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Nia JarrettJanuary 17, 2018Vertebrate Histology Q1: Describe thedifferent types of staining used in histology.

Explain in detail the process offunction of staining with Eosin and Hematoxylin stains?  Staining is a process used to highlight significant features of tissuesin addition to the enhancement of tissue contrast. In the medical world,staining is used for the medical diagnosis of tumors. This process is done byapplying a specific dye to the anterior and posterior borders to find diseased cellsthat don’t belong. In the biological world, staining is vital in marking cellsand detecting nucleic acids, proteins, cellular structures, organelles, and gelelectrophoresis for microscopic examination. On occasion, multiple stainingtechniques are vital for differential staining, multiple staining, or doublestaining.1)   The iron hematoxylinstain is used to distinguish the finer cytologic details within tissues such ascell membranes, mitochondria, chromosomes, spindle fibers, mucin, elasticfibers, and much more. Hematoxylin is one of the most commonly used dyes. Thedye itself originates from the heartwood of a logwood tree.

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It is known mainlyfor its extensive use in identifying morphological descriptions for protozoafound in the intestines of humans. While it’s commonly used in institutionsbuilt for research, it has never been used as a routine lab procedure due tothe difficulty in technique. The problem in technique stems from the controlrequired in the decolorization process. This is difficult due to the procedure beingdone under a light microscope. This process requires extensive knowledge in theuse of stains. Iron hematoxylin stains its structures a deep blue-black. It isalso useful in the study of mitosis. Below is a display of an amphibian liverstained with iron hematoxylin on medium power.

The nuclei within each cell ofthe liver is stained the deep-bluish color.  2)   TheMallory-Azan stain is of the trichrome method and uses 3 different colors. Inthis case, it stains cell nuclei, erythrocytes, fibrin, epithelial hyaline, andfibrinoids red. Collagen fibers, mucus, basophil cytoplasm, and fibrous hyalineare stained blue. If elastic fibers are by chance stained, they will be ayellow or pink color. This stain is great in identifying connective tissues,along with epithelium. From forensic practice, the stain differentiateschromophobe and basophilic cells in the hypophysis. The difference betweenHeidenhain’s adaptation and Mallory-Azan is that Heidenhain uses azocarmineinstead of acid fuchsin.

Below is a picture of fatty liver. Collagen fibers arein small areas and stained blue.  3)   Masson’strichrome is known as a connective tissue technique since it is primarily usedto identify supporting tissue elements such as collagen. Muscle and nuclei arestained red, while collagen itself is stained green.

Cytoplasmic componentswill be stained a purplish-red. The noticeable difference in the collagen fibersfrom other components are due to a marked difference in permeability. While thered acidic stain labels important features such as collagen fibers, phosphotungsticacid allows the less permeable structures to retain the red stain and while theremaining washes away, the light green dye takes its place. Masson’s trichrome playsan important role in muscular, cardiac, hepatic, and kidney pathologies. Masson’strichrome is also routinely used to observe the presence of fibrosis. Collagenin the main component targeted in the Masson stain, so excessive accumulationof collagen within the tissues is an accurate signal for the diagnosis offibrosis. While this is important, quantification of the disease itself isprone to variability and bias and requires special imaging tools to aid infurther knowledge of its quantity.

It’s also vital in detecting and analyzingtumors on hepatic and kidney biopsies. To the right is a picture of a mouselung using the Masson technique.  4)   Periodic-AcidSchiff (PAS) is a lab test that consists of oxidation and reduction reactionsthat result in turning basement membrane material, polysaccharides, and mucosubstancesa bright red color. PAS is primarily used to identify structures within thetissue that contain glycogen, mucus, membranes, and connective tissues in theskin.

It selectively stains complex carbohydrates or substances red. Collagenwithin in the tissue will stain pink while nuclei will stain blue. This processexposes the tissue to periodic acid. This acid then acts as an oxidizing agentand works to oxidize compounds that have hydroxyl groups or dialdehydes. Thesealdehydes react with Schiff’s reagent to release fuchsin and stain componentscontaining oxidizable compounds. These dialdehydes form an insoluble magentacolor when exposed to Schiff’s reagent. When concerning marrow and blood cells,glycogen is the target component. In the blood of normal individuals, thecytoplasm stains pink or red with a granular-like appearance in some cases.

Monocytecytoplasm stains pink, erythrocytes do not stain, and platelets stain as well.PAS also aids in the diagnosis of glycogen storage disease, Paget’s disease ofbreast, and fungal infection.  Below is apicture of tissue that has produced mucin from goblet cells.

The mucin isstained a purple color. 5)   Silverimpregnation consists of soaking tissue in silver for an extensive amount oftime to reveal reticular fibers. Reticular fibers are known to have a lowaffinity for silver salts.

This makes it a perfect pretreatment to enhance selectivity for impregnation tooccur. Treatment effects consist of creation of silver sites in reticular fibersand uptake with considerable amounts of silver by tissues in smaller forms. Myelinatedfibers are a light brown color and nerve cells are of a yellowish or browncolor. Unmyelinated axons along with reticular fibers appear to be dark brownor completely black. Right below the description shows a slide of a lymph nodethat has been treated with silver.

As you can see, the reticular fibers arestained black and the cortex a dark brown. 6)   Orcein isused to identify elastic fibers within tissue. Originally, Orcein was a textiledye in Egypt and was used specifically for dyeing silk and wool until the 20thcentury. The idea to integrate this into histology came about in 1878 as astain for cytoplasm.

The staining properties were unique; collagen and elastinwere strongly colored. The dye also reacted well with embryonic collagens, andfine fibers. Structures are typically stained a brownish-red while collagenfibers within the tissue are colorless or pale brown. In the picture below, weare looking at elastic fibers found in the dermis.

As you can see, the fibersare stained brownish red towards the top of the picture. 7)   Sudan blacktechnique is used to stain lipids a brownish-black color for light microscopy. Sudanblack stains a plethora of lipids including phospholipids, sterols, and neutralfat. It fixes to an undefined granule component and can’t be extracted fromstained granules by organic dye solvents.

Sudan black is not as lipid-specificas some may think but are still useful to stain other materials. An image oftissue containing fat cells are stained black.    8)    Hematoxylin and eosin are the most commonly used technique forhistology and pathology. Hematoxylin is a basic dye that stains nuclearcomponents of tissue blue.

It has little to no staining capacity and isoxidized to hematein. Hematoxylin staining calls for the use of a mordant. Thistypically stains nuclear components a dark blue.  Hematein is anionic and the tissue is alsoanionic.

This simply boils down to hematein having a poor affinity for tissueand making it inadequate as a nuclear stain without a 3rd element;which in this case is the mordant. The mordant is there to form the linkbetween the tissue and the stain. Eosin, on the contrary, is an acidic dye thatstains cytoplasmic and intercellular structures pink. This is the most suitablestain to work with hematoxylin. It has the ability to distinguish between thecytoplasm of different cells to the different types of connective tissueswithin the fibers and matrices. Eosin itself is negatively charged and reactswith positively charged components of tissue. Difficulties encountered withEosin is that the staining itself is intense and can make it difficult when obtainingaccurate differentiation.  H & E areversatile in that it can be used after any fixative.

In addition to that,H can be used as a progressive stain. That simply means that the tissueis soaked in the hematoxylin long enough to reach the goal anticipated.  Below is a picture of cardiac muscle. Itstains pink due to its ability of the structure to be negatively charged. Q2: Describe the different parts of light microscope.

Which part do youthink is the most important part of a light microscope? Explain you reason in detail?(3.5 points)                The parts of the microscope all work togethercollectively to make viewing the specimen under the microscope an efficientprocess. The eyepiece or ocular is the piece of the microscope where you look throughat the top to view the slide.

The standard magnifying power of an eyepiece isusually 10x or 15x. To get the total magnification, multiply the totalmagnification by the magnification used. The inter-ocular distance is known asthe distance between the eyes; this can also be called interpupillary distance.This piece can easily be adjusted, and the distance can vary from person toperson. The iris diaphragm determines how much light passes through the stageopening. It also adjusts both the contrast and the resolution of the specimen beingobserved; it’s more efficient at higher powers. The condenser is a lens that concentrateslight on the specimen and increases the resolution in the microscope.

This partcan be found in or below the stage. The coarse and fine focus knobs mainfunction is to focus the view on the specimen. The knobs are made together withthe fine focus knob protruding from the coarse focus knob. The coarse focusmoves the lens up and down, while the fine focus is used to fine-tune the focusof the specimen being viewed after using the coarse adjustment knob. Theobjective lenses on a microscope usually comes with 3 or 4. The powers are 4x(shortestlens), 10x, 40x, 100x(longest lens).

The higher powers are typically spring loadedto reduce the occurrence of damages to the microscope if it hits the slide bychance. If it does hit the slide, the lens simply retracts. Lenses aretypically color coded and can be interchangeable.

The shortest lens is the onewith the least power while the longest one has the most power. The intensitydetermines the intensity of the light that will pass through the specimen. Itis located at the base of the microscope. The base is the bottom of themicroscope. It supports the entire microscope and allows it to stand on itsown. The stage clips are used to hold the specimen in place while it is beingviewed. The body, also known as the arm, is used to support the eyepiece andobjective lens. The moving stage allows the viewer to move the stage in anydirection to view the specimen in its entirety.

 The most important partof the microscope in my opinion would be the condenser. With the ability tofocus light on the specimen and improve resolution there’s no denying that thispart is the most important. While the objective lens holds the title fordetermining how much the image is magnified, viewing at a higher resolution isnot possible without a quality condenser. A condenser shines light through thespecimen, sharpening the image and improving the resolution. Condensers aretypically most useful when viewed from 400x and above.

If they’re much lowerthan this, its possible that the microscope may not have a condenser. Othermicroscopes use concave lenses instead of condensers to focus light on the specimen. ReferencesNine, Jaf. “MICROSCOPEPARTS AND FUNCTIONS.” Microscope Parts and Functions, www.

amscope.com/microscope-parts-and-functions/.”Masson Trichrome.” Histalim, www.histalim.

com/accueil/activities/our-services/histology/masson-trichrome/.Alturkistani, Hani A, etal. “Histological Stains: A Literature Review and Case Study.” Global Journal of Health Science, Canadian Center of Science and Education, Mar. 2016, www.ncbi.nlm.

nih.gov/pmc/articles/PMC4804027/.Paxton, Steve, et al.”The Leeds Histology Guide.

” Histology Guide, 1 Jan. 1970,histology.leeds.ac.

uk/what-is-histology/histological_stains.php.Bellham, Sarah.”Histology Stains.

” Histology Dot, www.histology-world.com/stains/stains.htm.