Book Reviews

Plant Microtechnique and Microscopy. By Steven E. Ruzin.

Published by Oxford University Press, New York, July

1999, xi�322 pages. Softback. £32.50. ISBN 0-19-

508956-1.

Not since the classic do-it-yourself O'Brien and McCully was

published in 1981 have I come across such a comprehen-

sive book dedicated to techniques for plant light microscopy.

This volume is full of the widest range that you could

imagine of protocols for plants and originates from the

author's long experience in teaching plant microtechnique.

Half of the text could be considered by many to be old-

fashioned in concentrating on basic tissue preparation,

dehydration, embedding and sectioning followed by classical

staining techniques, but in this age of the analysis of

mutant phenotypes and transgenics these more traditional

techniques are once more in vogue, and this book is a rich

repository of useful protocols. Unfortunately, it is in the

more recent techniques of ¯uorescence microscopy and

immunocytochemistry that the book is at its weakest.

The book starts off with eight pages of `quick start'

protocols covering ®xation and embedding schedules and a

couple of basic staining procedures with cross-references to

other chapters. Whether this will prove to be useful, I'm not

really sure. I would like a bit more background information

before rushing into the laboratory and embedding my all-

important specimen! Chapter 2 covers the basics of light

microscopy, which is then continued in great detail at the

end of the book in an appendix. Perhaps it could all have

been in an appendix or in one longer chapter. Chapters 3±

10 then take us through all the techniques of ®xation,

embedding in different media, a myriad of staining proto-

cols, through to mounting media and troubleshooting. For

me, this is the most useful section in the book, and the only

real criticism I have to make is that the use of ultramicro-

tomes for cutting thick resin or cryo-sections for light

microscopy could have been covered.

Chapter 11 takes us through the more standard cyto-

chemical tests, such as Feulgen staining for nucleic acids,

and then on to enzyme cytochemistry. This is a very short

section with a few basic protocols and a conspicuous lack of

suggestions as to controls. Surely this important area of

histochemistry deserves a bit more attention. Fluorescent

dyes are then afforded a bit more space. However, I am

concerned about the accuracy of some of the information in

this section. For instance, a table tells us that plant Golgi are

stained by NBD ceramide. As far as I am aware, no-one has

managed to stain plant Golgi with a vital ¯uorescent probe

other than GFP constructs. Likewise, Cascade Blue is men-

tioned as a cytoplasmic probe and Lucifer Yellow for mem-

branes, whereas both these dyes are in fact sequestered, in

most instances into the vacuole. There is no mention at all

of the extremely useful FM dyes that are now becoming

popular as membrane tracers.

The ®nal chapter covers the localization of molecular

targets in tissues, namely immunocytochemical and in situ

hybridization technology. This is the most disappointing

chapter in the book, yet potentially the most useful. It is

clear that the author cannot be a user of such techniques

and has relied too heavily on protocols taken from the

animal, and not the plant, literature. There is too much

emphasis on enzyme detection techniques, which are not

that popular with plant immunocytochemists (why include

DAB detection of peroxidase here and not mention the

technique in the enzyme cytochemistry section?) and too

little information on ¯uorescence antibody labelling. The

®rst protocol is for PVA embedding; who in the plant world

uses this technique? There are the standard protocols for

staining wax-embedded material and butyl methyl metha-

crylate sections with enzyme and ¯uorescence detection.

Silver enhancement of gold labels is covered, but linked to a

wax-embedding protocol; why? This technique can be used

on material prepared in many different ways and, contrary

to the text, silver enhancement does not any longer have to

be carried out in the dark. The latter protocol again relies on

the animal literature. There is no reference to the wonderful

work of Tobias Baskin, who adapted the methacrylate tech-

nique for plant material. There is just a cursory mention of

labelling unembedded material, with one protocol on maize

rot tip squashes and not a single reference to any of the

thousands of papers that must have used the so-called

`square protoplast' technique. A delve into the literature

would have uncovered various other interesting and useful

immunotechniques for plants, such cryo-sectioning, or

freeze-shattering for whole tissue staining, or the interest-

ing combination of freeze-substitution and immuno¯uores-

cence, to mention but a few.

In situ hybridization fares a little better than immuno-

cytochemistry but the layout of this section is very strange,

with autoradiography slipped in between in situ on sections

and on whole-mount preparations, with the TUNEL assay

for apoptosis then creeping in before the chapter reverts

Journal of Microscopy, Vol. 197, Pt 3, March 2000, pp. 320±321.

q 2000 The Royal Microscopical Society320

back to ¯uorescence in situ. The book ®nishes with a useful

set of appendices on toxicity of the various reagents, buffer

recipes and list of manufacturers, before ®nishing with a

longish section on optics and lenses.

In conclusion, the idea behind the book is sound and it is

a useful laboratory aid, but it is let down by unnecessary

omissions and errors in the latter half.

CHRIS HAWES

Electron Microscopy Methods and Protocols. Edited by

M. A. Nasser Hajibagheri. Methods in Molecular Biology

Volume 117. Published by Humana Press, Totowa, New

Jersey, 1999. Ringbound. $89.50. ISBN 0-896-03640-5.

284 pages.

This is an ambitious book, covering a large range of

techniques based around electron microscopy, which could

be of interest to many researchers. In the Preface, the editor

indicates that the aim of the book is to appeal to both the

established electron microscopist as well as newcomers to

the ®eld.

Book reviewers are generally not encouraged to give long

lists of chapter headings ± but for a book such as this, a

short list is essential to give an idea of the scope of the

chapters. The ®rst chapter is on basic EM techniques,

followed by negative staining and preparation of frozen

hydrated thin ®lms. There are several chapters on aspects of

immunolabelling, as well as microwave processing, enzyme

cytochemistry, in situ hybridization, preparation of yeast for

EM, preparation of DNA and RNA and protein-DNA

complexes, and ®nally X-ray microanalysis. Altogether the

book contains a total of 15 chapters.

The format of each chapter is a brief Introduction,

followed by Materials and Methods, then Notes and ®nally

References. I confess to ®nding the Notes rather a

distracting format. In some chapters they seemed to contain

much of the meat of the text, and even ®gures and tables.

One chapter has 44 Notes and it is distracting to have to

constantly ¯ick back and forward from text to notes.

This is essentially a practical book. The methods described

appeared free from major pitfalls, although it would be a

rare person who claimed expertise in all the ®elds in this

book. However, a mental `trial run' of the protocols in the

chapters in the less familiar areas seemed to get me vaguely

where I thought I should be. There is obviously a lot about

colloidal gold labelling and a number of chapters describe

various methods for applying the techniques. They cover

the classic Tokuyasu method of preparing thawed cryosec-

tions, embedding in both LR Gold and Lowicryl resins and

high pressure freezing and freeze-substitution. It was good

to see a chapter on quanti®cation of immunogold labelling

alongside the `how-to-do-it' chapters. This is perhaps the

strongest section of the book, but as this is a relatively well-

established ®eld there is certainly nothing startlingly new

here. However, different laboratories will have different

approaches to these techniques and there is always some-

thing new to learn in this area.

The illustrations are a truly mixed bunch. They range

from the good, the good but printed too small, through

barely adequate line drawings to the almost black `guess

where the gold is supposed to be' type. Strangely, one or two

are repeated in colour, with the colloidal gold in pretty

colours, but the underlying morphology is still impene-

trable. The newcomer would also be forgiven for serious

confusion generated by colloidal gold shown as white rather

than the normal black dots on a very dark nucleus, with no

reference to the image manipulation needed to do this. This

is a shame, as the quality of illustrations is so important in

such a book. First-rate illustrations can grab your attention

and stimulate further interest in the technique. Indeed, for

the newcomer it is vital to show the sort of results that can

be achieved with any technique and they may provide help

when deciding which of several apparently similar approaches

should be chosen.

If there is a problem with the book (apart from the

illustrations and enough typographical errors to be notice-

able) it is that it is over-ambitious. Whilst some chapters

cover their ®eld well, there just is not enough room for every

chapter to cover the ground in suf®cient depth. To give just

one example, enzyme cytochemistry is covered in a chapter

of just seven pages. This gives a ¯avour of the technique,

with a couple of nice illustrations, but unless you are

interested in localizing acid phosphatase, you need to look

elsewhere for your methods and protocols. In such

circumstances, the lack of a comprehensive bibliography

to help track down further reading is disappointing.

For the newcomer to the world of modern electron

microscopy, the book covers a lot of ground and introduces

a good range of methodologies gathered together in one

place. Some of these introductions are a little cursory, and

will also fail to ful®l the needs of the more experienced

microscopist. Is it essential reading for all EM labs? Have a

look for yourself before you buy.

PAU L M O NAG H A N

BOOK REVIEWS 321

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