Nia useful in the study of mitosis. Below is

Nia Jarrett

January 17, 2018

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Vertebrate Histology

 

Q1: Describe the
different types of staining used in histology. Explain in detail the process of
function of staining with Eosin and Hematoxylin stains?

 

Staining is a process used to highlight significant features of tissues
in addition to the enhancement of tissue contrast. In the medical world,
staining is used for the medical diagnosis of tumors. This process is done by
applying a specific dye to the anterior and posterior borders to find diseased cells
that don’t belong. In the biological world, staining is vital in marking cells
and detecting nucleic acids, proteins, cellular structures, organelles, and gel
electrophoresis for microscopic examination. On occasion, multiple staining
techniques are vital for differential staining, multiple staining, or double
staining.

1)   
The iron hematoxylin
stain is used to distinguish the finer cytologic details within tissues such as
cell membranes, mitochondria, chromosomes, spindle fibers, mucin, elastic
fibers, and much more. Hematoxylin is one of the most commonly used dyes. The
dye itself originates from the heartwood of a logwood tree. It is known mainly
for its extensive use in identifying morphological descriptions for protozoa
found in the intestines of humans. While it’s commonly used in institutions
built for research, it has never been used as a routine lab procedure due to
the difficulty in technique. The problem in technique stems from the control
required in the decolorization process. This is difficult due to the procedure being
done under a light microscope. This process requires extensive knowledge in the
use of stains. Iron hematoxylin stains its structures a deep blue-black. It is
also useful in the study of mitosis. Below is a display of an amphibian liver
stained with iron hematoxylin on medium power. The nuclei within each cell of
the liver is stained the deep-bluish color.

 

2)   
The
Mallory-Azan stain is of the trichrome method and uses 3 different colors. In
this case, it stains cell nuclei, erythrocytes, fibrin, epithelial hyaline, and
fibrinoids red. Collagen fibers, mucus, basophil cytoplasm, and fibrous hyaline
are stained blue. If elastic fibers are by chance stained, they will be a
yellow or pink color. This stain is great in identifying connective tissues,
along with epithelium. From forensic practice, the stain differentiates
chromophobe and basophilic cells in the hypophysis. The difference between
Heidenhain’s adaptation and Mallory-Azan is that Heidenhain uses azocarmine
instead of acid fuchsin. Below is a picture of fatty liver. Collagen fibers are
in small areas and stained blue.

 

3)   
Masson’s
trichrome is known as a connective tissue technique since it is primarily used
to identify supporting tissue elements such as collagen. Muscle and nuclei are
stained red, while collagen itself is stained green. Cytoplasmic components
will be stained a purplish-red. The noticeable difference in the collagen fibers
from other components are due to a marked difference in permeability. While the
red acidic stain labels important features such as collagen fibers, phosphotungstic
acid allows the less permeable structures to retain the red stain and while the
remaining washes away, the light green dye takes its place. Masson’s trichrome plays
an important role in muscular, cardiac, hepatic, and kidney pathologies. Masson’s
trichrome is also routinely used to observe the presence of fibrosis. Collagen
in the main component targeted in the Masson stain, so excessive accumulation
of collagen within the tissues is an accurate signal for the diagnosis of
fibrosis. While this is important, quantification of the disease itself is
prone to variability and bias and requires special imaging tools to aid in
further knowledge of its quantity. It’s also vital in detecting and analyzing
tumors on hepatic and kidney biopsies. To the right is a picture of a mouse
lung using the Masson technique.

 

4)   
Periodic-Acid
Schiff (PAS) is a lab test that consists of oxidation and reduction reactions
that result in turning basement membrane material, polysaccharides, and mucosubstances
a bright red color. PAS is primarily used to identify structures within the
tissue that contain glycogen, mucus, membranes, and connective tissues in the
skin. It selectively stains complex carbohydrates or substances red. Collagen
within in the tissue will stain pink while nuclei will stain blue. This process
exposes the tissue to periodic acid. This acid then acts as an oxidizing agent
and works to oxidize compounds that have hydroxyl groups or dialdehydes. These
aldehydes react with Schiff’s reagent to release fuchsin and stain components
containing oxidizable compounds. These dialdehydes form an insoluble magenta
color when exposed to Schiff’s reagent. When concerning marrow and blood cells,
glycogen is the target component. In the blood of normal individuals, the
cytoplasm stains pink or red with a granular-like appearance in some cases. Monocyte
cytoplasm stains pink, erythrocytes do not stain, and platelets stain as well.
PAS also aids in the diagnosis of glycogen storage disease, Paget’s disease of
breast, and fungal infection.  Below is a
picture of tissue that has produced mucin from goblet cells. The mucin is
stained a purple color.

5)   
Silver
impregnation consists of soaking tissue in silver for an extensive amount of
time to reveal reticular fibers. Reticular fibers are known to have a low
affinity for silver salts.
This makes it a perfect pretreatment to enhance selectivity for impregnation to
occur. Treatment effects consist of creation of silver sites in reticular fibers
and uptake with considerable amounts of silver by tissues in smaller forms. Myelinated
fibers are a light brown color and nerve cells are of a yellowish or brown
color. Unmyelinated axons along with reticular fibers appear to be dark brown
or completely black. Right below the description shows a slide of a lymph node
that has been treated with silver. As you can see, the reticular fibers are
stained black and the cortex a dark brown.

6)   
Orcein is
used to identify elastic fibers within tissue. Originally, Orcein was a textile
dye in Egypt and was used specifically for dyeing silk and wool until the 20th
century. The idea to integrate this into histology came about in 1878 as a
stain for cytoplasm. The staining properties were unique; collagen and elastin
were strongly colored. The dye also reacted well with embryonic collagens, and
fine fibers. Structures are typically stained a brownish-red while collagen
fibers within the tissue are colorless or pale brown. In the picture below, we
are looking at elastic fibers found in the dermis. As you can see, the fibers
are stained brownish red towards the top of the picture.

7)   
Sudan black
technique is used to stain lipids a brownish-black color for light microscopy. Sudan
black stains a plethora of lipids including phospholipids, sterols, and neutral
fat. It fixes to an undefined granule component and can’t be extracted from
stained granules by organic dye solvents. Sudan black is not as lipid-specific
as some may think but are still useful to stain other materials. An image of
tissue containing fat cells are stained black.

 

 

 

8)    Hematoxylin and eosin are the most commonly used technique for
histology and pathology. Hematoxylin is a basic dye that stains nuclear
components of tissue blue. It has little to no staining capacity and is
oxidized to hematein. Hematoxylin staining calls for the use of a mordant. This
typically stains nuclear components a dark blue.  Hematein is anionic and the tissue is also
anionic. This simply boils down to hematein having a poor affinity for tissue
and 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 link
between the tissue and the stain. Eosin, on the contrary, is an acidic dye that
stains cytoplasmic and intercellular structures pink. This is the most suitable
stain to work with hematoxylin. It has the ability to distinguish between the
cytoplasm of different cells to the different types of connective tissues
within the fibers and matrices. Eosin itself is negatively charged and reacts
with positively charged components of tissue. Difficulties encountered with
Eosin is that the staining itself is intense and can make it difficult when obtaining
accurate differentiation.  H & E are
versatile 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 tissue
is soaked in the hematoxylin long enough to reach the goal anticipated.  Below is a picture of cardiac muscle. It
stains pink due to its ability of the structure to be negatively charged.

Q2: Describe the different parts of light microscope. Which part do you
think is the most important part of a light microscope? Explain you reason in detail?
(3.5 points)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The parts of the microscope all work together
collectively to make viewing the specimen under the microscope an efficient
process. The eyepiece or ocular is the piece of the microscope where you look through
at the top to view the slide. The standard magnifying power of an eyepiece is
usually 10x or 15x. To get the total magnification, multiply the total
magnification by the magnification used. The inter-ocular distance is known as
the distance between the eyes; this can also be called interpupillary distance.
This piece can easily be adjusted, and the distance can vary from person to
person. The iris diaphragm determines how much light passes through the stage
opening. It also adjusts both the contrast and the resolution of the specimen being
observed; it’s more efficient at higher powers. The condenser is a lens that concentrates
light on the specimen and increases the resolution in the microscope. This part
can be found in or below the stage. The coarse and fine focus knobs main
function is to focus the view on the specimen. The knobs are made together with
the fine focus knob protruding from the coarse focus knob. The coarse focus
moves the lens up and down, while the fine focus is used to fine-tune the focus
of the specimen being viewed after using the coarse adjustment knob. The
objective lenses on a microscope usually comes with 3 or 4. The powers are 4x(shortest
lens), 10x, 40x, 100x(longest lens). The higher powers are typically spring loaded
to reduce the occurrence of damages to the microscope if it hits the slide by
chance. If it does hit the slide, the lens simply retracts. Lenses are
typically color coded and can be interchangeable. The shortest lens is the one
with the least power while the longest one has the most power. The intensity
determines the intensity of the light that will pass through the specimen. It
is located at the base of the microscope. The base is the bottom of the
microscope. It supports the entire microscope and allows it to stand on its
own. The stage clips are used to hold the specimen in place while it is being
viewed. The body, also known as the arm, is used to support the eyepiece and
objective lens. The moving stage allows the viewer to move the stage in any
direction to view the specimen in its entirety.

 

The most important part
of the microscope in my opinion would be the condenser. With the ability to
focus light on the specimen and improve resolution there’s no denying that this
part is the most important. While the objective lens holds the title for
determining how much the image is magnified, viewing at a higher resolution is
not possible without a quality condenser. A condenser shines light through the
specimen, sharpening the image and improving the resolution. Condensers are
typically most useful when viewed from 400x and above. If they’re much lower
than this, its possible that the microscope may not have a condenser. Other
microscopes use concave lenses instead of condensers to focus light on the specimen.

 

References

Nine, Jaf. “MICROSCOPE
PARTS 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, et
al. “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.