HPCT 2ND AND 3RD WEEK Flashcards by Potato Patata

A clearing agent that is used when the tissue is to be cleared directly from water, as in a frozen section

Glycerin and Gum syrup

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Most commonly used clearing agent

Xylene

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Characteristics of a good clearing agent

Should be miscible with alcohol to promote rapid removal of the dehydrating agent from the tissue

Should be miscible and easily removed by melted paraffin wax and/or by mounting medium to facilitate impregnation and mounting of sections

Should not produce excessive shrinkage, hardening or damage of tissue

Should NOT dissolve aniline dyes

Should NOT evaporate quickly in a water bath

Makes tissue transparent

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Most important step in embedding

Orientation

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Most common, routinely used embedding media

Paraffin wax

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Used in tough tissues (embedding media)

Celloidin

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Used in tissues that we want to prevent dehydration histochemistry (embedding media)

Gelatin

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Embedding media for electron microscopy

Plastic

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Melting point of paraffin wax (Routine)

56C

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Process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cell

Staining

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Main reason why cells are stained

To enhance contrast and visualization of the cell or certain cellular components under the microscope

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Process whereby TISSUE CONSTITUENTS ARE DEMONSTRATED in sections by direct interactions with a dye or staining solution, producing coloration of active tissue component

Histological staining

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Process where by constituents of tissues are studied THROUGH CHEMICAL REACTIONS that will permit microscopic localization of specific tissue substance

Histochemical staining

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Combination of immunologic and histochemical techniques to detect phenotypic markers under microscope

Immunohistochemical staining

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Purified form of a coloring agent or crude dye that is generally applied in an aqueous solution

Histological stain

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Parts of the cells or tissue that are ACIDIC take more of (nucleic acid)

BASIC dye

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Parts of the cells or tissue that are BASIC take more of (Cytoplasm)

ACIDIC dye

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Process of giving color to the sections using aqueous or alcoholic dye solution

Only ONE dye is used, which is washed away after 30-60 seconds, prior to drying and examination

Direct staining

E.G Methylene blue

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Process whereby the action of the dye is intensified by ADDING other agent or mordant

Eg. Mordant, accentuator

Indirect staining

E.G Gram Stain

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Serves as a link or bridge between tissue and the dye to make staining reaction possible

INTEGRAL part of the staining reaction (staining won’t be possible without this)

Mordant

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Accelerates or hastens the speed of staining reaction by increasing staining power and selectivity

NOT ESSENTIAL to the chemical union of tissue and the dye

Accentuator

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Process whereby tissue elements are stained in a definite sequence

Staining solution is applied for specific periods of time or until the desired intensity of coloring of the tissue elements is attained

Once the dye is taken up by the tissue, IT IS NOT WASHED NOR DECOLORIZED

Progressive staining

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The tissue is first overstained to obliterate the cellular details, and excess stain is removed or decolorized from unwanted parts of the tissue

Most common example is H and E staining

Regressive staining

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Selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surround tissues

Differentiation/Decolorization

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Acts as differentiator for both basic and acidic dye by dissolving excess dye

Alcohol

May act as a differentiating agent

Mordant

Staining with a color that is DIFFERENT from that of the stain itself

Metachromatic staining

Tissues are stained in color shades that are SIMILAR to the color of the dye itself

Orthochromatic staining

Process where a specific tissue element is demonstrated, not by stains, but by colorless solutions of metallic salts which are reduced by the tissue, producing an opaque usually BLACK deposit on the surface of tissue or bacteria

Metallic Impregnation

Not absorbed by the tissue BUT HELD physically on the surface as precipitates

Metallic impregnating agent

Reduced by argentaffin cells, forming black deposits under microscope

Ammoniacal silver stain

Blueing agent examples:

Tap water, Saturated lithium carbonate, 0.5% Ammonia in distilled water, Ammonium hydroxide, Scott's Solution

Selective staining of living cell constituents

Vital Staining

Done by injecting the dye into any part of the animal body Example: Lithium, India Ink, Carmine

Intravital staining

Used to stain cells immediately after removal from the living body Example: Neutral red, Janus green, Trypan Blue, Thionine, Nile Blue, Toluidine blue

Supravital staining

What is the best vital dye?

Neutral red

Vital dye that can stain mitochondria

Janus Green B

Application with a different color for contrast and background

Counter staining

Red cytoplasmic stain

Eosin Y Eosin B Phloxine B

Yellow cytoplasmic stain

Picric acid Orange G Rose Bengal

Green Cytoplasmic stain

Light green Lissamine green

Red Nuclear stain

Neutral Red Safranin O Carmine Hematoxylin

Blue Nuclear stain

Methylene Blue Toluidine Blue Celestine Blue

Most widely used cytoplasmic stain

Eosin

One of the most valuable stains used for differentially staining connective tissues and cytoplasm An Acid dye and the terms acidophilic, oxyphilic, and eosinophilic are often used Routinely used in histopathology as a counterstain to hematoxylin

Eosin

A primary stain Basic dye Nuclear stain Nuclei: Blue to blue-black

Hematoxylin

Secondary stain (Counter stain) Acidic dye Cytoplasmic stain Cytoplasm: Pink

Eosin

A mixture of picric acid and acid fuchsin for the demonstration of CONNECTIVE TISSUES

Acid Fuchsin-Picric acid (Van Gieson's stain)

Fiber that can be stained by Van Gieson's stain

Collagen Fiber

Giving GREEN Fluorescence for DNA and RED Fluorescence for RNA

Acridine Orange

Permits discrimination between dead and living cells

Acridine Orange

Used for staining of hemoglobin

Benzidine

Used as a contrast stain for Gram's technique, AFB, Papanicolau method, Diphtheria staining

Bismarck brown

From Cochineal bugs / coccus cacti Chromatin stain for fresh materials in smear preparations Combined with aluminum chloride (Best's Carmine solution) for GLYCOGEN staining (bright red color)

Carmine

Best known as an indicator (pH Indicator) May be utilized as a stain for axis cylinders in embryos

Congo red

Congo red karjian's technique stains what?

Amyloid stain

OLDEN of all stain Stains amyloid, cellulose, starch, carotenes, and glycogen

Iodine

Iodine that is used for gram's wiegert method for staining microbes and fibrin

Gram's Iodine

Used as a test for glycogen, amyloid, and corpora amylacea

Lugol's iodine

Stains and demonstrate mitochondria

Janus Green B

Acts as a contrast stain for ascaris eggs and erythrocytes and as a bacteria spore stain

Malachite Green

Excellent stain for elastic fibers (Tanzer Unna Orcein method) Recommended in dermatological studies, demonstrate the finest and most delicate fibers in SKIN

Orcein

Iron stain May be used for microanatomical color contrast of specimen Used as an intravital stain for the circulatory system

Prussian Blue

Nuclear stain for fixed tissues Substitute for Thionine in fresh frozen tissue sections Recommended for NISSL GRANULE staining and chromaphilic bodies

Toluidine Blue

Tissue fixative and a decalcifying agent Acts as a contrast stain to acid fuchsin in demonstration of collagen using Van Gieson's stain Acts as a cytoplasmic stain in contrast to basic dyes Counterstain to crystal violet

Picric acid

Invented by Paldwell Trefall in 1881, The simplest among the different types of microtomes. This consists of a heavy base and two arms. The lower arm resting on pivots and a supporting column, and attached to the micrometer screw, at the base of which is found the ratchet wheel with feed mechanism.

Rocking Microtome

Invented by MINOT in 1885-86 to cut paraffin embedded tissue and is CURRENTLY the most common type used for both routine and research laboratories Knife is fixed in horizontal position Sections are cut between 3 and 5 um

Rotary Microtome

Developed by adams in 1789. Two models are available, one is Base-Sledge Microtome that consist of two movable pillars holding the adjustable knife clamps allowing to bet set an angle for cutting celloidin sections Base-Sledge microtome is favored in laboratories where very hard tissue or large blocks are usually sectioned The second type is the standard sliding microtome. It is different from the base sledge microtome because with this instrument, the block remains stationary while the knife is MOVED backward and forward. Developed mainly for cutting celloidin embedded tissue blocks and is MORE DANGEROUS because of the MOVABLE KNIFE.

Sliding Microtome

Invented by Queckett in 1848. Stage for block holder is hollow and perforated around its perimeter, attached to a reinforced flexible lead pipe thru which carbon dioxide passes from a cylinder. Used to cut undehydrated thin to semi-thin sections of fresh, frozen tissues, especially in instances when rapid diagnosis is required and demonstration of histological fat is needed

Freezing microtome

A refrigerated apparatus used for freezing the tissue into the block holder to the correct degree of hardness that allows for easier and faster sectioning Kept at temperature between -5C to -30C (Average is -20C) Capable of freezing fresh tissues within 2-3 minutes and cutting sections of 4um with ease

Cryostat

Equipped with a glass or gem grade diamond knife that is used to cut very thin sections typically 60 to 100 nanometer of tissue embedded in EPOXY resin Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a TRANSMISSION ELECTRON MICROSCOPE

Ultrathin microtome

Microtome knives: One sided of the knife is flat while the other is concave. LESS concave sides are recommended for cutting CELLOIDIN-EMBEDDED tissue blocks on a sliding microtome MORE concave sides are used to cut PARAFFIN sections on base-sledge, rotary or rocking microtome

Plane-concave knife (usually 25 mm in length)

Microtome knives: With bot sides concave, recommended for cutting PARAFFIN-EMBEDDED sections on a rotary microtome

Biconcave knife (usually 120 mm in length)

Microtome knives: Both sides are straight, recommended for frozen sections or for cutting EXREMELY HARD and TOUGH specimens embedded in paraffin blocks, using a base sledge type or sliding microtome

Plane-Wedge knife (Usually 100mm in length)

Angle formed between the cutting edges

Bevel angle Normally about 27-32 degree

For cutting serial sections of large blocks of paraffin embedded tissues

Rocking microtome

For cutting paraffin embedded sections

Rotary microtome

For cutting celloidin embedded sections

Sliding microtome

For cutting UNEMBEDDED sections

Freezing microtome

For cutting frozen section

Cryostat or cold microtome

For cutting sections for EM

Ultrathin Microscope

Substance which can be smeared on to the slides so that the sections stick well to the slides Choice of slide and adhesive will be influenced by the staining methods to be subsequently applied Not necessary for routine staining Essential for methods that require exposure of sections to acid and alkalis

Adhesives

Knives for EM Used for trimming and semi-thin sectioning of tissue block Washed with detergent, rinsed in distilled water, and alcohol, and dried with lint-free paper

Glass knives

Knives for EM Used to cut and type of RESIN BLOCK- brittle and expensive but very durable Cutting edge must be kept clean to avoid damage during sectioning

Diamond knives

Removal of gross nicks to remove blemishes, grinding the cutting edge of the knife on a stone (Carborundum)

Honing (HEEL TO TOE)

Types of hone For manual sharpening (Best results)

Belgium yellow

Types of hone Gives more polishing effect

Arkansas

Types of hone Much coarser; used only for BADLY NICKED KNIVES

Fine carborundum

The process whereby the BURR formed during honing is remove and cutting edge of the knife is polished

Stropping (TOE TO HEEL)

Has a sharp cutting edge that can cut 2-4 microns thick section with ease. Makes honing and stropping obsolete

Disposable blade

The thermostatically controlled type is preferable. Water from a hot water tap can be used although this can give rise to air bubbles The temperature of the water should be between 5-10C BELOW the melting point of the paraffin wax

Water bath

Temperature setting at the melting point of the wax so that no obvious damage is done to the sections and drying is complete in 30 minutes. Hot play may also be used instead of this equipment For more delicate tissues such as brain, a lower temp is used to avoid splitting and cracking of the section due to excessive heat 37C for 24 hours or longer is recommended

Drying oven or hot plate

A tool that is needed for handling section during cutting, and for removing folds and creases on the sections during "floating out" in water bath

Forceps

Polysaccharides of glucose, normally stored in the liver, heart, and skeletal muscles, but it may be abnormally present in certain diseases

Glycogen

Made up of hexosamines or mucus that is secreted by the goblet cells of intestinal mucosa, respiratory lining cells and certain glands

Mucin

Both glycogen and mucin are stained by

Periodic Acid-Schiff (PAS)

A histochemical stain that will demonstrate carbohydrates and other substances in the tissues Can help diagnose: Glycogen storage disease Tumors Fungal Infection Basement membrane disease (eg. Goodpasteur's syndrome) M6 Leukemia (Acute erythroleukemia/DiGuglielmo)

Periodic Acid Schiff

PAS and tissue chemical bond

Coulombic bonds

POS POSITIVE substance color: PAS nuclei color:

Positive substance: Red or Magenta Nuclei: Blue

PAS with diastase for glycogen Pas nuclei color:

Glycogen: Red Nuclei: Blue black

Best carmine color

Glycogen: Bright red granules Nuclei: Blue or grayish blue Mucin, fibrin: Weak red

Langhan's iodine method for glycogen (Carleton's method)

Glycogen: Mahogany brown Tissue constituents: Yellow

Other carbohydrate stains

Fresh frozen azure - A metachromatic staining for glycosaminoglycans Alcian blue technique Metachromatic toluidine blue staining Combined aldehyde fucshin-alcian blue Mucicarmine stain Hale's dialyzed (Colloidal) Iron technique Fluorescent acridine orange technique

Unilocular lipid-filled adipocytes that specialized in lipid STORAGE

White adipose tissue

Multilocular adipocytes that specialize in lipid BURNING

Brown adipose tissue

Appears as signet rings on H and E stain because large lipid droplet displaces the nucleus

Fat cells

Breakdown products within cells from oxidation of lipids and lipoprotein WEAR AND TEAR PIGMENT found most commonly in heart, liver, CNS, and adrenal cortex PAS positive and variably acid fast

Lipofuscin or lipochrome pigments

Not a real dye Gives color to lipids simply because they are more soluble in lipid medium of the tissues, than their medium of 70% alcohol E.G Sudan Black B, Sudan III, and Sudan IV

Oil soluble dyes (Lysochromes)

Stain that colors fat droplets BLACK MOST SENSITIVE OF THE OIL SOLUBLE DYES

Sudan Black

Recommended for staining triglycerides (Neutral lipids) giving them a deep RED stain

Sudan IV (Scharlach R)

First sudan dye to be introduced Good fat stain for CNS Gives less deep and LIGHTER ORANGE STAIN

Sudan III

Oil red O method in dextrin

Fat - Brilliant red Nuclei - Blue

Osmic acid stains for fats

Nuclei - Yellow-orange Fats - Black

Other lipid stain

Nile blue sulfate for fats Toluidine blue - acetone method for sulfatide Borohydride-Periodic Schiff (BHPS) method for glycosides

Sections must be left in the oven for a minimum of ___

30 minutes

Molecules of basic dyes has what charge

Positive charge

Cell wall and cytoplasm of bacterial cells has what charge

Negative charge

A 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

Indirect staining

A 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 are attained

Progressive staining

The tissue is overstained to obliterate the cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue Common example is H&E staining

Regressive staining

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

Differentiation (Decolorization)

Mordant that can oxidize hematoxylin to a soluble, colorless compound

Iron alum

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

Metallic Impregnation

Corner stone of tissue-based diagnosis

H&E

Stains nuclei and other parts blue in H and E

Hematoxylin (hematocleus)

Stain other structures and cytoplasm pink or red in H and E

Eosin (eosytoplasm)

Hematoxylin binds strongly to ___ and consequently binds to nuclear ___ and stains nuclei BLUE

Binds strongly to acids, and consequently binds to nuclear DNA

Most fixatives can be used in H and E staining except ___ that inhibits hematoxylin

Osmic acid solutions

H&E procedure

1. Clear paraffin embedded sections in first xylene bath for 3 mins 2. Transfer to second xylene bath for 2 to 3 minutes 3. Immerse in first bath of absolute ethyl alcohol for 2 mins 4. Transfer to a bath of 95% ethyl alcohol for 1 or 2 minutes 5. Rinse in running water for 1 minute 6. Stain with Harris alum hematoxylin for 5 mins (Ehrlich's hematoxylin requires 15-30 mins) 7.Wash in running tap water to remove excess stain 8. Differentiate in 1% acid-alcohol (1mL concentrated HCL to 99ml of 80% ethyl alcohol) for 10-30 seconds 9. Rinse in tap water 10. Blue in ammonia water (5 mins ave) or 1% aqueous lithium carbonate until the sections appear blue 11. Wash in running water for 5 mins 12. Counterstain with 5% aqueous eosin for 5 mins. If alcohol eosin is used, the time can be reduced to 30seconds or 1 min 13. If aqueous eosin is used, wash and differentiate in tap water under microscope control until the nuclei appear sharp blue to blue black and the rest of the tissue appear in shades of pink. If alcoholic solution is used, differentiate with 70% alcohol 14. Dehydrate, clear, and mount

Staining in frozen section

Hematoxylin-Eosin method Thionine method Polychrome Methylene blue method Alcoholic pinacyanol method ( Used for supravital staining of mitochondria and primarily for color sensitization in photography)

H&E staining of frozen section is regressive or progressive staining?

Progressive staining

Failure of staining may be due to?

Paraffin, Fixative, or decalcifying solution that has not been thoroughly washed out and removed.

A process whereby various constituents of tissues are studied thru chemical reactions that will permit microscopic localization of a specific tissue substance.

Histochemical staining (Histochemistry)

A combination of immunologic and histochemical techniques using a wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies to detect and demonstrate tissue antigens and phenotypic markers under the microscope

Immunohistochemical staining (IHC)

The last step in tissue processing that results in a permanent histological preparation suitable for microscopy

Mounting

Sections are fixed by leaving the slides in?

37C incubator overnight or by placing the slides in a wax oven at 56C-60C for 2 hours, or by drying the slides on a hot plate at 45C to 55C for 30 - 45 mins

CNS or brain tissue drying time and temperature

37C for 24 hours or longer

Alternative to drying

Use of adhesives

A substance which can be smeared on the slides so that the sections stick well to the slides.

Adhesives

If staining is to include antigen retrieval (IHC), enzyme pretreatment for in-situ hybridization (ISH), or prolonged incubation steps. What should we do?

Charged slides or adhesive must be used

Adhesives are essential for methods that require exposure of sections to?

Alkalis and acids (especially ammoniacal silver solutions)

Instances when sections may float from the slide

For urgent cryostat sections to be submitted for immunocytochemistry For CNS tissues For tissues containing blood clot For tissues which have been decalcified When sections are to be subjected to high temperature

Most commonly used adhesive

Albumin

Albumin solution is prepared by mixing equal parts of?

Glycerin, distilled water, and white of eggs. Which is then filtered through coarse filter paper and a crystal of Thymol is added

Disadvantage of albumin

Retains some of the stain and gives a dirty background

Cab be bought as a 0.1% solution and further diluted (1 in 10 with distilled water) when ready to use.

Poly-L-Lysine

Adhesive ability of this substance slowly loses its effectiveness

Poly-L-Lysine

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

Aminopropyltriethyoxysilane (APES)

It is invaluable in cytology particularly for cytosine preparation of proteinaceous or bloody material

Aminopropyltriethoxysilane

Mayer's Egg albumin formula

Egg white 50CC Glycerin 50CC Filer and add about 100 mg crystals of thymol to prevent the growth of molds

Most commonly used because it is very easy to make Convenient Relatively inexpensive adhesive

Mayer's Egg Albumin

Dried Albumin formula

Dried albumin 5gm Sodium Chloride 5gm Dissolve in 100cc of Distilled water and add crystals of thymol

Dried and stored in 70% alcohol until it is ready for staining

Dried Albumin

Gelatin 1% formula

Gelatin 1gm Distilled water 100mL Glycerol 15mL Phenol crystal 2 gm Adding up to 30mL of 1% aqueous gelatin to the water in a floating out bath and mixing it well is a most convenient alternative to direct coating of slides

Gelatin-formaldehyde mixture formula

1% gelatin 5mL 2% formaldehyde 5mL Coat the slides with the above mixture. Allow coated slides to dry at 37C for one hour or overnight before use

____ coated slides are very useful in cytology, particularly for cytospin preparations of proteinaceous or bloody material

APES (3-aminopropylethoxysilane)

A syrupy fluid applied between the section and the coverslip after staining, setting the section firmly, and preventing the movement of the cover slip

Mounting Medium

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

Mounting medium

Governs the contrast between the cellular detail and the background

Refractive index

The slide then may be incubated at ___C for __ - __ hours after mounting to harden the medium

37C for 12-24 hours

Setting may be hastened in a hot oven at __C for _ hours

50C for 2 hours

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 or for metachromic staining of amyloid

Aqueous mounting media

Usually made up of gelatin, glycerin jelly, or gum arabic (to solidify the medium), glycerol (to prevent cracking and drying of the preparation), sugar (to increase the refractive-index), and a preservative solution

Aqueous mounting media

Aqueous mounting media examples: Has a low RI, moderately transparent and evaporates easily, hence good only for temporary mounting. Does not allow tissue to be examined under OIO

Water

Aqueous mounting media examples: May also be used as a preservative. Refractive index is 1.46 Sets quite hard and will keep sections mounted for years, especially if sealed on the edged with paraffin wax MISCIBLE with water Inexpensive and non-poisonous Disadvantages is that it is difficult to prepare slides that are truly permanent in nature because it is a thick liquid, it can slowly run off a slide that is tilted

Glycerin

Glycerin Jelly (Kaiser's 1880) Formula and Refractive index

Refractive index: 1.47 Formula: Gelatin 10gm Glycerol 70mL Distilled water 60mL Phenol crystals 0.25gm Gelatin is added to distilled water and incubated in a water bath at 60C until dissolved. Glycerol and then phenol crystals are added and mixed. Stored in a ref at 40C. Heated at 60C for use

Standard mounting medium used when dehydration and clearing with xylene cannot be made (as in fat stains)

Glycerin Jelly

Has the highest index of refraction and thus provides the best viewing and may be optimal for critical or irreplaceable material

Pure glycerin

Often used as a mountant in immunofluorescence microscopy Alternative for glycerin jelly

PVA

Farrant's Medium Refractive index and formula

Refractive index is 1.43 Formula: Gum arabic 50gm Distilled water 50mL Glycerol 50mL Sodium merthiolate 0.025gm Dissolve gum arabic in distilled water with gentle heating and add glycerol and sodium merthiolate. Mix well and label

May be used as a substitute of sodium erthiolate for preservation of the medium

Arsenic trioxide

Addition of _____ in Farrant's medium will produce a neutral instead of an acid (pH7.2 > pH 4.4) and therefore will rase the Refractive index to 1.44

50gm potassium acetate

Apathy's medium Refractive index and formula

Refractive index 1.52 Formula: Pure gum arabic (crystals not powder) 50 gm Pure can sugar or sucrose 50gm Distilled water 50mL Thymol crystals 0.05gm

This medium is used for methylene blue-stained never preparations and as a general purpose aqueous mountant One of the most useful aqueous mountants for fluorescent microscopy, being virually non-fluorescent pH is 4.0 (Highly acidic) Does not require ringing

Von apathy's medium

Adding this into Von apathy's medium will raise the pH to near 7.0 and will prevent bleeding of metachromatic stains for amyloid

20g of calcium chloride or 10g of sodium chloride

Brun's fluid Formula

Glucose 24gm Glycerin 6mL Spirits of camphor 6mL Distilled water 84mL Mix, shake well, and filter. Store the solution in a well-stoppered bottle

Recommended for mounting frozen sections from water

Brun's Fluid

Frozen section that are mounted directly from water, or paraffin sections which require dehydration and clearing, usually should be mounted on?

Glycerin, Gum syrup, or Brun's Fluid

Used for preparations that have been dehydrated and cleared in xylene or toluene, and are recommended for MAJORITY of staining methods

Resinous mounting media

A natural resin extracted from the canadian tree, abus malsamea, usually dissolved in xylene in an incubator at 37C or paraffin oven at 58 C, and filtered obtaining the desired consistency by controlled evaporation of the solvent Refractive index is 1.524

Canada Balsam

Transparent, almost colorless oleoresin that adheres firmly to glass and sets to a hard consistency without granulation. Darkens slightly with age and slowly becomes acid because it oxidizes xylene causing gradual fading of many stains

Canada balsam

Recommended for whole mounts and for thick sections because it does not shrink much

Canada Balsam

This is a resinous medium recommended for small tissue sections but not for whole mounts because of shrinkage produced on drying Refractive index is 1.532 It is prepared by dissolving the common plastic, polystyrene in a suitable hydrocarbon solvent (usually xylene)

Dibutyl Phthalate and Xylene

A synthetic resin mixture in xylene, available in a pale yellow or colorless solution Dries quickly without retraction and preserves stains well Sections are Quickly mounted from xylene Refractive index is 1,52

XAM

A synthetic resin which is soluble in xylene (Used as a 60% solution in xylene) Generally preferred over DPX Refractive index is 1.544

Clarite or Clarite X

Generally suitable for all enzymatic label/chromogen combinations and fluorescent labels Specimens mounted in such media are mounted straight from the aqueous phase with no dehydration or clearing

Aqueous mounting medium

Aqueous mounting media for phycobiliprotein fluorescent labels (Phycoerythrin, Phycocyanin) must NOT contain ____ as this quenches the staining intensity

Glycerol

Exposure to _____ of most fluorescent labels results in diminished staining, a process known as photo bleaching

Excitation light

Most useful aqueous mountant for fluorescent microscopy with a refractive index of 1.52

Apathy's Medium

Stained section on the slide must be 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 for years to come

Cover slipping

When the mounting media is too thin what will form under the coverslip

Bubbles

Other than thin mounting media, what can cause bubbled appearance under the microscope

Contamination of clearing agents

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

Ringing

Ringing media used that is made up of two parts paraffin wax mixed with 4-9 parts powdered colophonium resin, heated and filtered

Kronig cement

After soaking the broken slide in xylene to remove the cover slip and placing it in incubator for 37C until all the mounant has been covered. The whole slide will be covered with what?

6 parts butyl acetate and 1 part durofix and left in the incubator for 30 mins until the mixture hardens into a film

Two types of trimming: Sides, tips, and bottom of the tissue are trimmed using a knife or a blade to form a truncated pyramid/ 4-sided prism

Coarse Trimming

Two types of trimming: Block is placed in the microtome - setting the adjuster at 15mm or by advance the block using the coarse feed mechanism where the surface is trimmed away until the entire tissue surface has been exposed

Fine trimming

Process wherein a process tissue is cut into uniformly thin slices using microtome to facilitate studies under the microscope

Sectioning or cutting

For paraffin embedded tissue blocks which may be cut by ROCKING and ROTARY microtome

Paraffin Sections

For celloidin embedded tissues which are usually cut by means of the SLIDING MICROTOME

Celloidin sections

Which may be cut from the tissues that have been fixed and frozen with CO2 or for fresh or fixed tissues frozen with CRYOSTAT

Frozen section

Thickness of paraffin section

4-6 um

Thickness of Celloidin section

10-15 um

Thickness of frozen section

10 um

Thickness of ultrathin section: Semithin

0.5-1.0 um Glass knives are used

Thickness of ultrathin section: Ultrathin

500-1200 A or 50-120 nm Diamond knives are used

Section cut is picked by:

Index finger Camel hairbrush Spatula Flat beaded forceps

Tissues which tend to crumble (Blood clot or bone marrow) or do not form a smooth flat surface can be section with ease, ____ into the block surface while the section is being cut slowly, to reduce the effect of static electricity

Exhaling gently

Generally, a ____ cutting stroke produces the best results and the least compression

Slow, uniform

Sections are removed in ribbons of ___ to allow easy location of serial sections

Ten

The sections are then floated out on a water bath set at 45-50C which is lower or higher than how many of the melting point of the wax?

6-10% lower than the melting point of the wax

Main goal of floating the tissue

To flatten the section

Sections should not be left on the water bath for how many seconds?

longer than 30 seconds because it will have an expansion and distortion of tissue

Faults or problems observed when cutting sections: Brittle or hard tissue

Reason: Prolonged fixation, Prolonged dehydration, prolonged clearing, prolonged paraffin infiltration, overheated paraffin, drying out of tissues before actual examination Remedy: Use tissue softeners (Phenol or molliflex)

Faults or problems observed when cutting sections: Clearing agent turns milky

Reason: Incomplete dehydration Remedy: Repeat dehydration with absolute alcohol

Faults or problems observed when cutting sections: Tissue smells of clearing agent

Reason: Clearing agent not removed, incomplete impregnation Remedy: Block is trimmed down nearest to the tissue. Remaining wax is melted on embedding oven and paraffin impregnation is repeated, changing the paraffin at least once before blocking

Faults or problems observed when cutting sections: Tissue is opaque

Reasons: Incomplete clearing Remedy: Repeat clearing. If already embedded, clear up to 12 hrs

Faults or problems observed when cutting sections: Tissue shrinks away from wax when trimmed

Reason: Insufficient dehydration, incomplete clearing and impregnation Remedy: Repeat the whole procedure

Faults or problems observed when cutting sections: Tissue is soft when block is trimmed

Reason: Incomplete fixation Remedy: Repeat fixation

Faults or problems observed when cutting sections: Airholes found on tissue during trimming

Reason: Incomplete impregnation Remedy: Repeat impregnation

Faults or problems observed when cutting sections: Frozen tissue crumbled and comes off the block holder when cut

Reason: Freezing is not adequate Remedy: Refreeze the tissue block

Faults or problems observed when cutting sections: Frozen tissue chips into fragment when cutting

Reason: Tissue is frozen too much Remedy: Warm the tissue with the fingers

Dyes that are obtained from plants and animals, previously utilized for dyeing of wool and cotton

Natural Dyes

A natural dye derived by extraction from the core or the heartwood of a mexican tree known as ______

Hematoxylin The tree is known as Hematoxylin Campechianum

The most valuable staining reagent used by the cytologist due to its powerful nuclear and chromatin staining capacity Its striking polychrome properties may be produced with proper differentiation Used after almost any fixative and is a permanent stain

Hematoxylin

Hematoxylin is a true basic dye?

Hematoxylin is not a true basic dye

Active coloring agent of hematoxylin

Hematin

How does hematin form?

Formed by the oxidation of hematoxylin, a process known as ripening Accomplished by exposing the substance to air and sunlight, thereby oxidizing hematoxylin

Artificial ripening of hematoxylin

By adding strong oxidizing agent such as hydrogen peroxide, mercuric oxide, potassium permanganate, sodium perborate, or sodium iodate

Recommended for progressive staining of tissues and are usually counterstained with Eosin, Congo red, and Safranin

Alum hematoxylin

Rapid ripening of Ehrlich's reagent is brought by the addition of?

Sodium Iodate

Harris' solution of hematoxylin is ripened with?

Mercuric Chloride

Iron hematoxylin compounds are used only for?

Differential or regressive staining using acid-alcohol for differentiating agent

Hematoxylin solution that is utilized for the study of spermatogenesis

Copper Hematoxylin solution

Used frequently in histology to examine thin sections of tissue

Hematoxylin and Eosin staining protocol

An old histologic dye extracted from coccus cacti / Cochineal bug

Cochineal dyes

Liquid that are extracted from cochineal bug/ coccus cacti is then treated with alum to produce what?

Carmine

Widely used as a powerful chromatin and nuclear stain for fresh material and smear preparation

Carmine (Cochineal dyes)

When carmine is combined with picric acid, it is extensively used in neuropathological studies

Picrocarmine

Carmine combine with aluminum chloride that is used for the demonstration of GLYCOGEN

Best's carmine stain

A vegetable dye extracted from certain lichens which are normally colorless, but when treated with AMMONIA and EXPOSED TO AIR, it will produce blue or violet colors

Orcein

A weak acid, soluble in alkali, and is mainly used for staining ELASTIC FIBERS

Orcein

Obtained from lichens, treated with lime and soda, and exposed to ammonia and air Not used as a cytological stain because of its poor staining property Used mainly as an INDICATOR

Litmus

Synthetic dyes are sometimes known as

Coal tar dyes

Originally manufactured from substances that have been taken from coal tar

Synthetic dyes

Derived from the hydro-carbon benzene and are collectively known as

Aniline dyes

Substances with definite atomic groupings and are capable of producing visible colors

Chromopores

Simple benzene compounds which contain such substances are known as

Chromogens

Before chromogen can be called a dye, it needs this substance to be added

Auxochrome

Active coloring substance is found in the acid component and the inactive base is usually the sodium salt of a sulfonate of rosaniline

Acid dyes

The only substance that can fix, differentiate, and stain tissue all by itself

Picric acid

Where the active coloring substance is found in a basic component that combines with the acid radical

Basic dyes

An example of a basic unclear stain that can be used as an indicator and as a dye

Methylene Blue

Tissues fixed in these fixatives usually favor staining with basic dyes

Mercuric chloride and formaldehyde

Formed by combining aqueous solutions of acid and basic dyes, capable of staining cytoplasm and nucleus simultaneously and differentially

Neutral dyes

Because of large molecular complexes, this dye is insoluble or barely soluble in water BUT they are usually soluble in alcohol

Neutral dye

A staining solution most commonly used for routine histologic studies

Hematoxylin

Mordants in hematoxylin used to demonstrate nuclear end cytoplasmic structures are?

Alum and iron

Among the many methods used to demonstrate mitochondria by light microscopy, the most permanent and the simplest is?

Regaud's modification of iron hematoxylin

Hematoxylin variation the is especially for the study of mitosis Can be used after almost any fixative

Haidenhain's Hematoxylin

One of the most valuable stains used for differentially staining connective tissues and cytoplasm Routinely used in histopathology and as a counterstain in hematoxylin

Eosin

Most commonly used eosin. It is readily soluble in water, less in alcohol Available in both aqueous and alcoholic solutions Showing a green yellow fluorescence especially in alcoholic medium

Eosin Y (Yellowish)

Eosin compound that has a very faint bluish cast

Eosin B

A stain that are based on a combination of eosinate (chemically reduced eosin) and methylene blue

Romanowsky stains

Preferred over H and E for inspection of blood cells because different type of leukocytes can be readily distinguished All are also suited to examination of blood to detect blood-borne parasites like malaria

Romanowsky stain

Mixture of picric acid and acid fuchsin for demonstration of CONNECTIVE TISSUE

Acid Fuchsin-Picric Acid (Van Gieson's stain)

May be used to stain collagen, smooth muscle, or mitochondria

Acid Fuchsin (Masson stain)

A basic acridine fluorochrome which permits discrimination between DEAD AND LIVING CELLS Green Fluorescence for DNA and red Fluorescence for RNA

Acridine Orange

Used to demonstrate deposits of calcium salts and possible sites of phosphatase activities

Acridine Red 3B

A complex, water-soluble phthalocyanin dye, similar to chlorophyll, which stains acid mucopolysaccharides by forming salt linkages with them Simple, produces a striking blue color, and it is resistant to various counterstaining procedure

Alcian Blue

Combined with PAS, as it stains acidic mucins BLUE, whereas PAS stains neutral mucins red

Alcian Blue

Forms an orange=red lake with calcium at pH of 4.2 Works best with small amounts of calcium (Such as in michaelis-gutman bodies) Used on the DUPON ACA analyzer to measure serum calcium photometrically

Alizarin Red S

A cytoplasmic stain used for counterstaining of epithelial sections

Aniline Blue

Nuclei are deep red; cytoplasm is pale red

Azocarmine

A plasma stain utilized also for deep staining of acid-fast organisms, for mitochondria, for differentiation of smooth muscles with the use of picric acid

Basic Fuchsin

Sodium metabisulphite 1gm 1N hydrochloric acid 10mL

Coleman's Feulgen Reagent

Sodium metabisulphite ANHYDROUS 1gm NORMAL hydrochloric acid 20mL

Schiff's Reagent

Basic fuchsin 0.5gm 95% ethyl alcohol 50mL DH2O 50mL

Mallory's Fuchsin stain

Concentrated HCL 1mL Paraldehyde 1mL 0.5% basic fuchsin in 70% alcohol 100mL

Aldehyde Fuchsin (Gomori's stain)

Used for staining hemoglobin

Benzidine

A contrast stain for gram's technique, in acid fast and papanicolau's method, and for staining diphteria organisms

Bismarck Brown

Used as a chromatin stain for fresh materials in smear preparations. Slightly soluble in water at a neutral reaction Usually kept in ammoniacal solution which changes its properties due to oxidation

Carmine

Most important component of carmine is?

Carminic acid

Carmine that is combined with aluminum chloride to stain GLYCOGEN

Best carmine solution

A mordanted dye acting as a basic dye and staining acidic substances

Carmalum Solution (Mayer's solution)

An oxazine dye used as an alternative to iron hematoxylin nuclear stain Produce a strong and precise nuclear stain that is resistant to decolorization by succeeding acid stains and solutions

Celestine blue

Best known as an indicator, may be utilized as a stain for axis cylinders in embryos

Congo red

Used as 4% aqueous solution in staining elastic tissues and myelin Used to identify deposits of protein in tissue called amyloid

Congo red

Commonly used in histology to stain nervous tissues Stains the acidic components of the neuronal cytoplasm violet specifically NISSL BODIES

Cresyl Violet

A nuclear or chromatin stain used for staining amyloid in frozen sections and platelets in blood Gentian violet is the staining solution formed by the mixture of this dye, methyl violet, and dexterin

Crystal violet

Routinely used for the identification of specific or highly selective cellular epitopes or antigens in frozen or paraffin-embedded tissues

Immunohistochemistry

Most common antibody in IHC

IgG

Produced by immunizing an animal with a purified specific molecule that contains the antigen of interest and collecting immunoglobulin-rich serum after the animal has produced humoral antibody against the antigen

Polyclonal antibodies

Most frequently used animal for polyclonal antibodies

Rabbit followed by goat, pig, sheep, horse, guinea pig

Animals immunized with the SPECIFIC immunogen will produce numerous clones of plasma cells that in turn will produce the antibody

Monoclonal antibodies

Animal used for monoclonal antibodies

Mice

Techniques that have been developed to produce monoclonal antibodies that do not cross-react with other molecules

Hybridoma and cloning techniques

IHC sections are fixed in

absolute methanol or acetone to prevent immunological activity and prevent destruction of some oft he labile antigenic sites

Pre-treated with proteolytic enzymes to break down formalin cross-linking, unmask and allow certain antigenic sites to be exposed

Formalin fixed paraffin sections

Proteolytic enzyme digestion is specially useful for demonstrating

Heavy chain immunoglobulins, complement and specific antigens such as cytokeratin in formalin-fixed paraffin embedded biopsies

Most common enzymes for proteolytic enzyme digestion is

Trypsin and protease

A simple laboratory technique that has become an essential part of many immunohistochemistry and in situ hybridization procedures, is a pretreatment method used to improve staining results

Heat-induced epitope retrieval

HIER heating sources

Microwave, vegetable steamer, pressure cooker, and water bath

A relatively new technique that involves the boiling of formalin-fixed deparaffinized sections in certain solutions, such as 0.01 M-citrate buffer (pH 6.0), EDTA at pH 8.0 or Tris Edta (pH 10.0)

Microwave antigen retrieval

Another alternative of antigen retrieval that appears to be less time consuming and allows for more consistent recovery of many antigens, compared to large batch microwave oven technique

Pressure cooking antigen retrieval

A highly sensitive marker for epithelial cells, and is present in epithelial tumors.

Keratin

More frequently found in carcinomas of the lung, breast, uterus, and ovaries Negative for CK20

CK7

More common in carcinomas of the colon and stomach Negative for CK7

CK20

a HMW protein that is helpful in determining the site of tumoe

Epithelial membrane antigen

An oncofetal antigen that is present in carcinomas of the gastrointestinal tract, pancreas, lung, breast, ovary, uterus, and cervix

Carcinoembryonic antigen (CEA)

CEA IS ___ in adenocarcinoma and ____ in mesothelioma

Positive in adenocarcinoma Negative in mesothelioma

Useful in distinguishing lung adenocarcinomas from mesotheliomas

TTF-1 Thyroid transcription factor -1

Extremely useful in the diagnosis of prostatic adenocarcinoma

PSA

A contractile intermediate filament protein present in muscle and some non-muscle tissue Sensitive marker for muscle differentiation and can be used to identify tumors derived from smooth, skeletal, and cardiac muscle

Actin

a 57kD intermediate filament protein present in normal mesenchymal cells and their neoplastic counterparts

Vimentin

Melanomas, and schwannomas always stain positive for

Vimentin

a 53kD intermediate filament expressed by smooth and striated muscle Highly specific for myogenic tumors, including leiomyoma, and rhabdomyosarcoma

Desmin

a 51 kD intermediate filament protein expressed by CNS glial cells, particularly astrocytes Widely used to confirm the diagnosis of astrocytoma

Glial fibrillary acidic protein

Expressed in cells of neural origin, particularly neurons, neuronal processes, peripheral nerves, sympathetic ganglia, adrenal medulla, and neuroendocrine cells

Neurofilaments

a low molecular weight CALCIUM-BINDING protein that is expressed in CNS

S-100 protein

Most common immunohistochemical markers used to assess proliferation of tumor cells

Ki-67 and PCNA

____ considered to be a more sensitive method than immunofluorescence

Chromogenic detection

More sensitive than the traditional direct technique, and is suitable for frozen section immunohistochemistry

Enhanced polymer one-step staining method

A technique in immunohistochemistry that is a 2 or 3 step procedure that involved application of the unconjugated primary antibody, followed by a labeled antibody directed against the first antibody

Indirect technique

Most commonly used enzyme for indirect antibody enzyme-complex techniques

Horseradish peroxidase

Remains a standard method among most laboratories, especially in combination with frozen section processing for immunofluorescence of renal and skin biopsies

Paraffin wax section immunoperoxidase technique

Most common chromogen

Diaminobenzidine (DAB)

Recommended technique for use on blood and bone marrow smears

APAAP

Cytologic collection and preparation Samples of endocervical canal

Endocervical brush

Cytologic collection and preparation For patients with hysterectomy

Vaginal scrape

Cytologic collection and preparation Used for vaginal hormonal evaluation

Lateral vaginal scrape

Cytologic collection and preparation Localization of vaginal adenosis

Four quadrant vaginal scrape

Cytologic collection and preparation Detection of herpetic lesions or carcinoma

Vulvar scrape

Pale pink cytoplasm and dark pyknotic nuclei

Mature superficial cells

Elongated cells with basophilic vacuolated cytoplasm

Intermediate cells

BOAT shaped intermediate cells with strong tendency to fold/curl on edges

Navicular cells

Round/oval boat shaped cells with translucent basophilic cytoplasm observed greatest at the center of the cell

Pregnancy cells

Round to oval (fried sunny side-up egg appearance)

Parabasal cells

Small cells, slightly cylindrical with less basophilic cytoplasm, occurring in tightly packed groups of 3 or more

Endometrial cells

Honeycomb appearance

Endocervical glandular cells

Found in puberty after menopause

Basal cells

Most common organism of the normal vaginal flora Maintains the acidic pH of vagina Stains BLUE to LAVENDER with pap's method

Doderlain Bacillus

Abnormal variation of squamous epithelial cells that are granular and irregular in appearance Indicative of bacterial vaginosis (G. vaginalis)

Clue cells

Wrinkled prune appearance surrounded by perinuclear halo Squamous cells that shows CPE of HPV

Koilocyte

Fern or palm leaf patter

Arborization

Basis of early detection of early pregnancy

Fern test

A dirty knife edge can cause this fault

Sections roll up