m are firmly embedded atthis stage, and the slides can be stored upright. Thosein Euparal, however, may be set round the edges only,and should be treated with care, or given furtherbaking.

For upright storage of slides, aluminium holdersare very convenient. Four slides are housed in eachholder. The holder is the size of an index card and can

be kept in a standard card filing cabinet.

Preparation of surfacesThe staining methods applied to sections can equalIywelI be used for surface preparations.

Leaf surfacesThe epidermis of most leaves can be readily removedby the scraping method. Only those leaves with veryprominent veins, or large, numerous hairs poseproblems, and demand a great deal of patience.

Material may be fresh, or washed after fixing. Asuitable piece is cut from the leaf (Fig. 1.6). Thesurface for study is placed face down on a glazed tileor glass plate. It is irrigated with a few drops ofsodium hypochlorite. One end is held securely with acork, and the other end scraped lightly with a safetyrazor blade. With practice a double-edged blade can

be used, but it is better to start off by using a single-edged blade. The blade is held at 90° to the leaf(Fig. 1.6). The gentle scraping is continued, addingextra hypochlorite as necessary, keeping the leaf wellirrigated. If the leafis not severed by a forceful scrape,you will end up with a thin, clear area which can becut off, placed in a cavity block for a few minuteswith sodium hypochlorite and then washed in a petridish of water. Loosely adhering cells can then bebrushed off with a camel-hair paint brush.

The preparation can then be viewed in waterunder the microscope. It is easy to see if enough hasbeen scraped away. Experience will soon enable youto judge when the end point is reached in scraping.Make sure the surface is placed the right way upbefore the final mounting.

With some material it is unnecessary to scrape theleaf, since the epidermis can be removed by peeling itoff the fresh leaf. This is done by folding the leaf tobreak the surface, and either stripping directly,pulling one part of the leaf downwards relative to theother, or by holding as thin a layer of the surface aspossible between forceps and peeling it back.

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Stem surfacesA thin strip of stem surface can be obtained by makingthe first L.S. cut on the microtome just pass throughthe surface layers. This requires careful microtomeadjustment, but is quite satisfactory.

Surface replicasSometimes it is not possible or desirable to remo ve theepidermis itself - a plant may berare, or there may belittle material readily available. A good imprint of thesurface can be obtained with a film of cellulose ace-

tate. It may be necessary to wipe the leaf surface withacetone to cIean it. Then clear nail varnish is brushed

on. More than one layer may be required. The driedfilm can be removed and mounted, preferably in amedium with a different refractive index or little willbe seen.

Although replicas can be made easily, using avariety of materials (Iatex, for example), 1 prefer towork with the epidermis itself. Replicas can tell uslittle about celI contents.

Cutícular preparatíonsVarious chemical treatments can be used to cause thecuticle to separate from the leaf. The cuticle is inmany respects better than the replica, but can be verydelicate.

One method is to digest away the leaf tissues usingnitric acid (care !). The cuticles will frequently fIoat

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place squareof leat on slide

hold the leaf with a cork

leaf irrigated in Parozone

Fig. 1.6 Prepari~g leaf surface for microscopy by the scraping method.

Materials and methods 7

scrape gentlywith razor blade

tri m off the th in part,transferto dish andbrush off loose cells.

stain, then mount the correct way up on a slide

to the surface, or, when the leafhas been fully washed,can be teased off the dissolving tissues.

Clearing materialWhole thin leaves or stems or flowers can be made

transparent by soaking them in chloral hydrate, wash-ing and soaking in sodium hydroxide solution,alternately, for several changes, with several hours ateach stage.

After the final washing, the organ can be carefullystained in safranin (1% in 5°% alcohol) dehydratedthrough a very gradual alcohol series and mounted.Veins and sclereids show up well.

{\lternatively, the preparation may be left un-stained for examination by various optical methods(p. q).

Standard levels (Fig. 1.7)When plants are being examined for comparativepurposes, either for identification or for taxonomicreasons, it is important that similarparts of the organsare looked ato The leaf, for example, is normallylooked at in transverse section across its broadestregion, or half way along the length of the lamina.The surface of the leaf is studied near the centralregion of the lamina. The margin may also beexamined.

Petioles should be examinedin T.S. just where thelaminabegins, half-way down its length and alsonearthe base. Stems are normalIy sectioned in the middleof the internode, or, in addition, at the node. Roots

are normally sectioned at a convenient level, sinceaccurately defined positions are harder to delimit.For very detailed studies, of course, sections are re-quired from many other levels, and sometimes serialsections are needed. These are particularly useful inthe study of no des, and shoot apices.

A simple infiltration techniqueFix in F AA for 48 hours; wash in running water for 1hour. Pass through alcohol series 5°, 7°, 9° and 95per cent and absolute 4 hours each; absolute alcohol-xylol 3 :1, 2 hours; absolute alcohol-xylol 1: 1, 2hours; absolute alcohol-xylol 1: 3, 2 hours. Xylol,two changes, 1 hour each. Xylol :52° wax 3: 1, 1: 1,1:3, pure wax two changes, 2 hours each, then over-night in pure wax, followed by one change for 2 hoursin pure wax. The normal embedding procedure in theem bedding oyen is then folIowed.

Electron microscopesThis book is not intended for people who are usingelectron microscopes, but some space must be givento a brief description of their uses.

There are two main types of electron microscope,the transmission microscope (TEM) and the scanningelectron microscope (SEM). Thin sections are exam-ined in the transmission microscope, or carbonreplicas produced from specimens can be used wheresurface features are to be studied. Electrons are madeto pass like a focussed light beam through the section.Some parts of the specimen are electro n-dense or areprepared by stains and fixatives to be electron-dense,

('

8 lntroduction

Standard levelssample forsurfaces

sectionpetioleat A, B-+e

sample forsurfaces,.1

:2

section for T.S.half way alonglamina

T.S.

R.L.S.T.L.S.

Secondary wood-cube cut from trunk

-half wayalong lamina

- T.S. near base

~ T.S. at nodefor leaf trace

Stem studies

Linear leaL

T.S.

~T.S.+ L.S.roots are cut

at convenientlevels Rhizomes pose problems

and are cut at convenientlevels

Fig. 1.7 Selectiori of standard levels for comparative work. For wood, a cube is prepared so that it gives transverse, tangentiallongitudinal and radiallongitudinal faces.

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whereas other parts are electron-opaque, permittingelectrons to pass and form an image either on aspecial fluorescent screen, or directly on a photo-graphic plate. The transmission microscope usese1ectromagnetic lenses to focus the electron beam,which has a very much higher resolving power than abeam of light. That is, it can make distinct pointswhich are very dose together on the object. Thelargest commorÍly available microscopes can resolvebetween points about 20 nm apart, and have theability to magnify above 500,000 x . Naturally, at suchhigh magnifications, only very small areas can be seenat a time. By comparison, the best light microscopeusing green light can give a maximum real magnifica-tion of about 1,200 X . Using ultra violet light, slightlyhigher magnifications can be obtained.

Methods of preparation of the specimen for trans-mission microscopes, are straightforward, but a veryskilled operator is needed to produce good results.Fixation of the specimen is very critical, for example.

The scanning electron microscope is used mostcommonly to examine the surface of specimens. Somespecimens can be examined fresh for a brief time, butmost are dehydrated carefully to minimize shrinkageand distortion, then coated with a very thin layer ofmetal, usually gold or goldjpalladium alloy. This

gives a better image and prevents contamination ofthe microscope by water.

Because of its relative ease of use, and since quitelow (1OX ) magnifications as well as those of up to orabove 180,000 x can be obtained on it, the instrumenthas a very wide use in applied plant anatomy. Cur-rently, resolution of 1,000 nm or even better than7°0 nm can be obtained as a matter of routine.

The specimen is bombarded with a focussed ray ofelectrons. The electrons are made to scan in parallellines over a rectangular area. Secondary electrons areemitted by the object, are collected by a series ofelectronic devices, and a synchronous image is dis-played on a small cathode ray tube. Most tubes areabout 10 cm square, and have about 1,000 lines. Thescreen itself is photographed to produce a permanentrecord.

Provided the coated specimen is kept dean anddry, it can often be used many times.

A great depth of field can be obtained with thisintrument, about 500 times that of a light microscope.Many surface patterns of leaves, seeds and fruits,spores, etc., are being seen and understood properlyfor the first time.

Of course, the cost will make it impossible formany people to own or even use an SEM, but once

characters have been seen and illustrated or des-

cribed, it is astonishing how many can be seen with agood dark field, epi-illuminating optical microscope.It is only specimens which have to be magnified aboveabout 1,200 times that cannot be interpreted with thelight microscope.

Extending the use of the student's microscopeIt is unusual for the student to have a high quality lightmicroscope, but the one available will serve well ifit iskept clean, and all the lenses are centred properly.

One simple way to make the microscope moreversatile is to make polaroid attachments. A disc ofpolaroid material mounted over the eyepiece, andanother fitted in the filter carrier, or in a holderbetween the mirror (or light source) and the micro-scope slide, turn the instrument into a polarizingmicroscope. Crystals become easily observable, as dostarch grains and details of cell wall structure.Figure 7.5 shows part of a stem section in polarizedlight. .

Thin cellophane placed. in one layer over the lowerpolaroid sheet (the analyser) will cause the light beamto become elliptically polarized. This phenomenongives a coloured background against which crystals,

. etc. will appear a different colour. Rotation of thepolaroid over the eyepiece (polarizer) will causechanges in the colours. This technique is useful for

Further reading 9

examining unstained, macerated material (p. 50),sclereids in cleared material (p. 41) or for looking athairs or surface details in preparations where stainingwould make the subject too dense.

Other optical techniquesOther optical techniques include phase contrast,anoptral contrast (Fig. 4.7) and dark ground.Fluorescence microscopy and interference micros-copy are also used in research.

The use of very thin (ultra microtome) sectionshas recently enabled the light microscopist to seedetails of cell walls otherwise obscure, and the studyof transfer cells (p. 13) is possible with such sections.

Further readingBradbury, S., 1973. Peacock's Elementary Microtechnique (4th edn

rev.), Edward Arnold, London.

Foster, A. S., 195°. Practical Plant Anatomy, Van Nostrand,New York and London.

Gurr, E., 1965. The Rational Use 01Dyes in Biology, Leonard Hill,London.

Purvis, M. J., Collier, D. c., & Walls, D., 1964. LaboratoryTechniques in Botany, Butterworths, London.