Microscope preparation

• جلا ةيبونجلا ةينقتلا ةعما


  Technical Institute - Basra
• ةيبطلا تاربتخملا تاينقت مسق


  Department of Medical Laboratory Techniques
• Microscopic Slides / practical
• Lecturer


  Jassem M. Abd Ali
• Methods


  • P.W Method
  • Freezing method
  • Celloidin Method
  • Electron Microscope Method
• Paraffin wax methods


  1. Fixation

  2. Washing

  3. Dehydration

  4. Clearing

  5. Infiltration

  6. Embedding

  7. Cutting

  8. Staining

  9. Mounting & Covering
• Staining


  To stain tissue section with stain to easily examination by using Hematoxylin and eosin (H&E) are most commonly used stains in histology and histopathology. Hematoxylin stains nucleus with blue color, while eosin stains the cytoplasm with pink color.

  
• Staining methods


  • Vital staining
  • Routine staining
  • Special staining

  
• Special staining types


  • Schmoral stain
  • Verhoeff stain
  • PAS: Periodic acid Schif
  • Feulgen
  • Sudan III
  • Best carmine stain
• Staining reactions


  • Direct staining
  • Indirect staining
  • Regressive stain
  • Progressive stain
• Mordant


  Substance which combine with tissue and stain, liking the two and causing reaction between them
• Dye classification by reference


  • Natural dyes
  • Synthetic dyes
• Dye classification by reaction


  • Basic dye
  • Acidic stain
  • Neutral dyes
• Decolorization


  Removal is stain or excess stain from tissue section
• Differentiation


  Removal of excess stain from tissue until color is retained only by the elements to be studies
• Counter stain


  Used one or more additional staining to show the other components of the tissue

  
• Difficulties in staining


  • Staining solution may have deteriorated
  • Staining solution not be ripe
  • Decalcifying solution or the fixation may not have been washed out
• Mounting and covering


  1. Mounting of section

  2. Cover slipping
• Properties of mounting medium


  • Permeates the tissue space
  • Dissolved in a solvent that will be miscible with clearing agents
  • Will have a suitable index of refrection(1.52)
• Type of cover slips


  • Circles
  • Squares
  • Oblongs
• Harris Hematoxyline stain


  HX powder, Absolute alcohol, Aluminum ammonium sulphate, Distilled water, Mercuric oxide
• Eosin stain


  0.5% watery, 0.5% alcoholic
• Differentiation solution


  1% acid alcohol (100 ml): HCL concentration 1 ml, 70% Ethanol 99ml
• DPX


  Synthetic mounting medium used in mounting & covering, but not commonly used in lab. Consists of Distrene, Dibutyl phathalate, Xylene
• Procedure of staining


  1. De-waxing

  2. Hydration

  3. Staining nucleus with Hematoxyline

  4. Staining cytoplasm with Eosin

  5. Dehydration

  6. De-alcoholization

  7. Mounting & covering

  
• Paraffin wax method processes


  • Fixation
  • Washing
  • Dehydration
  • Clearing
  • Infiltration
  • Embedding
  • Trimming
  • Cutting
  • Floating
  • Mounting
  • Drying
  • Staining and examination
• Freezing method


  • Advantage: Diagnosis in a few minutes, good for fatty/lipoid materials, good for central nervous system and enzymes, water in tissue gives support

  Disadvantage: Almost impossible to obtain serial section, structure details tend to be distorted
• Differences between Paraffin wax method and Freezing method


  • Supporting media
  • Ribbon/section
  • Fixed/unfixed tissue
  • Microtome used
  • Section thickness
• Frozen section thickness


  10 - 15 micron
• Mounting media


  1. Gelatin 15g

  2. Distilled water 100ml

  3. Glycerin 100ml
• Disadvantages of freezing method


  • Almost impossible to obtain serial sections
  • Lack of embedding mass causes distortion of structural details
  • Staining of unfixed tissue rarely as satisfactory as fixed tissue
• Differences between paraffin wax and freezing methods


  • Supporting media
  • Continuous ribbon
  • Tissue fixation
  • Microtome used
  • Section thickness
  • Mounting media
• Decalcification


  1. Tissue fixed and dehydrated before decalcification

  2. Decalcifying fluid removes calcium salts

  3. Decalcification should not disrupt tissue structure or affect staining
• Decalcifying fluids


  • Nitric acid
  • Nitric acid formalin
  • Formic acid
  • Trichloroacetic acid
• Decalcification


  • Temperature below 10C slows process, 40-60C speeds it up but can damage tissue
  • Best temperature is 10-30C
• Bone tissue preparation


  1. Paraffin wax after decalcification

  2. Freezing after decalcification
• Decalcification technique


  1. Suspend tissue in 50-100x volume of 5% nitric acid, change solution once-twice daily

  2. Test progress of decalcification regularly

  3. Transfer to 70% alcohol when complete to wash out acid and start dehydration
• Bone tissue preparation procedure


  1. Fixation in 10% formalin

  2. Washing

  3. Cutting to 4-5mm size

  4. Decalcification

  5. Dehydration

  6. Clearing

  7. Paraffin wax infiltration

  8. Blocking

  9. Cutting and mounting

  10. Staining (Hematoxylin-eosin, Schmoral, Giemsa)
• Schmoral stain is the best stain for bone tissue
• Electron microscope


  Instrument used to magnify small objects, with magnifications 10-500x higher than light microscope