What is Rocking microtome , write its advantages ?
Cambridge rocking microtome
The instrument is named because arm has to move in a specific rocking motion while cutting the sections. It is a simple machine in which the knife is held by means of microtome thread. The rocking microtome was designed specifically for cutting paraffin wax sections but in an emergency we can also use frozen section by inserting a wooden block in which the tissue is frozen.
It cuts the sections between 1 to 20 microns. The knife of rocking microtome is fixed with the edge, while the object is moved against this knife in circular motion, producing a sharply curved surface to the block with each stroke the tissue holder automatically moves vertically towards the life. Cutting stroke is Spring operated and is easy to handle. Precautions :
Microtome must be placed on a solid non-slippery surface to allow a better hold.
Advantages of Cambridge rocking microtomes
The cost of a knife and microtome is low.
Celloidin embedded tissues can be sectioned easily.
Why clearing is necessary in tissue processing ?
Clearing is an importnat step in tissue processing for light microscopy. The purpose of clearing is to remove dehydrating agents from tissues and to prepare the tissues for impregnation with the embedding agent.
Xylene is used most common \clearing agent worldwide
What is principle of PAS stain ?
PAS - McMANNUS' PERIODIC ACID SCHIFF'S - GLYCOGEN
PURPOSE:
Glycogen is present in skin, liver, parathyroid glands and skeletal and cardiac muscle. The PAS stain is used for demonstration of basement membranes, fungus secreting adenocarcinoma from undifferentiated squamous cell carcinoma, and mucosubstances secreted from the epithelia of various organs. A routine stain for liver and kidney biopsies.
PRINCIPLE:
The PAS stain is a histochemical reaction in that the periodic acid oxidizes the carbon to carbon bond forming aldehydes which react to the fuchsin-sulfurous acid which form the magenta color.
Briefly decribe artifacts of fixation ?
Following are the artifacts of fixations such as :
Formaldehyde fixative give brown pigmentation to tissues
Mercuric chloride leaves a black precipitate of mercury in tissue
Some fixation produce shrinkage in tissue & some causes swelling
Due to poor penetration of fixatives in biochemical molecules like glycogen diffuse from unfixed parts giving false localization or considerable loss . This term is know as streaming artifact and most probably observed in glycogen.
What is Vapour fixation ?
Vapour fixatives may be used to fixed cryostat -cut section of fresh tissues and section or block of frozen dried tissue.
Formaldehyde vapours are genrated from heated paraformaldehyde is a very high reactivity.
Cryostat sections mounted on slides may be placed in a closed vessels above the formaldehyde & the vessels placed in an oven at 60-70 degree celcius fpr two hours
Using this method we have produced sections showing excellent preservation of glycogen with a very good morphology details.
Enlist various things required for gross section of tissue ?
Following are the materials required to make a gross section of tissue such as : Microtome
Microtome Knife Water bath Forceps
Freezing plate
Glass slides
Give principle of aldehyde fixatives ?
Crosslinking fixatives – aldehydes
Crosslinking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. Preservation of transient or fine cytoskeletal structure such as contractions during embryonic differentiation waves is best achieved by a pretreatment using microwaves before the addition of a cross linking fixative.
The most commonly used fixative in histology is formaldehyde. It is usually used as a 10% neutral buffered formalin (NBF). Since formaldehyde is a gas at room temperature, formalin – formaldehyde gas dissolved in water (~37% w/v) – is used when making the former fixative. Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine. Its effects are reversible by excess water and it avoids formalin pigmentation. Paraformaldehyde is also commonly used and will depolymerise back to formalin when heated, also making it an effective fixative.
Another popular aldehyde for fixation is glutaraldehyde. It operates similarly to formaldehyde, causing the deformation of proteins' α-helices. However glutaraldehyde is a larger molecule than formaldehyde, and so permeates membranes more slowly. Consequently, glutaraldehyde fixation on thicker tissue samples can be difficult; this can be troubleshot by reducing the size of the tissue sample. One of the advantages of glutaraldehyde fixation is that it may offer a more rigid or tightly linked fixed product—its greater length and two aldehyde groups allow it to 'bridge' and link more distant pairs of protein molecules. It causes rapid and irreversible changes, is well suited for electron microscopy, works well at 4 oC, and gives the best overall cytoplasmic and nuclear detail.
These crosslinking fixatives, especially formaldehyde, tend to preserve the secondary structure of proteins and may also preserve most tertiary structure.
Working Principle of Phase Contrast Microscopy with help of diagram ?
Write the principle of reticulin stain ?
Reticulin fibers have little natural affinity for silver solutions. On Treatment with potassium permaganate it produces sensitised sites on fibers where deposition can be initiated
The silver is in the form of readiliy able to precipitate as metallic silver. The optimal pH for maximum uptake of silver deposition of silver in the form of metal.
Reaction of fixatives with lipids ?
Phosholipid are fixed by aldehydes.
Formaldehyde reacts with unsaturated fatty acids hence less lipid can be demostrated in tissue stored in it for a long time.
Mercuric chloride reacts with lipid to form complexes.
ultrastructural demonstration of lipids -Post fixing in imidiazole-osmium tetroxide
Enlist various chemical used in tissue processing ?
Tissue processing involves many steps & different kinds of chemical are used of each steps here is the detail of tissue processing and chemical used in tissue processing :
POST FIXATION OF TISSUE
DEHYDRATION
CLEARING
IMPREGNATION & INFILTRATION
1.DEHYDRATION
The first stage in tissue processing is dehydration (the removal of water). In tissues, water is present in both free and bound forms and needs to be removed before processing can continue. Dehydration is usually carried out using alcohols (such as ethanol) but these can dissolve certain cellular components such as lipids. Although dehydration can also cause tissue shrinkage, the stage is necessary in all infiltration methods, except where tissues are supported by an aqueous embedding medium (such as water-soluble waxes).
In paraffin wax processing, dehydration from aqueous fixatives such as formalin is usually initiated in 70% alcohol before progressing through 90%-95% to absolute alcohol before proceeding to the clearing stage. However, direct transfer to 95% alcohol is often performed if tissues are adequately fixed. Duration of dehydration is dependent on tissue thickness; the thicker the block, the longer the time. Generally, blocks 1 mm thick should receive up to 30 minutes while blocks 5 mm thick require up to 90 minutes or longer in each change.
EXAMPLES OF DEHYDRATING AGENT
Acetone
This is a colourless flammable liquid with a characteristic odour, low toxicity and is freely miscible with water and organic solvents. Acetone is fast and effective as a dehydrant and may also act as a coagulant secondary fixative. Acetone is ideal for fatty tissue samples and they can be transferred directly from acetone into paraffin wax.
Alcohols
These are clear, colourless, flammable and hydrophilic liquids that are miscible with water and most of the organic solvents. In addition to their role as dehydrants, alcohols also act as secondary coagulant fixatives during tissue processing. The most commonly used alcohol used in tissue processing is ethanol.
Butanol
This alcohol is mainly used for plant and animal tissues. N-butanol causes less hardening and shrinkage than ethanol but is poorly miscible with water and paraffin wax so longer times are required. It is flammable, has a penetrating odour and is an eye irritant. Iso-butanol has similar properties but is a less costly substitute for n-butanol.
Ethanol (ethyl alcohol)
This is a rapid and efficient dehydrant and is the most commonly used. Dehydration is usually initiated in 75% alcohol with progress through 90%-95% ethanol before several changes of absolute ethanol to complete dehydration. Progressive removal of bound water from carbohydrates and proteins during prolonged immersion in absolute ethanol causes tissues to harden excessively and become brittle. Colloid, blood, collagen and yolky tissues are particularly affected.
Dioxane
Also known as diethylene dioxide, this is a colourless, flammable liquid that produces less shrinkage and hardening than with ethanol. The liquid is miscible with water, most organic solvents and paraffin wax and is excellent for tissues that have been excessively hardened by conventional processing. Dioxane has a rapid but gentle action and tissues are able to remain in it for long periods without harm.
Isopropanol
This is completely miscible with water and most organic solvents and is fully miscible with molten paraffin wax. Isopropanol shrinks and hardens tissues and is used to dehydrate hard, dense tissues. It is less severe than ethanol but tissues may be transferred from 60%-70% to absolute isopropanol to minimize shrinkage. Isopropanol can be used as a xylene substitute.
Methanol
This reagent is a good ethanol substitute but is rarely used because it is volatile, flammable and costly. Methanol tends to harden tissues more than ethanol and is a poor lipid solvent.
Phenol
This consists of clear hygroscopic crystals and is also available in a liquefied form. Phenol is soluble in water, alcohol and most organic solvents. However, phenol develops a pink discolour on exposure to air and light so containers must be protected from light and tightly sealed.
2.CLEARING
Clearing is the transition step between dehydration and infiltration with the embedding medium. Although tissues are water-free following dehydration, infiltration with wax cannot be carried out because wax and ethanol are largely immiscible. Many dehydrants are immiscible with paraffin wax and a solvent (clearing agent or ante medium) miscible with both the dehydrating agent and the embedding medium is used to assist the transition between these steps. The term clearing arises because some solvents have a high refractive index. When dehydrated tissues are placed into these reagents, they are rendered transparent. This property is used to determine the endpoint and duration of the clearing step since the presence of opaque areas indicates incomplete dehydration. Clearing agents are fat solvents and therefore remove fat from the tissue. It must be noted that shrinkage occurs when tissues are transferred from the dehydrating agent to the clearing agent and from the clearing agent to wax. In the final stage shrinkage may result from the extraction of fat by the clearing agent. Xylene is the most popular clearing agent and several changes of it are required to completely displace the ethanol. The choice of a clearing agent depends upon the type of tissue processor used, the processing conditions such as temperature, safety factors and cost.
Examples of clearing agents
Amyl acetate,methyl benzoateandmethyl salicylate
These are chiefly used as nitrocellulose solvents in double embedding techniques. They have low toxicity, but their strong penetrating odours necessitate good laboratory ventilation. They are ideal for manual processing as tissues may be left in them for extended periods without hardening. These esters are difficult to eliminate from paraffin wax and should be extracted from tissues with one or two brief changes of toluene or similar solvent before passing through two or three changes of wax. Methyl benzoate and methyl salicylate render tissues completely transparent and are used for clearing helminthes parasites for examination and whole mounting. Methyl salicylate clears tissues from 96% ethanol, hardens less and has a more pleasant odor than methyl benzoate. It causes minimal tissue shrinkage and hardening and tissues can remain in it indefinitely without harm. This ester is one of the best though expensive transition solvents.
Benzene
Benzene is more gentle and rapid than xylene and toluene and is probably the best transition solvent However, its toxicity and carcinogenicity has preclude its use in histology.
Butyl acetate
This is used as a xylene substitute and nitrocellulose solvent.
Carbon tetrachloride
Because of its high toxicity it is now rarely used in histology.
Cedarwood oil
This is largely composed of cedrene, rapidly clears tissues from 95% alcohol, hardens tissues the least of all the transition solvents, but is difficult to eliminate from tissues during wax infiltration. It is particularly useful for processing dense tissues such as uterus or scirrhous carcinomas, and has a role in forensic histopathology in processing the hardened skin margins of electrical burns and bullet wounds. Tissues can remain in cedarwood oil indefinitely without harm. Low viscosity refined oil should be used for clearing. Formation of crystalline cedrol in cedarwood oil can be overcome by the addition of 1 ml xylene or toluene to 80 ml cedarwood oil. Cedarwood oil is expensive, but exhausted oil can be restored by filtering, then heating to 60°C under vacuum for 30-60 minutes.
Chloroform
This is an expensive, volatile but slowly penetrating solvent. It causes less brittleness than xylene and is often used on dense tissues such as uterus. However, it attacks some plastics and sealants so is not generally recommended for enclosed processors.
Limonene(d-limonene)
This is derived from citrus fruit and is a component of various proprietary blends of transition solvents such as Histoclear and Citroclear marketed as xylene substitutes. It is less viscous than cedarwood oil and is similar to the esters in clearing action and in elimination from wax. Limonene may cause allergic skin reactions.
Methyl benzoateand methyl salicylate
See Amyl acetate.
Terpenes
Terpenes are isoprene polymers found in essential oils originally derived from plants, though some are now synthesized. They are the earliest transition solvents to be used in histology and include turpentine and oils of bergamot, cedarwood, clove, lemon, origanum and sandalwood. In general the natural oils are not highly pure compounds but contain several substances. Many terpenes clear tissues and celloidin sections from 80%-95% alcohol, render tissues transparent and have a slow gentle non-hardening action. Most are generally regarded as safe though some have particularly strong odours which can be overpowering, requiring good laboratory ventilation.
Tissue penetration is aided and shrinkage minimized by diluting viscous terpenes. Terpenes have low evaporation rates and are difficult to eliminate from paraffin wax, necessitating one or two 30 minute changes of toluene or similar solvent to remove the terpene before infiltration with wax. Brief immersion in toluene does not negate the effectiveness of the terpene. Alternatively, tissues are given three, four or more changes of wax until the terpene has been eliminated. Although biodegradable, terpenes are not water miscible and should not be flushed away with water, but disposed of by recycling or incineration.
TRICHLOROETHANE :
This is commonly used as a xylene substitute and is a component of agents such as Inhibisol and CNP30. These solvents are stable to light but tend to slowly liberate hydrochloric acid on contact with water. Because of their high volatility, members of this group may achieve and exceed maximum allowable concentrations in poorly ventilated laboratories far more rapidly than xylene under the same conditions.
XYLENE & TOULENE
These agents clear rapidly and tissues are rendered transparent, facilitating clearing endpoint determination. Concerns over the exposure of personnel to xylene relate mainly to the use of the solvent in coverslipping rather than in processing and xylene substitutes can be used in these circumstances. Xylene hardens tissues fixed in non-protein coagulant fixatives and prolonged clearing in the solvent should be avoided. Industrial grade xylene may contain nearly 25% of other solvents such as ethyl benzene, with traces of benzene, odorous mercaptans and hydrogen sulphide. Only the sulphur and benzene-free solvent-grade xylene should be used for histological purposes.
INFILTRATION AND EMBEDDING
Infiltration
This is the saturation of tissue cavities and cells by a supporting substance which is generally the medium in which they are finally embedded. The most common agent of choice is paraffin wax which is molten when hot and solid when cold. An infiltrating and embedding medium should ideally be molten between 30°C and 60°C and suitable for sectioning. Additionally, the properties of the medium should be similar to those of the tissues to be sectioned with regard to density and elasticity. Various substances have been used to infiltrate and embed tissues in readiness for eventual section cutting or microtomy.
Embedding
Paraffin embedding is the standard method used in histology laboratories to produce blocks of tissue for section cutting (microtomy). This process is usually carried out using an embedding centre and involves surrounding the tissues by a medium such as paraffin wax which when cooled and solidified will provide sufficient support for section cutting or microtomy (see stages below). The production of properly oriented and accurately labelled blocks is one of the essential skills of trained histologists and includes knowledge and understanding in areas such as tissue sampling, identification and human anatomy.
What is vaccuum impregnation ?
The use of vacuum infiltration is often used to help complete impregnation of tissues with wax. This is carried using a molten wax or other medium under reduced pressure. Vacuum assistance helps to not only reduce the time tissues are subjected to heat but it also assists in the complete removal of any remaining solvent. Modern tissue processors are equipped to deliver vacuum and pressure during tissue processing.
How you fixed a bone in Histopathology lab ?
Unless immediate diagnosis is needed using cryomicrotomy, all bone specimens must be totally fixed before subjecting them to any decalcification and processing procedures. Complete fixation helps protect bone and surrounding soft tissue from the damaging effects of acid decalcification.
Ten percent neutral buffered formalin (NBF) is suitable for both paraffin and non-tetracycline labeled bone. It should be noted that fixation proceeds faster by reducing the size of the bone, opening the bone, and removing excess skin and soft tissue surrounding the lesion. Large specimens can be bisected or reduced in size by sawing into multiple slabs, and immersed into fixative immediately, or no longer than 48 hours after initial fixation. Once cut into smaller pieces, the samples should be placed into fresh fixative.
For MMA (Methylmethacrylate) embedding, 10% NBF is generally used for fixation. Alcoholic formalin or 70% ethanol fixation is the fixative of choice for tetracycline-labeled bone.
Alcohol-based fixatives are not recommended for bone destined for acid decalcification as alcohol can slow or prevent decalcification. Fixatives containing chloroform (Carnoy’s) and mercury (B5, Zenker’s, Susa), including substitutes of them, should be avoided for specimens to be radiographed since those fixatives tend to make bone radio-opaque and unsuitable for specimen interpretation.
How to prepare Mayer's hematoxylin?
1. Add 50.0 g of aluminum ammonium sulfate to 800 mL double-distilled H2O. Stir, but do not use heat to assist dissolving.
2. Add 1.2 g of hematoxylin crystals and stir until dissolved.
3. Add 0.2 g of sodium iodate and 1.0 g of citric acid. Stir until dissolved.
4. Add 50 g of chloral hydrate and stir overnight at room temperature.
Chloral hydrate is a controlled substance; check with your laboratory safety officer regarding purchase and appropriate/safe storage.
5. Bring the total volume up to 1000 mL (this usually takes ∼100 mL H2O). Filter and store in a dark bottle at room temperature.
Write uses of Alcian blue in the histology laboratory ?
Alcian Blue: Diagnostic Applications
Alcian blue is used in the histology laboratory to demonstrate acid mucins which can be secreted by various connective and epithelial tissue tumors. Acid mucosubstances are stained turquois-blue. The alcian blue stain is most commonly used on tissue samples obtained from the gastrointestinal (GI) tract and is useful in diagnosing pathological processes such as Barrett's esophagus. Using alcian blue solutions of varying pH (1.0 and 2.5) also helps differentiate various types of acid mucosubstances. Alcian blue may be used in combination with the PAS staining procedure, so that both acid and neutral mucins can be demonstrated in the same tissue sample. Alcian blue will stain acidic mucins blue and PAS will stain neutral mucins rose red. This technique is also useful in diagnosing diseases of the GI tract.
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