Histotechnology: Routine Staining Part 1

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Questions and Answers

What are Autochrome Groups?

Groups responsible for tissue to bind firmly to a given dye

What is the significance of Amino Group in dyes?

Anionic auxochrome

Non-significant group

Cationic auxochrome (correct)

Neutral chromophore

Chromogen consists of a chromophore + ________.

auxochrome

Match the following dye classifications with their examples:

Natural Dyes = Hematoxylin, Cochineal, Saffron Fluorescent Dyes = Aniline dyes, Toluidine Blue, Azure-eosinate Synthetic/Artificial Dyes = Coal Tar Dyes, Acridine orange Signup and view all the answers

What is the purpose of staining in histology?

To outline tissue and cellular components Signup and view all the answers

In progressive staining, tissues are overstained and excess dye is selectively removed until the desired intensity is attained through ______ staining.

regressive Signup and view all the answers

What is the purpose of regressive staining in tissue staining?

Selective removal of excess stain from the tissue during staining process. Signup and view all the answers

What are examples of acid differentiators used in staining methods?

Both a and b Signup and view all the answers

Nuclei are stained with basic dye (blue) to exhibit __________.

the nucleus Signup and view all the answers

Chromophores are the group on the Benzene ring which alters the light resonance properties of the compound.

True Signup and view all the answers

What is the staining mechanism primarily based on in histological techniques?

Alteration of contrast and color Signup and view all the answers

Meta-chromatic staining is when certain dyes exhibit a color similar to the stain itself.

False Signup and view all the answers

Match the following staining categories with their actions in tissues:

Acidic dyes = Stains cytoplasm, acidophil granules, etc. Basic dyes = Stains nucleus, basophil granules, etc. Romanowsky dyes = Consist of mixtures of basic and acidic dyes Signup and view all the answers

What is the intermediary between dye and tissue that increases the affinity between them?

Polyvalent metal ion Signup and view all the answers

Which of the following should NOT be used to indicate any substance that improves in staining like actions of accentuators and accelerators?

Mordant Signup and view all the answers

What type of staining reaction involves tissue being penetrated by dye solution?

Direct Staining Signup and view all the answers

Match the staining agents with their examples:

Accentuator = KOH in Leoffler’s Methylene blue Accelerator = Chloral Hydrate Agents = Tannic acid Signup and view all the answers

Study Notes

Routine Staining Part 1

Introduction to Staining

Begins with the complete removal of paraffin in fixed sections of slides
Dewaxing starts with heating the slides in 60°C for at least 30 minutes to soften the wax
Slides are then immersed in a couple of changes of xylene, followed by descending concentrations of alcohol to distilled water prior to primary staining

Steps Prior to Staining

Slide preparation involves fixation, dehydration, clearing, and impregnation/infiltration
Manual embedding technique involves using a pop-out mold to make a tissue block from the processed tissue using paraffin wax
Important factors in manual embedding include centering or orientation of the tissue block

Cutting and Mounting

Familiarize yourself with the parts of the microtome, its uses, and how to use it properly during cutting
Expected to produce tissue ribbons as the final product
Manual method of getting individual tissue sections from the ribbon involves using a floatation bath
Albumin is used as an adhesive to the tissue section to prevent it from washing out during the staining process

Staining Mechanism

Staining promotes easier optical differentiation and identification of cell and tissue components
Treating tissue or cells with a series of reagents to acquire a color, making the stained element transparent
No particles are seen, and the stained element must be transparent to have a good slide

Principles of Histological Technique

Alteration of contrast and color are used to distinguish tissue components
Nuclei are acidic, anionic, or negatively charged, and stain with basic dyes (blue) to exhibit a basophilic reaction
Cytoplasm is amphoteric, and its staining depends on the pH of the solution

Dyes/Stains

Chemical substances used to achieve visible color contrast in the microscopic picture of a prepared tissue
Have twin properties of a color band and the ability to bind to tissues
All dyes are organic compounds, mostly derivatives of coal tar or benzene

Integral Components of Dyes

Chromophores: the group on the benzene ring that confers color, altering the light resonance properties of the compound
Auxochromes: groups responsible for the tissue to bind firmly to a given dye, possessing a net charge
Autochrome groups: amino, hydroxyl, and carboxyl groups, which are important cationic and anionic auxochromes

Classification of Dyes

Based on pH: acidic, basic, and neutral dyes
Based on structure: azo, quinone, and oxazine dyes
Based on use: histological, histochemical, and immunological dyes

Qualities of a Successful Staining

Specificity/selectivity: the ability to stain specific parts or target parts for the pathologists to observe
Sensitivity: the ability to catch even the smallest amount of the target component### Routine Staining Part 1

Categorization of Staining

Based on how they are used in the lab:
- Intravital staining: injecting dye into a living animal or person to exhibit a tumor marker
- Supravital staining: staining living cells immediately after removal from the living body
Based on how it is done in the lab:
- Progressive staining: staining until the desired intensity of color is attained, then excess dye is removed selectively
- Regressive staining: overstaining, then removing excess dye to differentiate structures

Types of Staining

Vital staining: demonstrates cytoplasmic and nuclear structures, used for identification of specific structures
Routine staining: used in a day-to-day basis, stains various tissue elements with little differentiation except for nucleus and cytoplasm
Selective/special staining: has little or no affinity for tissue elements, used for identification of specific structures

Staining Methods

Direct staining: uses simple aqueous or alcoholic solutions of dye
Indirect staining: uses a mordant or accentuator to intensify the action of the dye
Meta-chromatic staining: uses certain dyes to exhibit a color different from the stain itself
Counter-staining: provides contrast and background to the tissue component in target

Differentiation and Decolorization

Selective removal of excess stain from the tissue during regressive staining
Differentiation is controlled by following exact times specified or by microscopic examination of the section
Differentiation is carried out by:
- Washing a section in a simple solution
- Using weak acids, bases, oxidizing agents, and mordants
- Exposure to air may oxidize and improve the process

Mordants and Accentuators

Mordant: a substance that enhances the combination of the dye with the tissues, forming a colored "lake" tissue-mordant-dye complex insoluble in aqueous and alcoholic solvents
Accentuator: a substance that increases the speed and selectivity of the dye, but does not participate in the staining reaction
Examples of mordants: alum, iron, and polyvalent metal ions
Examples of accentuators: KOH, phenol, glacial acetic acid, and aniline

Requirements in Staining

All glassware should be thoroughly cleaned to eliminate any carry-overs
Correct solvent should be used
Silver and osmic acid solutions should be kept in dark bottles to prevent oxidation
Solutions like dilute ammonia should be freshly prepared due to its volatility
Constituents of stain dissolved should follow the formula
Alcoholic solutions of the stain should be kept in dark stoppered bottles

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Description

Learn about the general procedure of routine staining in histotechnology, including dewaxing and introduction to staining techniques.