Book Review: Developmental and Structural

Plant Microtechnique and Microscopy. Ruzin, Steven E. 1999. ISBN 0-19-508956-1 (pbk U.S.$45.00). 322 pp. Oxford University Press.198 Madison Avenue, New York, New York, 10016. - Interest in plant microtechnique and microscopy is seeing a resurgence in recent years. In part this is due to innovations in instrumentation but it is primarily the result of the wedding of microtechnique and microscopy to molecular techniques thus permitting visualization of genes or gene products in situ. This manual is a needed replacement for Berlyn and Miksche's Botanical Microtechnique and Cytochemistry (1976) and O'Brien and McCully's The Study of Plant Structure. Principles and Selected Methods (1981).

The general organization is quite traditional with the exceptions of chapters one and 10. The opening chapter, Quick Start, provides abbreviated protocols for eight frequently used techniques. In addition to traditional paraffin and plastic embedding schedules, it includes modified techniques employing the microwave oven to speed processing. Of course it also contains protocols for immunolocalization and in situ hybridization. The final quick start procedure is adjusting for Kšhler illumination to optimize performance of the light microscope. These abbreviated procedures are meant primarily for knowledgeable practitioners, but each step of the schedule is cross referenced to the appropriate section and chapter of the main text. Chapter 10 collects in one place the problem solving tips and notes mentioned in previous chapters, as well as additional suggestions. For instance he cautions against simply uprooting specimens if you are interested in studying root material, because the vascular cylinder easily can be separated from the cortex. Another example is that Toluidine Blue O stained tissue embedded in methacrylate will be destained if Permount or Plastic UV Mount (Polysciences) is used to mount the cover glass.

Chapter two deals with optical microscopy and along with Appendix VI, Optics, provides a comprehensive introduction to the theory and practice of microscopy. It begins with a brief history of the compound microscope and proceeds through modern, infinity corrected systems providing the basic theory and application of common, and some uncommon, types of light microscopy. For each example, the optical principles are clearly explained, set-up procedures are presented, step-by-step, and examples are provided to illustrate suitable materials for each technique. I was particularly pleased to see an explanation and set-up procedure for Rheinberg illumination. This is an inexpensive technique no longer used by researchers but one that should be much more widely used in teaching laboratories. Cellophane filters are cut to match the numerical aperture of the lens (the procedure for doing this is explained) and when placed correctly will result in 'optical staining' with the background one color and different shades of reflected light of contrasting color illuminating the specimen. As an added advantage, if phase contrast objectives are used, even without the phase condenser, a multicolored 'phase contrast' will be achieved!

Subsequent chapters cover the typical sequence of specimen preparation: chemical fixation; tissue dehydration; infiltrating and embedding tissue; sectioning and mounting; and staining. Like the rest of the book, the text is clearly and well-written and the coverage is complete and precise. For instance, in formulations there is no question of whether measurements are (v/v) or (w/v); it is specifically stated. Numerous citations to recent primary literature are presented throughout which will be of great benefit to the researcher fine-tuning his or her technique to a particular research organism or problem. These chapters are filled with details and advice from an experienced practitioner. He uses numerous tables to present alternative reagents, stains and dyes and summarize their advantages and uses. A delightful surprise in these chapters is the interjection of the authors personality. For instance, the last recommendation for fixing plant tissues is an emergency fixative for field collections: 'Just about any strong alcoholic beverage: gin, vodka, even wine will preserve field collected material (Carlquist, 1996).' In the chapter on infiltrating and embedding he presents illustrated directions for the 'microtechnique origami method' of constructing paper boats for paraffin embedding. Under trouble-shooting, in the sectioning and mounting section, he gives sage advice developed, no doubt, through long experience - - 'When all else fails, section some other day.' Finally, to end the chapter on staining, Ruzin presents a table containing directions for removing various of the common stains from hands and clothing. He ends with a caveat: '...in some traditional circles, stained hands are thought to be a sign of microtechnique prowess.'

Chapters eight and nine deal with alternative methods of microtomy and some special methods. At one time several of these methods were mainstays of plant microtechnique, but they had fallen out of favor. Today, they are seeing renewed application so it is fortunate to have them 'revived' in this text. The technically simplest, but often most difficult to successfully apply, method of microtomy is free-hand sectioning. Although Ruzin suggests that double-edge teflon-coated blades are best, I still prefer the old Gillette Blue Blades - - if they can be found. The key , as Ruzin notes, is to avoid the industrial single edge blades which are simply not very sharp. This chapter also covers operation of the Vibratome on fresh or agarose-embedded specimens and use of the freezing microtome, or Cryostat, on quick-frozen material. Although both of the latter have had regular use in studies of living materials or in cytohistochemical techniques during the past few decades, the flowering of molecular biology has resulted in renewed interest in their application. The final instrument is the sliding, or sledge, microtome which remains unrivaled for wood studies, either whole mount sections or plastic embedded. The special methods chapter concentrates on clearing and maceration techniques. It includes the best of the traditional techniques, such as clearing with NaOH and chloral hydrate, as well contemporary variations of Herr's (1982) 4-1/2 technique. Similarly, recent modifications of traditional macerating techniques are described for both woody and non-woody tissues. Two interesting methods are saved for last - preserving color in whole mounted specimens and repairing broken microscope slides. Again, these will be of little use to most researchers, but they may be invaluable to botany teachers.

Chapter 11 concentrates on histochemistry and cytochemistry. Again, Ruzin presents both the best of the traditional techniques as well as more recent innovations. A very useful table of 15 vital dyes is presented early in the chapter. This lists not only the working concentrations and specific targets, but lists one or more primary references. All the usual cytochemical reactions are presented: Aniline Blue for callose, Periodic acid-Schiff for carbohydrate, Phloroglucinol for lignin, etc. The section on cytochemical localization of enzymes includes standard protocols for acid phosphatase, alcohol dehydrogenase, alkaline phosphatase, cytochrome oxidase, catalyase, glucuronidase, lipases, pectinase and peroxidases. The second half of the chapter deals specifically with fluorescence techniques. Although not mentioned in the microscopy chapter, the principles of fluorescence microscopy are adequately covered in the Optics appendix. An extensive table of fluorescence dyes and their cellular targets are presented along with a number of specific protocols. Again, the numerous citations of primary literature will be invaluable to researchers fine-tuning a schedule to their material. Indeed, throughout the book Ruzin encourages the reader to take the written procedures as a starting point, but to modify them as necessary for the material.

While the entire book will be an excellent text for new students learning microtechnique from scratch, the last chapter, Localization of Molecular Targets in Tissues, will be the most useful for my generation of researcher. We received a solid foundation in the traditional techniques but established our research programs before molecular techniques took hold. Here is a concise road map to get us back on track and up-to-speed. The chapter begins with several pages of introduction to the principles of immunolocalization followed by nine specific protocols illustrating different applications. As usual, a section of notes and practical tips follow the formal schedules.

Unfortunately there is a major deficiency in the text which will significantly reduce its usefulness. While great care is taken to provide the details necessary to produce quality specimens and obtain optimal microscopic images, there is nothing on image capture. Traditionally this would include only photomicrography on film media, but even this is lacking. With the choices available today, film, analog, and digital cameras, and the software available for enhancement and manipulation, there is an urgent need to compare these tools, note their strengths and weaknesses, and provide succinct descriptions of their optimal use. Perhaps this is the topic of a follow-up volume that is hopefully in preparation. —Marshall Sundberg, Emporia State University

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