What is interference Microscope ? In phase contrast microscopy, the specimen retards some light rays with respect to those which pass through the surrounding medium. The resulting interference of these rays provides image contrast but with an artifact called the ‘phase halo’. In the interference microscope the retarded rays are entirely separated from the direct or reference rays, allowing improved image contrast, color graduation, and quantitative measurements of phase change (or ‘optical path difference’), refractive index, dry mass of cells (optical weighing), and section thickness.
Classify Clearing Agent used in Hitological techniques ?
CLEARING AGENTS are used throughout the histology lab in the processes of tissue and slide preparation—to remove alcohol and other dehydrants from tissues prior to embedding (usually in paraffin wax), and from finished slides prior to mounting. They are also used after sectioning to remove paraffin wax after cutting on the microtome.Following are the clearing agent used in Histology lab such as : Xylene Toulene Chloroform
Xylene Substitute
Citrus fruit Oil
Stain used in histopathology lab ???
Mainy stained are used in histopathology lab to enhance the visibility & contrast of specimen.following are some stain with their priniciples 1) Hematoxylin and Eosin
This is a good general stain and is widely used. Hematoxylin acts as a basic stain via a substance called hematein which is formed in solutions of hematoxylin. It stains nucleic acids in the nucleus (chromatin and nucleolus) and cytoplasm (ribosomes) blue. Eosin is an acid aniline dye which stains the more basic proteins and other materials pink or red. It is thus mainly a cytoplasmic stain. Collagen and red blood cells stain pinkish or reddish with eosin.
2) Iron Hematoxylin
In this stain iron acts as a mordant to bind the hematoxylin. It is a good nuclear stain, rendering the chromatin and nucleolus black. Centrioles and basal bodies are black. The terminal web also binds this stain. Mitochondria, if adequately preserved, are black. A counterstain (eosin, acid fuchsin, etc.) is used to stain the cytoplasm.
3) Mallory's Trichrome Connective Tissue Stain
A stain composed of aniline blue, acid fuchsin, and orange G. It is a good stain to use for distinguishing cellular from extracellular components. Collagen fibers stain an intense blue. Mucus and ground substance take on varying shades of blue. Cytoplasm and neuroglia stain red. Elastic fibrils, red blood cells and nucleoli stain pink or yellow.
4) Osmic Acid or Osmium Tetroxide
Used in light microscopy to show fat, myelin and the Golgi complex, all of which stain black. It is also a fixative used routinely in electron microscopy.
5) Mallory's Phosphotungstic Acid Hematoxylin (PTAH)
Stains muscle deep blue, showing cross striations of skeletal muscle especially well. The ground substance of cartilage and bone stains yellowish to brownish-red; coarse elastic fibers stains purple.
6) Periodic Acid-Schiff (PAS)
Structures rich in glycogen and glycoproteins stain red or purple with this technique. The periodic acid attacks, 1,2 glycols and 1,2 amino alcohols, oxidizing the adjacent groups to aldehydes, which are detected by their reaction with Schiff's reagent to form a reddish color. If the treatment with periodic acid is carried out for more than 10 minutes, other polysaccharide containing molecules will also be oxidized, e.g. proteoglycans and glycolipids. Many PAS stained tissues are also stained with hematoxylin, hence PAS-H.
7) Elastic Tissue Stains
Orcein: Stains elastic fibers brownish red. Collagen fibers are pale brown or colorless.
Weigert's resorcin-fuchsin: Stains elastic fibers dark blue-black.
Verhoff's: Stains elastic tissue black. Neuroglia, myelin and fibrin are pink.
Note: The stain is designated as "elastic tissue stain" when it is not known which of these methods was used.
8) Silver Techniques
Romanes : A method for staining nerve fibers in paraffin sections with silver. It involves the use of silver chloride. Nerve fibers stain purple to black, nuclei red, neurofibrils purple, keratin yellow and bone cells black. The 'staining' is based on the reduction of silver salts by some substance(s) present in these elements.
Gomori : A silver impregnation of reticular fibers using silver nitrate that gives black reticular fibers.
Note: The stain is designated as "silver technique" when it is not sure which of these methods was used.
9) Nissl :
In this staining procedure a basic dye, such as thionin or gallocyanin, stains basophilic materials, such as RNA (e.g. Nissl bodies of nerve cells or other ribosomal aggregates), bright blue. Cartilage ground substance and the granules of mast cells stain metachromatically with thionin.
10) Romanovsky :
The Romanovsky-type stains are neutral dyes used primarily in the staining of blood smears. In solution the dye contains ions (eosin-, methylene blue+, and the azures+ which are oxidation products of methylene blue) and neutral salts (eosinates of methylene and the azures). These stains are not stable in water, so they have to be used immediately after mixing. Alternatively, an alcoholic solution of the dye may be applied, but diluted with water immediately afterwards. Romanovsky dyes stain the granules of acidophilic and neutrophilic leukocytes red, the granules of basophilic leukocytes blue, red blood cells red, plasma cells and lymphocytes blue.
What are the 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.
write application / Use of exfolliative cytology ?
Exfolliative cytology is a simple non-aggressive technique that is well accepted by the patient, and that is therefore an attractive option for the early diagnosis of oral cancer, including epithelial atypias and especially squamous cell carcinoma.
How Brain is fixed ?
The problem of fixing a whole brain is to render it firm enough to investigate the neuroanatomy and to produce sections to show histopathology and to respond to immunochemistry if required. Conventionally this fixation takes at least 2 weeks. Adickes et al. (1997) proposed a perfusion technique which allows all of the above to be accomplished and the report issued in 5–6 days. This method depends on the perfusion of the brain via the middle cerebral arteries. Fixatives may also be enhanced by the use of microwave technology . Alcoholic formalin should not be used for fixation if immunohistochemistry is to be performed using biotin-avidin (streptavidin) methods .
What is Cryostat ?
A cryostat (fromcryomeaning cold andstatmeaning stable) is a device used to maintain low cryogenic temperatures of samples or devices mounted with in the cryostat.
The cryostat is a refrigerated cabinet in which a specialty microtome is housed. All the controls for the microtome are operated outside the cabinet. The first cryostats were introduced in 1954. Improvements in design have facilitated sectioning and safety.
To produce thin, high-quality, frozen sections, the tissue must be properly frozen and embedded correctly, the conditions of the cryostat must be optimal, the block temperature must be correct for the tissue being cut and the blade must be clean and properly secured. The best-quality frozen sections are produced from fresh unfixed tissue which has been rapidly frozen.
What is impregnation and infiltration of tissue ?
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.
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.
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 BIOPSY ?
Biopsy A biopsy is a medical test commonly performed by a surgeon, interventional radiologist, or an interventional cardiologist. The process involves extraction of sample cells or tissues for examination to determine the presence or extent of a disease.
Following are the types of biopsy such as :
Needle biopsy.
Ultrasound-guided biopsy.
Aspiration biopsy (FNA fined needle biopsy)
Surgical biopsy.
What is Progressive and Regressive Staining ?
Progressive staining occurs when the hematoxylin is added to the tissue without being followed by a differentiator to remove excess dye. Modified progressive staining occurs when a mild differentiator is used not to remove excess stain from the nuclei, but rather removes background staining.
Regressives staining ,A type of staining in which tissues are over stained and excess dye then removed selectively until the desired intensity is obtained.
Differentiation, In regressive staining differentiation is the removal of washing out of the excess stain until the colour is retained only in the tissue components to be studies.
What is Papanicolaou stain & Its uses ?
Papanicolaou stain (also Papanicolaou's stain and Pap stain) is a multichromatic (multicolored) cytological staining technique developed by George Papanicolaouin 1942. ThePapanicolaou stain is one of the most widely used stains in cytology, where it is used to aid pathologists in making a diagnosis
The classic form of Pap stain involves five dyes in three solutions:
A nuclear stain, haematoxylin, is used to stain cell nuclei.
First OG-6 counterstain. The Orange G is used to stain keratin. Its original role was to stain the small cells of keratinizing squamous cell carcinoma present in sputum.
Second EA (Eosin Azure) counterstain, comprising of three dyes; the number denotes the proportion of the dyes, eg. EA-50, EA-65.
Eosin Y stains the superficial epithelial squamous cells, nucleoli, cilia, and red blood cells.
Light Green SF yellowish stains the cytoplasm of all other cells. This dye is now quite expensive and difficult to obtain, therefore some manufacturers are switching to Fast Green FCF, however it produces visually different results and is not considered satisfactory by some.
Bismarck brown Y stains nothing and in contemporary formulations it is often omitted.
What is Imunohistochemistry ?
Immunohistochemistry is the most common application of immunostaining. It involves the process of selectively identifying antigens in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
Immunohistochemistry (IHC) is an important application of monoclonal as well as polyclonal antibodies to determine the tissue distribution of an antigen of interest in health and disease. IHC is widely used for diagnosis of cancers; specific tumor antigens are expressed de-novo or up-regulated in certain cancers.
Classify fixatives ?
For answer watch my video an note the flowchart given in description of video ,here is the link of my lecture video :
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