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Histological techniques for life science researchers

It is the preparation of tissues for microscopic examination.
It is an effective diagnostic tool in clinical pathology.
Histological preparations reveal normal tissue structure, tissue abnormalities and cancerous conditions.

Histological techniques for life science researchers

  1. 1. Presented by Dr. B.Victor., Ph. D.Email : Blog:
  2. 2. Presentation outline Meaning and branches of histotechnology. Major steps in tissue processing Fixative –definition, kinds, characteristics. Micro-anatomical fixatives Principles of tissue fixation. Processing of tissues and tissue sections. Paraffin wax technique Types of microtomes Staining-types, double stains, acid stains, basic stains. Clearing tissue sections.
  3. 3. Meaning of histotechnology It is the preparation of tissues for microscopic examination. It is an effective diagnostic tool in clinical pathology. Histological preparations reveal normal tissue structure, tissue abnormalities and cancerous conditions.
  4. 4. Branches of histotechnology Histology- the microscopic study of the normal tissues. Histopathology – the microscopic study of tissues affected by disease. Histochemistry – the techniques provide information on the chemical composition of parts of tissues. Cytochemistry – the techniques provide information on the chemical composition of parts of cells.
  5. 5. Steps in the processing of tissues1. Fixation – preservation of tissues in its original condition.2. Dehydration – removal of water from tissues.3. Clearing – infiltration of paraffin solvent.4. Embedding – infiltration of paraffin wax.5. Microtomy – preparing thin slices of tissues.6. Staining – colouring of tissues.7. Mounting – arranging tissues on slides.
  6. 6. What is a fixative ? A fixative is described as a chemical substance which will preserve the shape, structure, relationship and chemical constituents of tissues and cells after death.
  7. 7. Purpose of fixing agents1. To kill and preserve living tissues.2. To stabilize the tissue and cell structure for subsequent treatments( wax embedding, sectioning, mounting).3. To prepare tissue for staining and optical contrast.4. To harden the tissue for section cutting
  8. 8. Requirements of a good fixative1. Penetrate the tissue and cells rapidly and evenly.2. Prevent autolysis and bacterial decomposition.3. Preserve tissues in their natural state and fix all chemical cell components ( proteins, carbohydrates, fats etc.,)4. Preserve cell volume.
  9. 9. Requirements of a good fixative(Cont’d)5.Avoid excessive hardness of fixed tissue.6.Allow enhanced optical differentiation by staining.7.Make the cellular components insoluble to liquids used in tissue processing.8.Be nontoxic and non-allergenic9.Providing iso-osmotic conditions to the tissues.
  10. 10. General principles of fixation Amount of fixing fluid should be approx. 10 to 20 times more than the volume of tissue held in a container with a required fixation time. Temperature has an important effect. A lower temperature retard fixation – reduce autolytic reaction. A higher temperature will decrease the required for fixation but will increase autolysis.
  11. 11. Methods of Fixation Fixation by heat - this denatures and coagulates proteins resulting in some distortion but is useful in fixing smears. Cryostat (freezing) fixing - it does not denatures proteins and minimizes distortion. useful in locating particular chemicals - histochemistry Fixation by chemicals - chemical fixatives are used.
  12. 12. Kinds of fixatives FixativesTemperature Chemical fixation fixation Simple fixatives Compound fixatives Micro-anatomical Cytological fixatives fixatives Nuclear fixatives Cytoplasmic fixatives
  13. 13. Common fixatives Routine Special Fixatives fixatives Primary Secondary fixatives fixatives Simple Fixation fixatives mixtures
  14. 14. Simple fixatives or primary fixatives or unmixed fixativesFormalin Ethyl alcohol non-coagulant fixative  Colourless liquid Acidic, cheap,  Reducing agent easy to prepare, Osmium tetroxide relatively stable  Strong oxidizing agent  Expensive, poor penetrationMercuric chloride Potassium dichromate Coagulant fixative,  Strong fixative black precipitate in tissues  Fix lipidsGlacial acetic acid Trichloro acetic acid Protein precipitant,  Protein precipitant Colourless solution Picric acid Pungent smell  Protein precipitant  Used as saturated solutions
  15. 15. Compound fixatives or fixationmixtures Micro-anatomical fixatives Compound Nuclear fixatives fixatives Cytological fixatives Cytoplasmic fixatives
  16. 16. Micro-anatomical fixatives 10% formal saline 10% neutral buffered formalin Heidenhain’s Susa Formal-sublimate Formal-saline sublimate Zenker’s fluid Helly’s fluid Bouin’s fluid Gendre’s fluid
  17. 17. Nuclear fixativesFlemming’s • Chromic acid (1%)-15 ml • Aqueous osmium tetroxide(2%)-4 ml fluid • Glacial acetic acid- 1 mlCarnoy’s • Absolute alcohol- 60 ml • Chloroform – 30 ml fluid • Glacial acetic acid – 10ml
  18. 18. Cytoplasmic fixatives Flemming’s • Chromic acid (1%) – 15 mlFluid without • Aqueous osmium tetroxide (2%)-4 ml Acetic acid • Mercuric chloride – 5 gm • Potassium dichromate -2.5 gmHelly’s fluid • Sodium sulphate – 1.0 gm • Distilled water -100 ml • 5 ml of 40% formalin before use. Formal • formalin Saline 10% • Sodium chloride
  19. 19. Category of fixatives Mercury • Zenker’s fluid fixative Chromate • Helly’s fixative fluid Picric acid • Bouin’s fixative fluid Alcohol • Carnoy’s fluid Fixative
  20. 20. Physico-chemical features offixatives  Degree of ionization  Oxidation-reduction potential  Reactions with proteins, lipids, carbohydrates  Rate of penetration  Shrinkage or swelling  Degree of hardening  Methods of washed out  Effect on staining  Compatibility with other fixatives
  21. 21. Processing of specimens Processing Of tissuesProcessing of specimens Processing of Tissue sections
  22. 22. Processing of animal tissues Fix in appropriate fixative Wash in water / iodine alcohol Partially dehydrate in 30, 50, % grades of alcohol – 30 -90 mts each Store in 70 % or 80% alcohol
  23. 23. Processing of plant tissues Fix in FAA (formalin-acetic acid-alcohol) Wash in 50% alcohol Partially dehydrate in 10, 20, 30, 40, 50 & 60% Alcohol grades – 20 mts each Store in 70% alcohol
  24. 24. Paraffin wax technique oftissue blocks Dehydration-the alcohol method -the acetone method - the dioxane (diethylene dioxide) method Clearing- de-alcoholisation -clearing agents- xylene, benzene, toluene, chloroform. Embedding- blocking – out in wax-wax impregnation
  25. 25. Paraffin wax technique oftissue blocks wax-wax impregnation- It can be cold wax infiltration and melted wax infiltration. Complete wax infiltration is essential for the production of good sections. Hard tissues requires a higher melting point wax. Number wax changes and time in each wax change, depend upon the density and size of the tissue. Embedding media – wax, gelatin, celloidin, polyester wax.
  26. 26. Cutting of tissue sections ormicrotomy Rotary microtome Ordinary Rocking microtome microtome Freezing Sliding microtome Microtome/ microtome cryostat Ultra - microtome
  27. 27. Microtomes The microtomes cut the tissue at a pre- determined uniform thickness. These instruments are designed for the accurate and serial cutting of thin slices of tissue. Several models are available – sliding, rotary, rocking ultra- thin microtomes.
  28. 28. Cambridge rocking microtome  It consists of a heavy base and two arms ; the lower arm rests on a column and supports the upper, both being pivoted on knife edges which acts a fulcrum.  The upper arm carries the block holder.  There is an adjustable cord.  The feed mechanism is graduated in units of 1or 2 um.
  29. 29. The rotary microtome  The section cutting is effected by the vertical rise and fall of the object against an fixed knife edge.  The block holder is equipped with adjustable screws.  The block is parallel to the microtome knife.  The knife holder is movable.
  30. 30. The sliding microtome The block remains stationary, while the microtome knife moves during the process of sectioning.
  31. 31. The freezing microtome  The optimum cutting temperature is -20 degree Celsius.  The freezing of the tissues is done by the carbon dioxide gas.
  32. 32. The cryostat  Sectioning is done on unfixed tissue.  The microtome is housed in a deep freezer cabinet.  The temperature can be maintained between -15 to -30 degree Celsius.
  33. 33. Paraffin processing of tissue Post-fixation Initial fixation treatment dehydration Wax De- Completeinfiltration alcoholization dehydration Wax Trimming the block microtomyembedding
  34. 34. Processing of paraffin sections Hydration Down-grading –Drying of sections De-paraffinization Graded alcohol series Staining Dehydration De-alcoholization -primary staining Up-grading- And clearing -counter-staining Graded alcohol series Mounting Observation In a medium with In a microscope Cover glass
  35. 35. Staining Staining is used to obtain contrast between the constituent parts of a tissue section. The depth of colouration is affected by chemical affinity, density, and permeability. Certain stains are metachromatic –i.e. they are capable of imparting one colour to certain constituents and another to others.
  36. 36. Mordanting The salts of certain metals are capable of radically alter the behavior of particular stains. These salts are called ‘mordants’. A mordant is capable entering into chemical combination with a stain. The resulting substance is called ‘a lake’.
  37. 37. Kinds of staining Progressive staining Retrogressive stainingThe tissue is left in the The tissue is overstain until the desired stained and decolorized depth of color is using differentiating obtained solution Direct staining Counter staining The stain combines Two stains are applied directly with cell one by one with proper structures destaining in-between.
  38. 38. Kinds of staining -2Acidic Basic Neutralstains stains stainsA colored organic A colored organic A colored organic acid combined base combined acid is linked with a metal. with uncolored chemically to a acetate, chloride colored organic or sulphate base. radical. These aredissolved in water or alcohol. They are dissolved They tend to stain in absoluteThey tend to stain nucleus. alcohol. the cytoplasm.
  39. 39. Common double stainsAnimal • Borax carmine and eosin Y • Haematoxylin and eosin Y • Haematoxylin and vantissues Geison stain Plant • Haematoxylin and eosin • Safranin and light greentissues • Safranin and haematoxylin
  40. 40. Good general stain for animal tissuesstain solvent effectPico carmine water Nucleus-red Cytoplasm-yellowBorax carmine alcohol Nucleus-pinkDelafield alcohol Nucleus - bluehaematoxylinHaemalum water Nucleus - blueEosin Y Water or alcohol Cytoplasm - pink
  41. 41. Clearing agent • It makes the processedClearing tissue transparent. agent • Removes alcohol from tissue sections.
  42. 42.  Dr.B.Victor is a highly experienced professor, recently retired from the reputed educational institution- St. Xavier’ s College, Palayamkottai, India-627001. He was the dean of sciences, IQAC coordinator and assistant controller of examinations. He has more than 32 years of teaching and research experience He has taught a diversity of courses and guided 12 Ph.D scholars. He is an expert in histological techniques and photo micrography. send your comments to :
  43. 43. Thank you for watching
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It is the preparation of tissues for microscopic examination. It is an effective diagnostic tool in clinical pathology. Histological preparations reveal normal tissue structure, tissue abnormalities and cancerous conditions.


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