HPCT - MICROTOME AND STAINING

Microtomy

Processed tissue is trimmed and cut into uniformly thin slices called sections. Facilitates studies under microscope. Cutting tools: knife, glass, or diamond blade. Microtome: basic instrument used.

Block holder

Where tissue is held in position.

Knife carrier and knife

Cutting sections.

Teeth/pawl, Ratchet Feed wheel and Adjustment screw

Lines up the tissue block in proper position with the knife, adjusting the proper thickness of the tissue for successive sections. Used for proper orientation of tissue adjacent to the cutting knife.

Rocking (cambridge) microtome

For cutting serial sections of large blocks of paraffin embedded tissues. By Paldwell Trefall. Simplest microtome. Consists of heavy base and two arms. Lower arm resting on pivots and supporting column. Attached to the micrometer screw at the base of which is found the ratchet wheel with feed mechanism. Upper arm carries the block holder on one end by means of screw connected to a lever by a piece of nylon thread. 10-12 UM. Disad: restriction in terms of size and reorientation.

Rotary (Minot) Microtome

For cutting paraffin embedded sections. Most common type used for both routine and research laboratories. For paraffin embedded tissues. Operated with staged rotary action wherein cutting is also part of the rotary motion. Can be manual, semi-manual, automated. 3-5um (paraffin) or thinner (synthetic resin)> Excellent serial sections. More stable.

Disadvantage: More complex design

More expensive

More dangerous. Heavier knife produces lesser vibration. Adjustable cutting angle

Sliding microtope

For cutting celloidin embedded sections. By Adams.

Base-sledge microtome.

Consists of 2 movable pillars holding the adjustable knife clamps. The adjustable nature of these clamps allows the knife to be set at an angle. Adjustable cutting angle. Block holder - set on a heavy metal base under the knife. Heavier than standard sliding microtome. 24 cm knife. Mainly for hard tissue or large blocks. Ideal for resin embedded decalcified bone. Electrically driven for modern models.

Standard sliding microtome

Stationary block while knife is moving backward and forward. Adjustable cutting angle for celloidin embedded tissue blocks. Both for large and small tissues. Recommended for extremely hard and tough tissue blocks.

Freezing microtome

For cutting unembedded frozen sections. By Queckett. For rapid diagnosis. Used to demonstrate fat, neurological structures, heat labile tissue constituents. Parts: moving bulb and stationary tissue block. Lever operated bulb that allows the release of rapid intermittent burst of carbon dioxide. Freezes the block holder and tissue using carbon dioxide. Recommended for undehydrated thin to semi-thin sections of fresh frozen tissues.

Cryostat (Cold) Microtome

For cutting frozen sections. Rotary microtome inside a cold chamber. -5 to -30C. Adjustable by use of thermostat. Freezes the tissue in 2-3 minutes. Cuts the tissues with thickness of 4 um. Prepares tissue for fluorescent antibody staining and histochemical enzyme studies. Most commonly used for urgent biopsies during intraoperative diagnosis.

Ultrathin microtome

Can produce very thin section. Equipped with glass or gem grade diamond knife. Tissue thickness: 60-100 nm. Epoxy resin tissues or 0.5-1um. Tissue stained with aqueous solution of heavy metal salt. Examined under TEM.

Plane concave knife

Length: 25 mm. Flat on one side; concave on the other side. Less concave side: for celloidin embedded tissues. More concave side: For paraffin embedded tissues such as base-sledge, rotary and rocking microtome.

Biconcave knife

Length: 120 mm. Concave on both sides. Recommended for paraffin embedded tissues such as rotary microtome.

Plane-wedge knife

Length: 100 mm. Straight on both sides. For frozen sections or extremely hard and tough specimens embedded in paraffin.

Honing

Sharpening of badly nicked knives with blunted ends. Ensure optimum sectioning of tissue blocks and prevents gross irregularities on tissue sections. Jagged edges produces striations and tears in tissue sections

Honing (Hard sharpening)

Involves removal of gross nicks on the knife edge or also called as coarse honing. To remove blemishes and grinding the cutting edge of the knife on a stone to acquire an even edge. Degree of sharpness = finesses of the abrasive used in sharpening. Makes use of hone (carborundum)

Belgium yellow

Manual sharpening gives best result. Used when the cutting edge has been rendered blunt or nicked. Gives the best result.

Arkansas

More polishing than Belgium yellow.

Fine carborundum

More coarse than the other two. Used forr badly nicked knifes followed by either of the first two knife sharpeners.

Xylene

Used to moisten the surface to remove scattered small particles of stone or metal.

Diamanthine

Used for final polishing, this is a powder that produces a fast cutting abrasive for polishing pivots and jewels. Leaves a mirror finish.

Stropping

Burr formed during honing is removed and cutting edge of the knife is polished. Burr is the bit of waste metal forming at the edges of our knives. Polishes and sharpens the cutting edge.

Disposable blades

Cheaper than conventional steel knives. No longer commonly used in the modern la. Sharp cutting edge: cut 2-4um thickness. Magnetic knives: used for cryostat.

Glass knives

For trimming and sem-thin sectioning for electron microscopy. Commercially available in 40 × 2.5 cm plate glass strips. Washed with detergent and rinses with distilled water & alcohol then later on, dried with lint free paper. Stored in dust free boxes.

Diamond knives

Cuts resin blocks for electron microscopy. May be brittle and expensive but very durable. Cutting edge must be kept clean.

Section cutting

Creates sequence of connected sections which are called ribbons.

Mounting

Last step in tissue processing. After draining, sections are fixed on slides by heat. 37C overnight. Wax oven at 50-60C for 2 hours. Hot plate at 45-55C for 30-45 mins. Delicate tissues for 37C for 24 hours.

Adhesives

Substances which can be smeared on top of the slides so that the sections stick well to the slides. Not necessary for routine staining provided that the slides are clean and free from grease.

Albumin

Most commonly used adhesive. Equal part glycerin + distilled water + egg white + crystal of thymol. Disadvantage: retains some stain giving dirty background. Thymol resistant organims contaminate gram stained sections.

Mayers egg albumin

50 cc egg white + 50 cc of glycerin +100 mg crystal of thymol. Most commonly used because it is very easy to make, convenient. and relatively inexpensive.

Dried albumin

Dried and stored in 70% alcohol before staining. 5g dried albumin + 5g NaCl + dissolved in 100 ml distilled water + crystal of thymol.

1% Gelatin

1g gelatin + distilled water + 100 ml glycerol + 2g phenol crystal. Add up to 30 ml of 1% aqueous gelatin in floating out bath. Most convenient alternative to direct coating of slides.

Gelatin-formaldehyde mixture

5 ML 1% gelatin+ 5 ml 2% formaldehyde. Dry the slide coated with this at 37C for 1 hr or overnight before use.

Poly-L-Lysine

Bought at 0.1% solution and diluted in distilled water. Loses effectiveness after few days. Must be used within a few days since effectiveness slowly decreases over time. Widely used section adhesive in immunohistochemistry.

Aminopropyltriethoxysilane

Better section adhesive and coated slides can be stored fro a long time. Very useful in cytology. Can be stored for a long time.

Aqueous mounting media

Mounting sections from distilled water when stains would be decolorized or removed by alcohol and xylene. Made up of gelatin, glycerin jelly or gum arabic. Glycerol prevents cracking and drying of the preparation. Sugar increases refractive index.

Water

Low refractive index. Moderately transparent and evaporates easily, hence is good only for temporary mounting. Prevents tissue to be examined under OIO.

Glycerin

May also be used as preservative . High index of refraction and provides greater visibility if diluted with water. Suitable for semi-permanent mounting medium with RI of 1.46. Sets hard and keeps sections mounted for years. Standard mountant for fat stains. Difficult to prepare slides that are truly permanent. Can be attacked by microorganisms. Also known as Kaiser’s 1880. Jelly has RI of 1.47

Farrant’s medium

RI of 1.43. 50g gum Arabic + 50 ml dist water + 50 ml distilled water + 50 ml glycerol + 0.025 g Na merthiolate/ arsenic trioxide. Addition of potassium acetate raising RI to 1.44. Not solidify upon storage. Takes longer time to solidify and may require ringing.

Apathy’s medium

RI of 1.52. 50 pure gum arabic + 50g pure cane sugar or sucrose + 50ml distilled water + 0.05g thymo crystal. Used for methylene blue stained nerve preparation. General purpose aqueous mountant. One of the most useful aqueous mountnats for fluorescent micrscopy. Not compatible with normal histological stains.

Bruns fluid

24g glucose + 6 ml glycerine + 6ml of spirits of camphor + 84 ml distilled water. Recommended for mounting frozen sections from water.

Resinous mounting media

Used for preparations that have been dehydrated and cleared in xylene or toluene. Recommended for majority of staining methods.

Canada Balsam

RI of 1.524. Natural resin extracted from Canadian tree Abus balsamea. Dissolved in xylene in 37C or paraffin oven at 58C. Filtered to obtain the desired consistency. Transparent, almost colorless that adheres firmly to glass and sets to a hard consistency without granulation. Recommended for whole mounts and for thick section because it does not shrink too much. Sets hard without granulation; but expensive.

DPX (Dibutyl Phthalate and Xylene)

RI of 1.532. Recommended for small tissue sections but not for whole mounts. Colorless neutral medium in which most standard stains are well preserved.

XAM

RI of 1.52. Synthetic resin mixture in xylene, pale yellow or colorless solution. It dries quickly without retraction, and preserves stains well. Sections here are quickly mounted from xylene.

Clarite X

RI of 1.544. Synthetic resin which is soluble in xylene. Preferred over DPX.

Floatation water bath.

Black inner wall. Temperature must be 6-10C lower than the melting point of wax.

Paraffin oven

Used to dry the specimen after fishing out. Temperature is maintained at 2-5C above the melting point of the paraffin wax. 62-65C. Overheatign causes dissolution of important components.

Staining

Process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cells. Nuclei are stained with basic dyes by forming dye salt union. Cells are colorless and transparent. Enhances contrast and visualization of the cell or certain cellular components; Highlights metabolic processes. Differentiates between live and dead cells. Demonstrates the relationship between internal and external structures of the cells. Identify different type of cells.

Histological staining/Microanatomical

Tissue constituents are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue component. Demonstrates the general relationship of tissues and cells with differentiation of nucleus and cytoplasm. Microanatomic stains, bacterial stains, and specific tissue stains.

Histochemical staining (Histochemistry)

Constituent of tissues studied through chemical reactions that will permit microscopic localization of a specific tissue substance. Perl’s prussian blue and PAS.

Immunohistochemical staining

Immunologic + histochemical techniques. Labeled antibodies to detect antigens/phenotypic marker. Used to identify abnormal cells found in cancerous tumors. Localization of bionmarkers and differentially expressed proteins in different parts of a biological tissue. Detection of specific molecular markers. Senstive to fixative selection and duration.

Direct staining

Process of giving color to the sections by using aqueous or alcoholic dye solutions. Only one dye is used. Ex, Methylene blue

Indirect staining

Process whereby the action of the dye is intensified by adding another agent or mordant. Allows subsequent counterstaining and dehydration. Mordant ex. Potassium alum for ehrlich hematoxylin, iron for Weigert hematoxylin. Accentuator: e. potassium hydroxide for Loeffler's MB, phenol for

Carbol thionine and Carbol fuchsin

Progressive staining

Process whereby tissue elements are stained in definite sequence. Specific periods of time or until the desired intensity is attained. Not washed or decolorized. Relies on selective affinity of dye to cellular elements.

Regressive staining

2 steps. 1st: overstained. 2nd: removed or decolorized excess stain from unwated parts of the tissue.

Differentiation/Decolorization

Selective removal of excess stain from the tissue during regressive staining. Used in simple and differential stains.

Metachromatic staining

Use of specific dyes which differentiate substances by staining them with a color that is different from that of the stain itself. Used in cartilage, connective tissues, epithelial mucins, mast cells granules, and amyloid. Belongs to the thiazine and triphenylmethane groups.

Metallic impregnation

Use colorless heavy metal salts which are selectively precipitated on certain cellular and tissue components. Produces opaque, black deposits on tissue surface. Utlized for silver staining of the CNS and reticulin fibers. Silver nitrate and gold/Gold chloride (Most commonly used embedding medium, but also stain). Ammoniacal silver - for argentaffin cells.

Vital staining

Selective staining of living cell constituents. Demonstrating cytoplasmic structures by phagocytosis of the dye particle. Staining of pre-existing cellular components. Excluded by living cells but taken up by dead cells. Ex. staining of RES with trypan blue. Reveals cytological details that might otherwise not be apparent. Nucleus of living cells is resistant to vital stains.

Intravital staining

Injecting the dye into any part of the animal body. Ex: Lithium, carmine, and india ink.

Supravital staining

Examines living cells that have been removed from an organism. Enter and stains living cells. NMB and BCB for detection of reticulocytes. Neutral red-best vital dye.

Counterstaining

Application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated. Similar to differential staining. Utilizes different colors of stains.

Coplin jar

Slotted jar used for few samples.

Slotted staining dishes

Different solutions are poured. Slides are placed on end singly or in staggered fashion. For many samples

Metal or glass staining racks or carrier.

Slides are transferred to appropriately sized glass dishes containing the staining solutions.

Acid dyes

Where coloring substances are found in the acid component and soidum is the agent used to remove the base radical. Most frequently are sodium salts (orange G, picric acid).

Basic dyes

Where the coloring substance is found in the basic component, the acid radical usually taken by sulfuric or hydrochloric acid. Most frequently are chloride salts.

Neutral dye

Capable of staining cytoplasm and nucleus simultaneously and differentially. Soluble in alcohol but insoluble in water.

Natural dyes

Cochineal dyes, logwood dyes, and vegetable extracts. Can be from insects, vegetables, or plants.

Synthetic dyes.

Aniline or coal tar dyes. Derived from hydrocarbon benzene and are known as aniline dyes.

Hematoxylin.

Natura dye derived from the heartwoo of a mexican tree known as Hematoxylin campechianum. COnsidered as the most valuable stain by cytologist. Hematein is the active coloring agent

Alum Hematoxylin

Recommended in progressive staining of the tissue. Usually the counterstain is eosin, and n some cases Congo red and sfaranin. Used in routine H and E. Mordant: Potassium alum. Produces good nuclear stain: Red

Iron hematoxylin

For differential and regressive staining. Used as oxidizing and mordant. Weigert - Ferric chloride. Heidenhain - ferric ammonium sulfate.

Tungsten hematoxylin

Mallory’s PTAH. For staining of muscle striations.

Copper hematoxylin

For spermatogenesis

Cochineal dyes

An old histologic dye drived from extract from the female cochineal bug, which is treated with alum to produce carmine. Powerful chromatin and nuclear stain. With picric acid → Picrocarmine for neuropathology

Orcein

Natural vegetable dye extracted from certain Licehns. Can produce blue or violated after treated with ammonia an oxidizing. Weak acid. Soluble in alkali. Mainly used for elastic fibers. Demonstration of HbsAg.

Saffron

Derived from the dried stigmata of Crocus sativus or Saffron crocus.

Eosin

Counterstain used for alum hematoxylin. Most commonly used cytoplasmic stain. Commonly used as a background for contrasting stains. Red acid dye, anionic dye. Best staining occurs at 4.6 to 5.

Eosin Y

Most commonly used. Yellowish in appearance but output has green yellow fluorescence in alcoholic medium.

Bluish

Also called imperial red. Faint bluish cast.

Ethyl eosin

Substitute for Eosin Y as a counterstain in H&E method.

Methylene blue

Commoon basic nuclear stain employed with eosin to provide marked differentiation of various structures of the tissue. Used as is

Methylene violet

Metachromatic formed whenever MB is heated in fixed alkali or alkali carbonate. For detection of RNA.

Toluidine blue

Used as a substitute for thionine in fresh frozen tissue section. Used to check for dysplasia, carcinoma, mast cells.

Crystal violet

Used for staining amyloid in frozen sections and platelets in blood. Used in hematology. Can also be used in gram stain reaction.

Aniline blue

Cytoplasmic stain used for counterstaining of Epithelial sections

Carbol fuchsin

Basic fuchsin. Acid fast organism. Can be used in hot method/Ziehl Neelsen method or coldmethod/kinyoun

Coleman’s feulgen reagent

Mitochondria. Good for highlighting DNA

Schiff’s reagent

Smooth muscle. Results in magenta like or violent in color. Detects presence of aldehydes

Mallory fuchsin

Also known in Mallory’s trichrome staining → visualization of connective tissues

Aldehyde fuchsin

Also known as Gomori stain. For muscle tissue.

Van gieson’s stain

Demonstration of connective tissue

Giemsa stain

Used for staining blood to differentiate leukocytes

Celestin blue

Resistnat to strong acid dyes. Nuclear stain.

Methyl green

Used to stain chromatin

Bismarck brown

Serves as contrast for Gram staining. Stain that does not stain. Used for tissues, acid mucin, cartilage, and bones.

Prussian blue

Colored salt of ferric ferrocyanide. Stains anything that has iron