HP Q and A
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HP Q and A

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Questions and Answers

Which of the following fixatives does not possess the property of becoming part of the tissue?

  • Formalin (correct)
  • Ethanol
  • Mercuric chloride
  • Osmium tetroxide

    • Best results in fixation are obtained through what type of solutions?

  • Hypertonic
  • Isotonic
  • Slightly hypertonic (correct)
  • Slightly hypotonic

    • Which of the following is added to osmium tetroxide fixatives for electron microscopy?

  • Glucose
  • Levulose
  • Sucrose (correct)
  • Fructose

    • The temperature utilized in Autotechnicon is?

    <p>0-4°C</p> Signup and view all the answers

    The following are not characteristics of a good fixative, except:

    <p>It should enter the tissue slowly for even penetration</p> Signup and view all the answers

    For fixation of tissues with tuberculosis, formalin must be heated at what temperature?

    <p>60°C</p> Signup and view all the answers

    What is the maximum effective concentration of a fixative?

    <p>20x the volume of the tissue to be fixed</p> Signup and view all the answers

    What is the ideal number of hours for fixation?

    <p>24 hours</p> Signup and view all the answers

    Formaldehyde is commonly used in routine fixation at a concentration of ________.

    <p>10%</p> Signup and view all the answers

    Which of the following is a characteristic of a good fixative?

    <p>It should preserve the chemical constituents of cells</p> Signup and view all the answers

    Which of these options are correct? (Select all that apply)

    <p>Calcium Acetate Formalin</p> Signup and view all the answers

    Which fixative has the most stable effect in tissues?

    <p>10% Formol-Saline</p> Signup and view all the answers

    What is one advantage of using 10% Formol-Saline?

    <p>Preserves plasma proteins better.</p> Signup and view all the answers

    Which method is the most ideal and reliable for decalcification?

    <p>X-ray method</p> Signup and view all the answers

    What is a disadvantage of using Formol Corrosive?

    <p>Forms mercuric/black deposits.</p> Signup and view all the answers

    10% Formol-Saline is excellent for preserving lipids.

    <p>False</p> Signup and view all the answers

    Chemical method is not recommended for routine purposes.

    <p>False</p> Signup and view all the answers

    How long should tissues typically be fixed in 10% Formol-Saline?

    <p>24 hours.</p> Signup and view all the answers

    What is the purpose of adding 0.1% urea to nitric acid during decalcification?

    <p>To prevent yellow color formation.</p> Signup and view all the answers

    The formula for Gendre’s fixative includes 95% ethyl alcohol saturated with _______.

    <p>picric acid.</p> Signup and view all the answers

    The decalcification time for Phloroglucin Nitric Acid is _____ hours.

    <p>12-24</p> Signup and view all the answers

    Match the following fixatives with their primary use:

    <p>10% Formol-Saline = Pathology and histochemical examination Mercuric Chloride = Preservation of cell detail in tissue photography Alcoholic Formalin = Cytologic examinations Glutaraldehyde = In electron microscopy</p> Signup and view all the answers

    What is the recommended decalcification time for Perenyi’s fluid?

    <p>1-3 days</p> Signup and view all the answers

    What is the primary purpose of Helly's solution?

    <p>To fix tissues in pituitary gland, bone marrow and blood-containing organs.</p> Signup and view all the answers

    What should be used inside a fume hood during decalcification?

    <p>Nitric acid</p> Signup and view all the answers

    Alcoholic formalin is ideal for preserving iron-containing pigments.

    <p>False</p> Signup and view all the answers

    The precipitate formed during the Oxalate test indicates the presence of _____ in acid solutions.

    <p>calcium</p> Signup and view all the answers

    Which fixative is recommended for renal tissues?

    <p>10% Neutral Buffered Formalin</p> Signup and view all the answers

    Which of the following is a commonly used dehydrating agent?

    <p>Alcohol</p> Signup and view all the answers

    What is the effect of transferring tissue directly from formalin to higher grades of alcohol?

    <p>It could lead to distortion of tissues.</p> Signup and view all the answers

    What should be done to remove black deposits caused by mercuric chloride?

    <p>Use 0.5% iodine in 70% ethanol followed by sodium thiosulfate.</p> Signup and view all the answers

    The concentration of formaldehyde used in a fixative is approximately?

    <p>20%</p> Signup and view all the answers

    What is one common use for chromate fixatives?

    <p>Preservation of Chromaffin tissues.</p> Signup and view all the answers

    What is the first and most critical step in histotechnology?

    <p>Fixation</p> Signup and view all the answers

    What is the purpose of decalcification?

    <p>To remove calcium or lime salts from calcified tissues.</p> Signup and view all the answers

    What is the primary aim of fixation?

    <p>To preserve the morphologic and chemical integrity of the cell as life-like as possible.</p> Signup and view all the answers

    Dehydration involves adding fluid to tissues.

    <p>False</p> Signup and view all the answers

    Which of the following processes is NOT part of fresh tissue examination?

    <p>Fixation</p> Signup and view all the answers

    Which is the correct sequence of steps in processing preserved tissues?

    <p>Fixation → Dehydration → Clearing → Infiltration</p> Signup and view all the answers

    Leaving a tissue specimen in air can cause it to ______.

    <p>dry-out</p> Signup and view all the answers

    What happens to cells when they are placed in a hypotonic solution?

    <p>Cells swell and can burst</p> Signup and view all the answers

    Match the following post-mortem changes with their descriptions:

    <p>Algor mortis = Cooling of the body Rigor mortis = Stiffening of the skeletal muscles Livor mortis = Post-mortem lividity or discoloration Autolysis = Tissue destruction by enzymes</p> Signup and view all the answers

    What is a disadvantage of using fresh tissue examination?

    <p>Tissues are not permanent and can change</p> Signup and view all the answers

    What is the ideal size of tissue to be fixed?

    <p>Not more than 2cm² in diameter and 4mm thick.</p> Signup and view all the answers

    The two most commonly used fixatives for general use are __ and __.

    <p>10% Formol Saline, Zenker's Fluid</p> Signup and view all the answers

    Which of these is an excellent microanatomic fixative for the pituitary gland, bone marrow, and blood-containing organs? (Select all that apply)

    <p>Lillie's alcoholic lead nitrate formalin</p> Signup and view all the answers

    What is the chemical name for general picric acid fixatives?

    <p>2,4,6-trinitrophenol</p> Signup and view all the answers

    Picric acid is highly explosive when dry.

    <p>True</p> Signup and view all the answers

    For how long should fixation with Bouin's solution be done?

    <p>6-24 hours</p> Signup and view all the answers

    Formol corrosive is optimal for fixing kidney structures.

    <p>False</p> Signup and view all the answers

    Which fixative is known to destroy cytoplasmic structures such as mitochondria?

    <p>Osmium Tetroxide</p> Signup and view all the answers

    What is the primary advantage of using alcohol fixatives?

    <p>May be used both as a fixative and dehydrating agent</p> Signup and view all the answers

    Which fixative is recommended for fixing chromosomes, lymph glands, and urgent biopsies?

    <p>Carnoy's Fluid</p> Signup and view all the answers

    Formol calcium remedied by using __.

    <p>Gendre's</p> Signup and view all the answers

    What is the effect of prolonged exposure to acid vapors when using Osmium Tetroxide?

    <p>Causes eye irritation or black osmic oxide deposition in the cornea</p> Signup and view all the answers

    Match the following fixatives with their notable characteristics:

    <p>Gendre’s = Best for preserving connective tissue mucin Zenker's Fluid = Recommended for fixing nucleoproteins Bouin's Solution = Fixation of embryos and delicate structures Alcoholic Picroformol = Excellent for glycogen preservation</p> Signup and view all the answers

    Alcoholic fixatives are contraindicated when lipids are to be studied.

    <p>True</p> Signup and view all the answers

    Which of the following fixatives is recommended for rabies diagnosis?

    <p>Picric acid</p> Signup and view all the answers

    Which of the following fixatives is recommended for demonstrations of mitochondria, chromatin, Golgi bodies, and mitotic figures?

    <p>Regaud’s</p> Signup and view all the answers

    Which of the following fixatives is NOT recommended for the demonstration of cytoplasmic structures such as mitochondria?

    <p>Flemming’s without acetic acid</p> Signup and view all the answers

    Which of the following is used to remove excess Osmium tetroxide black precipitate?

    <p>Flemming’s w/o acetic acid</p> Signup and view all the answers

    Picric acid fixative is known to be affected by what factor?

    <p>Large and thick tissues</p> Signup and view all the answers

    Which of the following fixatives is most rapid and specially recommended for fixation of lymph glands?

    <p>Newcomer’s</p> Signup and view all the answers

    Which of the following decalcifying agents is recommended for urgent biopsies?

    <p>Formol-nitric acid</p> Signup and view all the answers

    Which of the following statements regarding hydrochloric acid is false?

    <p>It is good for nuclear staining.</p> Signup and view all the answers

    What is the ideal time required for decalcifying tissue?

    <p>24-48 hours</p> Signup and view all the answers

    What is the most common chelating agent used to facilitate decalcification?

    <p>EDTA</p> Signup and view all the answers

    Study Notes

    Histopathology & General Pathology Overview

    • Histopathology focuses on the preparation of tissue specimens for microscopic examination.

    Methods of Fresh Tissue Examination

    • Fresh tissue specimens are immersed in isotonic solutions for dissection and microscopic analysis.
    • Various techniques include:
      • Teasing: Dissecting tissue to examine under microscopes.
      • Squash preparation: Forcing compressed tissue between slides for surface viewing.
      • Crushing: Vital dyes placed at the slide interface for absorption.

    Fresh Tissue Examination Advantages and Disadvantages

    • Advantages: Examines living tissues allowing observation of cellular activities (mitosis, motion).
    • Disadvantages: Fresh tissues are non-permanent and undergo changes post-mortem.

    Primary and Secondary Signs of Death

    • Primary signs include CNS, respiratory, and cardiovascular failure.
    • Secondary signs occur post-somatism, including:
      • Algor mortis: Cooling at 1-1.5°C/hr.
      • Rigor mortis: Stiffening of muscles.
      • Livor mortis: Post-mortem low visibility.

    Post-Mortem Changes

    • Autolysis involves cell breakdown by enzymes leading to liquefaction.
    • Putrefaction involves microbial decomposition, producing odors.

    Fixation in Histotechnology

    • Definition: Preservation of tissue structure and chemical integrity.
    • Importance: First critical step in histotechnique; stabilizes proteins for analysis.
    • Fixatives aim to: Preserve as lifelike as possible, harden tissues for cutting, and inhibit decomposition.

    Characteristics of a Good Fixative

    • Cheap, stable, safe to handle, and effective in preventing cellular autolysis.
    • Ideally should quickly kill cells, minimize shrinkage, and allow thorough penetration in tissue.

    Main Factors in Fixation

    • pH: Ideal range is 6.0-8.0 for effective fixing.
    • Temperature:
      • Usual: Room temperature (18-30°C).
      • Higher temperatures can accelerate fixation for urgent samples.
    • Thickness:
      • For electron microscopy: 1-2 mm2.
      • For light microscopy: ≤0.4 cm (4mm).

    Steps in Processing Preserved Tissues

    • The standard workflow includes fixation, dehydration, decalcification (if needed), clearing, infiltration, and embedding.

    Fixation Mechanisms

    • Additive: The fixative becomes part of the tissue (e.g., formaldehyde).
    • Non-additive: Alters tissue composition without becoming part of it (e.g., alcoholic fixatives).

    Ideal Size and Duration for Fixation

    • Maximum size: 2 cm² and 4 mm thick.
    • Ideal fixation duration: 4-6 hours, extending to 6-18 hours for some tissues.

    Summary Review Points

    • The primary aim of fixation is to facilitate easy cutting and to maintain the tissue's physical and chemical characteristics.
    • Best results in fixation are achieved with additive mechanisms that incorporate the chemical into the tissue’s structure.### Fixation Principles
    • Optimal results from slightly hypertonic solutions (400-450 mOsm).
    • Hypertonic solutions cause cell shrinkage, while isotonic (340 mOsm) and hypotonic solutions lead to swelling and suboptimal fixation.
    • Added sucrose to osmium tetroxide fixatives improves electron microscopy outcomes.

    Fixative Concentrations and Duration

    • Formaldehyde fixation typically at 10%, while glutaraldehyde is used at 3%.
    • Most formalin fixatives require up to 24 hours; buffered formalin can range from 2 to 6 hours or up to 1 week.
    • Electron microscopy fixation ideally lasts 3 hours, with prolonged fixation risking tissue shrinkage and hardening.

    Speed and Autolysis Prevention

    • Immediate fixation of specimens post-removal prevents autolysis and decomposition.
    • Fixation can inhibit microbial growth and neutralize drugs.

    Penetration and Volume

    • Formaldehyde penetrates tissue at approximately 1mm/hr.
    • Traditional fixative volume should be 10-25 times the tissue volume, with a maximum effective concentration of 20 times.
    • Osmium tetroxide needs only 5-10 times due to cost.

    Characteristics of Good Fixatives

    • A good fixative gradually kills cells, makes cellular components soluble, enters tissues slowly for even penetration, should be stable, and not excessive.

    Types and Classification of Fixatives

    • Simple Fixatives: Composed of a single component, including aldehydes (formaldehyde and glutaraldehyde), metallic fixatives (mercuric chloride and chromic acid), and alcohol.
    • Compound Fixatives: Mixture of two or more substances for optimal results.

    Routine and Specific Fixatives

    • Microanatomical Fixatives: Allow the study of tissue structures, including 10% neutral buffered formalin.
    • Histochemical Fixatives: Preserve specific cell parts, such as nuclear structures (e.g., Helly's, Orth's).
    • Cytological Fixatives: Typically contain acetic acid for preserving nuclear structures.

    Removal of Formalin Pigments

    • Methods for removing pigments include Lillie’s, Kardasewitsch’s, and the use of saturated alcoholic picric acid.

    Advantages and Disadvantages of Fixatives

    • Formaldehyde: Best for CNS tissues; stable with reduced shrinkage; suitable for histochemical analysis.
    • Glutaraldehyde: Good protein cross-linking but leads to more stable tissues; used for light microscopy.
    • Metallic Fixatives: Such as mercuric chloride, ideal for cell detail but can produce black precipitates.

    Practical Considerations

    • Autopsy and surgical specimens should be fixed quickly to limit decomposition.
    • Surgical tissues should be sterilized before fixation to prevent surface drying.
    • Hollow organs require specific packing with fixative-soaked materials.

    Additional Notes on Fixation

    • Glycogen-containing tissues should not use water due to solubility in aqueous solutions.
    • Prolonged fixation may lead to tissue shrinkage or changes in cellular structure.### Acidic Solutions and Fixatives
    • Glacial acetic acid is an acidic solution with a pH of 7.
    • Disadvantages include time-consuming preparation and corrosiveness to metals, resulting in black granular deposits.
    • Fixation time varies between 4-24 hours, reducing PAS positivity for mucin and diminishing myelin reactivity in some staining methods.
    • Glacial acetic acid is inert to neutral fats and phospholipids.

    Removal of Black Deposits from Mercuric Chloride

    • Use 0.5% iodine in 70% ethanol for 5-10 minutes followed by water, then 5% sodium thiosulfate to decolorize.
    • Alcoholic iodine is utilized for excess mercury removal, with decolorizing through alcohol in subsequent stages.

    Formol and Mercuric Chloride Fixatives

    • Formol is recommended for routine post-mortem tissues, excelling in silver reticulin methods and fixing lipids.
    • Disadvantages of Formol include tissue thickness restriction (maximum of 1 cm) and the formation of mercuric black deposits.
    • Non-frozen sections and impaired determination of tissue decalcification highlight the limitations of mercuric chloride fixation.

    Alcoholic Formalin and Gendre's Fixative

    • Gendre’s fixative comprises 95% ethyl alcohol saturated with picric acid and enhances immunoperoxidase studies.
    • It provides dual fixation and dehydration, making it effective for small tissue samples like liver and spleen.
    • Disadvantages include gross tissue hardening and poor preservation of iron pigments.

    Special Formalin Fixatives

    • Cajol’s formol ammonium bromide is excellent for nervous tissue and recommended for Trichrome staining, while Baker's formol calcium is used for lipid preservation.
    • Helly's solution serves as an excellent microanatomic fixative for pituitary gland and blood-containing organs.
    • Heidenhain's Susa is ideal for tumor biopsies with minimal tissue shrinkage.

    The Effects of Mercuric Chloride Fixatives

    • Mercuric chloride fixatives are known to produce black precipitates; recommended for tumor biopsies.
    • Each fixative has differing types of tissue compatibility, such as Zenker’s for blood-containing organs and Helly's for nervous tissue.

    Chromate Fixatives

    • Introduced among the most potent oxidizing agents used for precipitating proteins while preserving carbohydrates.
    • 10% BNF is widely used, with mercuric chloride serving as a nuclear fixative.

    Picric Acid Fixatives

    • Strong picric acid solutions effectively demonstrate glycogen and are recommended for delicate structures and biopsies.
    • The yellow dye characteristic prevents overlooked tissue, while formaldehyde can stabilize some fixatives for longer periods.
    • Bouin's solution specifically preserves soft tissues with minimal distortion.

    Osmium Tetroxide

    • Osmium provides excellent fixation for electron microscopy, allowing ultrathin sectioning.
    • It is also a yellow-stained solution, which aids in fragmentary biopsies but can inhibit hematoxylin, complicating counterstaining.

    These notes contain key details about different fixatives, their advantages, disadvantages, and specific uses with a focus on their role in histological preparation and staining techniques.

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