Insect Histology

Practical Laboratory Techniques

Pedro Barbosa, Deborah Berry, Christina K. Kary

• Publisher: Wiley-Blackwell
• Year: 2014
• Print ISBN:
  9781444336962
  9781444336955
• Edition: 1
• eBook ISBN:
  9781118875001
• Topic: Biological Sciences
• Subtopic: Entomology
• Index: Biological Sciences

This helpful guide is self-contained, holding all of the information you will need for the preparation of histological slides of all types of insect tissues. It explains the essential procedures for their preparation, providing step-by-step information on each phase, for example, on fixation, dehydrating, clearing, embedding, and staining.

The laboratory manual explains the cutting-edge methodology carried out today, and provides a thorough collection of both historically established and modern insect histology techniques side-by-side. It is the first book to present these various techniques appropriate to insects.

Key issues covered include:

• the use of genetic markers in insect histology;
• important approaches for the preparation of tissues and organs for SEM and TEM Fluorescence;
• new immunohistochemical techniques;
• histological problems encountered in insect tissues such as sclerotized chitin, yolk-laden eggs, chromosomes, genitalia, etc., and how to deal with them.

Photographs show the likely results of procedures, components of the process, and advantages of the use of particular approaches or compounds. As a comprehensive resource, this book is for students and researchers in entomology, systematics, developmental biology, insect cell biology, and morphology, wanting a clear introduction to the procedures for the histological display of insect tissues.

Chapter 1
Problems of sclerotized chitin: Softening insect cuticle

1.1 Introduction

The softening and processing of heavily sclerotized specimens for subsequent histological preparations is one of the major problems in insect histology. Many approaches to the solution of this problem have been suggested. Attempts to soften and otherwise alter sections with sclerotized chitin have been incorporated at every procedural level of histological methods. Suggestions have been made for changes in fixation, clearing, mounting, and embedding. Others have also attempted prefixation, postfixation, premounting, presectioning, and so on, as additional steps geared towards improving the quality of sections.

Aside from the more detailed procedures and specific compounds that are recommended in the following pages there are other simple general methods recommended. These techniques represent basic procedures that have been used independently or in conjunction with other methods. One of the most widely used procedures is the treatment of insect specimens with sodium or potassium hydroxide. These chemicals soften sclerotized portions of specimens and dissolve the soft internal tissues. They are generally used either cold or warm at a 10% concentration. These substances are also frequently used in the preparation of insect specimens for taxonomic study.

The use of hypochlorite of soda is another alternate for softening chitin. It is suggested for the preparation of all stages, that is, larvae, pupae, and adults. The insect is usually placed in boiling hypochlorite of soda (about 25% in distilled water). It is usually left in the solution for about 24 hours or more. A third, widely used approach is the use of tenerals or newly moulted specimens. In this way, the specimens are used before the cuticle has hardened.

The elimination of certain chemical agents which tend to harden insect tissues can also be helpful. Occasionally, it is best merely to avoid long exposures to hardening compounds. For example, to avoid excess hardening, short exposures or avoidance of the higher concentrations of ethanol will aid in preventing its hardening effects. The use of n-butyl or t-butyl alcohol as a substitute dehydrating agent may avoid the hardening of tissues. Similarly, prolonged exposure to certain chemicals or fixatives containing chemicals such as acidified dichromate, mercuric chloride or chromic acid is not recommended. Prolonge...

Table of contents

• Cover
• Title Page
• Copyright
• Preface
• Acknowledgements
• Introduction
• About the companion website
• Chapter 1: Problems of sclerotized chitin: Softening insect cuticle
• Chapter 2: Fixation
• Chapter 3: Dehydrating, clearing, and embedding
• Chapter 4: Staining
• Chapter 5: Immunohistochemical techniques
• Chapter 6: Use of genetic markers in insect histology
• Chapter 7: Fluorescence
• Chapter 8: Mounting
• Chapter 9: Preparation of whole mounts
• Chapter 10: Preparation of whole mounts for staining
• Chapter 11: Preparation of genitalia, mouthparts and other body parts
• Chapter 12: Preparation of chromosomes
• Chapter 13: Preparation of other specific insect organs and tissues
• Appendix: Dissecting fluids and saline solutions
• Index
• End User License Agreement