Histology Staining Techniques

Introduction

• Course topic: Histology
• Instructor: Dr. Dalia Eita
• Location: Mansoura National University

Agenda

• Methods for preparing light microscopic sections
• Principles of staining with Hematoxylin & Eosin
• Types of stains
• Types of microscopes

Learning Objectives

• Demonstrate tissue fixation and embedding procedures
• Demonstrate common staining methods, their rationale, and the usefulness of cytochemical and histochemical techniques
• Differentiate between light and electron microscopes

Special stains for organic components: Carbohydrates

• Periodic acid-Schiff (PAS) stains carbohydrates
  • Reacts with carbohydrates to produce free aldehyde groups
  • Combined with Schiff's reagent to produce a magenta color
• Best Carmine stains glycogen
  • Specific stain for glycogen
  • Stains glycogen granules red

Special stains for organic components: Lipids

• Frozen sections are used
• Sudan III stains lipids orange
• Sudan black stains lipids black
• Osmic acid stains lipids black

Other methods of staining

• Vital stains stain living cells (e.g., trypan blue or Indian ink stains phagocytic cells for observing them in living tissue)
• Metachromatic stains stain certain components differently from the dye's color (e.g. granules of mast cells stained reddish purple with toluidine blue)
• Enzyme histochemistry often uses frozen sections to demonstrate enzyme activity (e.g., alkaline phosphatase enzyme in kidney tubules)

Quiz: PAS Stain

• PAS stain for carbohydrates produces a magenta color

Microscopy: Types of Microscopes

• Light microscopes (student microscopes)
  • Magnification: 40x, 100x, 400x, 1000x
• Electron microscopes
  • Magnification: up to 50,000x

Microscopy: Resolution Power

• Resolution power measures the smallest distance between particles that can be distinguished under the microscope.
  • Light microscope resolution power: ~0.2 μm (200 nm)
  • Electron microscope resolution power: ~1 nm
• Magnification of light microscope = Magnification of Objective Lens x Magnification of Ocular Lens

Practical Microscopy Class

• Instructions for using a microscope:
  • Turn the microscope on
  • Place the slide on the stage and secure
  • Rotate the low-power objective
  • Focus the slide using the coarse adjustment knob
  • Switch to medium & high power objectives, adjust using fine adjustment knob only

Micro technique for Electron Microscopy

• Ultrathin sections (50-100 nm): Obtained using an ultramicrotome
• Staining with heavy metals (lead citrate & uranyl acetate)
• Electron micrograph:
  • Dark components = electron-dense
  • Light components = electron-lucent

Scanning Electron Microscope(SEM)

• Allows 3-dimensional visualization of the surface of a fixed specimen

This quiz focuses on histology techniques, particularly the principles and methods of tissue staining, including Hematoxylin & Eosin, PAS, and special stains for carbohydrates and lipids. Test your knowledge on various staining methods and their applications in microscopic analysis. Perfect for students of histology at Mansoura National University.