HPCT 2ND AND 3RD WEEK Flashcards
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
Most commonly used clearing agent
Xylene
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
Most important step in embedding
Orientation
Most common, routinely used embedding media
Paraffin wax
Used in tough tissues (embedding media)
Celloidin
Used in tissues that we want to prevent dehydration histochemistry (embedding media)
Gelatin
Embedding media for electron microscopy
Plastic
Melting point of paraffin wax (Routine)
56C
Process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cell
Staining
Main reason why cells are stained
To enhance contrast and visualization of the cell or certain cellular components under the microscope
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
Process where by constituents of tissues are studied THROUGH CHEMICAL REACTIONS that will permit microscopic localization of specific tissue substance
Histochemical staining
Combination of immunologic and histochemical techniques to detect phenotypic markers under microscope
Immunohistochemical staining
Purified form of a coloring agent or crude dye that is generally applied in an aqueous solution
Histological stain
Parts of the cells or tissue that are ACIDIC take more of (nucleic acid)
BASIC dye
Parts of the cells or tissue that are BASIC take more of (Cytoplasm)
ACIDIC dye
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
Process whereby the action of the dye is intensified by ADDING other agent or mordant
Eg. Mordant, accentuator
Indirect staining
E.G Gram Stain
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
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
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
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
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
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
- Clear paraffin embedded sections in first xylene bath for 3 mins
- Transfer to second xylene bath for 2 to 3 minutes
- Immerse in first bath of absolute ethyl alcohol for 2 mins
- Transfer to a bath of 95% ethyl alcohol for 1 or 2 minutes
- Rinse in running water for 1 minute
- 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 - Differentiate in 1% acid-alcohol (1mL concentrated HCL to 99ml of 80% ethyl alcohol) for 10-30 seconds
- Rinse in tap water
- Blue in ammonia water (5 mins ave) or 1% aqueous lithium carbonate until the sections appear blue
- Wash in running water for 5 mins
- Counterstain with 5% aqueous eosin for 5 mins. If alcohol eosin is used, the time can be reduced to 30seconds or 1 min
- 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
- 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
