COMPREHENSIVE HISTOPATHOLOGY FINALS

mounting

The last step in tissue processing that results in a permanent histological preparation suitable for microscopy, after adhesion of the sections onto the slide and appropriate staining of the tissue

smooth teasing needle

After cutting, sections are floated out on a water-bath. Bubbles accumulating under the ribbon may be removed with a ____________, care being taken not to tear the section.

37C overnight

Drying conditions: Incubator

56-60C for 2 hours

Drying conditions: Wax oven

45-55C for 30-45 minutes

Drying conditions: Hot plate

37C for 24 hours or longer

Drying conditions: Delicate tissues (e.g., CNS tissue or brain)

adhesive

Another alternative to drying is by the use of an ________. An ________ is a substance which can be smeared on to the slides so that the sections stick well to the slides.

• Cryostat sections for immunocytochemistry

• CNS tissues

• Tissues containing blood clot

• Decalcified tissues

• High temperatures

Certain instances when sections may float from the slide (5)

poly-L-lysine

• A favorite adhesive, can be bought as a 0.1 % solution and further diluted (1 in 10 with distilled water) when ready to use (final dilution of 0.01%).

• This is widely used as a section adhesive in immunohistochemistry.

• With time, the adhesive ability of this substance slowly loses its effectiveness. Therefore, the coated slides should be used within a few days.

3-Aminopropyltriethoxysilane (APES)

• A better section adhesive and coated slides can be stored for a long time.

• It is invaluable in cytology particularly for cytospin preparation of proteinaceous or bloody material.

95% alcohol bath

Instead of a water bath, celloidin sections using egg albumin as an adhesive must be transferred from:

30 ml, 1% aqueous gelatin

Adding up to _____ of _____________ to the water in a floating out bath and mixing it well is a most convenient alternative to direct coating of slides.

mounting medium

• This bonds specimen, slide and coverslip together with a clear durable film.

• It is usually a syrupy fluid applied between the section and the coverslip after staining, setting the section firmly, preventing the movement of the coverslip.

• It protects the stained section from getting scratched, to facilitate easy handling and storage of the slides, and to prevent bleaching or deterioration due to oxidation, thereby preserving the slides for permanent keeping.

• This also helps prevent the distortion of the image during microscopic examination.

Refractive index

_______________ is important because it governs the contrast between the cellular detail and the background, and also the transparency of the observed sample against the bright field of the microscope.

higher

The mounting media must always have an RI ( higher / lower ) than the mounted sample to impart more transparency.

37C, 12-24 hours

The slide may then be incubated at _____ for ________ after mounting, to harden the medium.

2 days / 48 hours

Do not store mounted slides vertically for _______ if cured at room temperature.

Excessive mounting medium

Causes oozing of the mountant out of the sides of the cover glass

Excessive blotting

Causes drying of the section, causing shrinkage and cracking of the specimen

Excess xylene, Too little mounting medium

Causes bubble formation on the section after putting mountant (2)

50C, 2 hours

Setting of the mountant may be hastened in a hot oven at _____ for _______.

Aqueous

________ mounting media are used for mounting sections from distilled water when the stains would be decolorized or removed by alcohol and xylene as would be the case with most of the fat stains (Sudan methods) or for metachromatic staining of amyloid.

• Generally suitable for all enzymatic label/chromogen combinations and fluorescent labels (no dehydration and clearing).

gelatin, glycerin jelly, gum arabic

Components (3) of aqueous media for solidification:

glycerol

A component of aqueous media that prevents cracking and drying of the preparation:

sugar

A component of aqueous media that increases refractive index:

water

• An aqueous medium that has a low refractive index, is moderately transparent, and evaporates easily, hence is good only for temporary mounting.

• Does not allow tissues to be examined under the oil immersion lens.

• Slides cannot be stored over extended time periods, as this medium evaporates.

glycerin

• Refractive index of 1.46

• An aqueous medium that may also be used as a preservative.

• This is usually regarded as the standard mountant for fat stains.

• It has a high index of refraction and provides greater visibility if slightly diluted with water.

• This is a very suitable semi-permanent mounting medium, sets quite hard, and will keep sections mounted for years, especially if sealed on the edges with paraffin wax.

• It is miscible with water, is inexpensive, and is non-poisonous.

• It is also not necessary to treat the specimens with alcohol or organic solvents, which may introduce artifacts and remove pigments.

phosphate buffered glycerol

• Refractive index of 1.47

• This mountant is commonly used to mount sections for immunofluorescence.

glycerin jelly

• Refractive index of 1.47

• The standard mounting medium used when dehydration and clearing with xylene cannot be made (as in fat stains).

• Stains tend to fade using this mountant.

pure glycerin

• This mounting medium has the highest index of refraction and thus provides the best viewing and may be optimal for critical or irreplaceable material, because old material is easily retrieved with hot water or steam.

• The disadvantage is that it should be melted before use.

polyvinyl alcohol

• Refractive index of 1.5

• This is often used as a mountant in immunofluorescence microscopy and has been recommended as an alternative for glycerine jelly.

• The mountant is not set in the desired amount of hardness and therefore requires "ringing."

• It is commonly used for fluorescent labels with paraphenylene-diamine as an antifading agent.

Farrant’s medium

• Refractive index of 1.43

• This gum arabic medium does not solidify upon storage and therefore does not need to be heated before use. However, it takes a longer time to harden and may therefore require ringing.

• Arsenic trioxide may be used as a substitute of sodium merthiolate for

  preservation of the medium.

• Addition of 50 gm. potassium acetate will produce a neutral (pH 7.2) instead of an acid (pH 4.4) medium, and therefore, will raise the refractive index to 1.44.

Apathy’s medium

• Refractive index of 1.52

• This medium is used for methylene blue-stained nerve preparations and as a general purpose aqueous mountant.

• It is one of the most useful aqueous mountants for fluorescent microscopy, being virtually nonfluorescent.

• It is not compatible with normal histological stains. The pH of the medium is near 4.0 (highly acidic) so stains fade or bleed into the medium.

• Raising the pH to near 7.0 will prevent "bleeding" of metachromatic

  stains for amyloid.

• The medium sets quite hard, has a higher refractive index, and does not require ringing.

Brun’s fluid

• This mountant is recommended for mounting frozen sections from water.

• Frozen sections that are mounted directly from water or paraffin sections which require dehydration and clearing may also use this medium.

Resinous

_________ media are used for preparations that have been dehydrated and cleared in xylene or toluene, and are recommended for majority of staining methods.

undecalcified bones, electron microscopy

The most important synthetic resins are used for embedding __________, and for ________________.

Canada balsam

• Refractive index of 1.524

• It is a transparent, almost colorless oleoresin that adheres firmly to glass and sets to a hard consistency without granulation.

• It is recommended for whole mounts and for thick sections because it does not shrink much.

• However, it darkens slightly with age and slowly becomes acid because it oxidizes xylene, thereby causing gradual fading of many stains.

• The harmful solvents which, constitute a health hazard such as xylene, may limit the use of this mounting medium.

• The medium can only be neutralized (via calcium carbonate) temporarily since the mixture becomes acidic and changes into a brown color upon storage.

• The solution acidifies and darkens with age and upon exposure to sunlight, for which reason it should be kept in a dark glass bottle. Stains are usually not preserved due to acidity on prolonged exposure.

Abus balsamea

Canada balsam is a natural resin extracted from what Canadian tree?

37C, 58C

Canada Balsam is usually dissolved in xylene in an incubator at _____ or paraffin oven at _____.

DPX (Dibutyl Phthalate and Xylene)

• Refractive index of 1.532

• This is a resinous medium recommended for small tissue sections but not for whole mounts because of shrinkage produced on drying; hence, it should be used in excess amounts.

• It is a colorless, neutral medium in which most standard stains are well preserved. It is prepared by dissolving the common plastic, polystyrene, in a suitable hydrocarbon solvent (usually xylene).

• It tends to set quickly and, in doing so, often retract from the edge of the coverslip.

• It has a greater advantage over Canada balsam in that slides can be cleaned of excess mountant simply by stripping it off after cutting around the edge of coverslip.

XAM (Xylene Alternating Media)

• Refractive index of 1.52

• This is a synthetic resin mountant in xylene, available in a pale yellow or colorless solution.

• It dries quickly without retraction, and preserves stains well.

• Sections are quickly mounted from xylene.

Clarite (or Clarite X)

• Refractive index of 1.544 (highest RI among resinous mountants)

• This is a synthetic resin which is soluble in xylene (used as a 60% solution) and is generally preferred over DPX.

glycerol

Aqueous mounting media for phycobiliprotein fluorescent labels (phycoerythrin, phycocyanin) must not contain _______ as this quenches the staining intensity.

photo bleaching

Exposure to excitation light of most fluorescent labels results in diminished staining, a process known as _____________.

cover slipping

The process by which the stained section on the slide is covered with a thin piece plastic or glass to protect the tissue from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section.

ringing

The process of sealing the margins of the cover-slip to prevent the escape of fluid or semi-fluid mounts and evaporation of mountant, to fix the coverslip in place, and to prevent sticking of the slides upon storage.

paraffin wax

_________ may be applied with a ringing iron and is satisfactory as a temporary ringing agent.

Kronig cement

This ringing medium is made up of two parts paraffin wax mixed with 4-9 parts of powdered colophonium resin.

Durofix

This is a cellulose-based adhesive that can also be used for ringing.

Clarite or Permount

Mounting a broken slide onto another clean xylene-moist slide with either a drop of which two (2) mounting media may be sufficient for immediate examination while a new section is being cut and stained?

staining

This is the process whereby tissue components are made visible in microscopic sections by direct interaction with a dye or staining solution.

histologic stain

A ___________ is the purified form of a coloring agent or crude dye that is generally applied in an aqueous solution.

mordant

• This is a chemical compound that reacts with the stain to form an insoluble, colored precipitate on the tissue and make the staining reaction possible.

• This is a substance that combines with the tissue and the staining solution, forming a "bridge" that allows the staining reaction to take place.

2 times, 1-2 minutes

For sections up to 10 um thick, deparaffinization is done by immersing the paraffin section in a solvent, usually xylene, for how many times and how long?

sections to water

Rehydration is necessary when using aqueous stains and is the exact reverse of impregnation. This process may be summed up by what phrase?

acid differentiator

If drying is not complete, the section (or part of it), especially from bone and nervous tissue, may become detached from the slide during the process of staining, usually after adding the _____________.

sections to alcohol

Rehydration is not necessary when using alcoholic stains and may thus be summed up by what phrase?

second

Which change of xylene ( first / second ) will also raise the refractive index of the glass slide, thereby reducing light refraction during microscopic examination?

True

True or False: The stained section may be left in xylene for an indefinite period of time until it is finally mounted on the slide.

True

True or False: The section should not be allowed to stay in alcohol for a long time.

False

True or False: The stained section may be left in alcohol for an indefinite period of time until it is finally mounted on the slide.

False

True or False: The section should not be allowed to stay in xylene for a long time.

30 minutes

Sections must be left in the oven for a minimum of ________ before they are finally stained to avoid floating off.

histological staining

This is the process whereby the tissue constituents and general relationship between cell and tissue are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue component.

direct or simple staining

This is the process of giving color to the sections by using only one aqueous or alcoholic dye solution, which is washed away after 30–60 seconds, prior to drying and examination.

• Methylene blue is an example.

indirect staining

This is the process whereby the action of the dye is intensified by adding another agent or a mordant which serves as a link or bridge between the tissue and the dye, to make the staining reaction possible.

• Entails the formation of a tissue-mordant-dye complex.

• Essential to staining.

accentuator

This staining component is not essential to the chemical union of the tissue and the dye. It does not participate in the staining reaction, but merely accelerates the reaction.

True

True or False: Basic dyes are positively charged, while acidic dyes are negatively charged.

potassium hydroxide (KOH)

What is the accentuator in Loeffler's methylene blue?

phenol

What is the accentuator in carbol thionine and carbol fuchsin?

progressive staining

• This is the process whereby tissue elements are stained in a definite sequence, and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained.

• Once the dye is taken up by the tissue, it is not washed or decolorized.

• The differentiation or distinction of tissue detail relies solely on the selective affinity of the dye for different cellular elements.

• Disadvantage: requires frequent monitoring.

regressive staining

• With this technique, the tissue is first overstained to obliterate the cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue until the desired intensity of color is obtained.

• Routine H & E staining is an example.

• Disadvantage: prone to excessive staining and over-decolorization.

differentiation or decolorization

This is the selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues.

pre-mordanting

This is a type of mordanting when the tissue is at first treated with mordant followed by the dye (e.g., Heidenhain’s iron hematoxylin).

meta-mordanting

• This is a type of mordanting when the mordant is combined with the dye upon usage (e.g., alum hematoxylin solutions).

• The most common type.

post-mordanting

• This is a type of mordanting when the dye is applied first before the mordant.

• Not used in histopathology staining.

• acid/base wash (1% acid OH)

• oxidizing agents (e.g., potassium permanganate)

• excess mordant

• replacement with another dye

Methods of differentiation (4)

metachromatic staining

• The staining technique that entails the use of specific dyes which differentiate particular substances by staining them with a color that is different from that of the stain itself.

• This is particularly employed for staining cartilage, connective tissues, epithelial mucins, mast cell granules, and amyloid.

• This is caused by Van der Waals aggregation and light absorption shifts.

orthochromatic staining

• The staining technique employed by most dyes wherein tissues are stained in colors or shades that are similar to the color of the dye itself.

True

True or False: The azures or toluidine blue are usually more effective metachromatic stains than methyl violets (e.g., crystal violet).

False

True or False: The azures or toluidine blue are more effective metachromatic stains for amyloid and amyloid deposits than methyl violets (e.g., crystal violet).

Alpha, Blue

Dye form: Monomer; Type & Observed Color: _______

Beta, Purple

Dye form: Dimer/Trimer; Type & Observed Color: _______

Gamma, Red

Dye form: Polymer; Type & Observed Color: _______

True

True or False: Increased dye concentration enhances metachromasia.

False

True or False: Increased pH enhances metachromasia.

False

True or False: Increased temperature enhances metachromasia.

True

True or False: Using an aqueous medium enhances metachromasia.

metallic impregnation

• This is a process where specific tissue elements are demonstrated, not by stains, but by colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black deposit on the surface of the tissue or bacteria.

• Such stains are not absorbed by the tissue but are held physically on the surface as precipitates or reduction products.

gold chloride, silver nitrate

What are the two (2) most valuable metals for metallic impregnation?

• metal containers and instruments

• formalin-laden atmosphere

• sunlight

Things to avoid when handling sections for metallic impregnation (3)

sodium chloride, hydrochloric acid

All unused metallic impregnation reagents should be immediately inactivated by _________ or dilute _________ solution and discarded.

vital staining

• This is the selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle (cytoplasmic phagocytosis) or by staining of pre-existing cellular components.

• The usual purpose is to reveal cytological details that might otherwise not be apparent; however, staining can also reveal where certain chemicals or specific chemical reactions are taking place within cells or tissues.

False

True or False: Vital stains are excluded by dead cells but are taken up by living cells.

Trypan blue

The vital stain for the reticulo-endothelial system:

propidium iodide

The vital stain for eukaryotic cells:

True

True or False: The nucleus of a living cell is resistant to vital stains, and therefore is not demonstrated.

True

True or False: Demonstration of nuclear structures during vital staining suggests permeability of the membrane to the dye, signifying the death of the cell.

intravital staining

This type of vital staining is done by injecting the dye into any part of the animal body (either intravenous, intraperitoneal or subcutaneous), producing specific coloration of certain cells, particularly those of the reticulo-endothelial system.

lithium, carmine, India ink

Common intravital dyes (3)