Answersheet Histopath

Bellerry C.
Bernante BSMT 3C
ANSWERSHEET HISTOPATH
4D’s model
1.Explain the importance of tissue processing
Tissue processing must be done precisely since poorly processed tissue badly affects the
section cutting and staining. Its basic aim is to remove water from the tissue section and them
impregnate the tissue with another medium which can give it support.
2. Make a concept map on tissue processing
FIXATION DEHYDRATIO CLEARING INFILTRATIO

N N
Reflection:
Tissue processing for light microscopy
According to the video, there is a view down a microscope of a thin section of kidney.
The kidney has been processed sectioned and stained, to allow us to study its cellular structure. If
you try to cut a piece of fresh tissue and look at it with a microscope all that can be seen as a
structuralist blob, there are several obvious problems. It is not permanent so the tissue decays, it
is a difficult to cut sections thin enough to allow light to pass through the section and it lacks
color, these difficulties are overcome by fixation provision of a support medium for the tissue
and staining. To preserve fresh tissue, it is placed in fixative for a length of time suitable for the
size of the piece and the type of fixative used. An example would be a specimen of this size will
be fixed in formalin for approximately 36 hours. The fixed tissue is transferred into a labeled
plastic cassette that is then placed in a stainless steel carrier, it is hooked onto the arm of the
automatic tissue processor. The carrier is lowered into the first bar of 70% alcohol, the timer is
set the processor holds beakers of alcohol of increasing concentration as the machine goes
through its cycle. The tissue is dehydrated gradually to avoid excessive shrinkage. Paraffin wax
is not soluble in alcohol, so after dehydration the tissue is exposed to a solvent that is miscible
with both alcohol and paraffin wax. These solvents are known as clearing agents because they
also make the tissue translucent. The last two Baker's in the machine contained the clearing
agent, this laboratory uses a non-toxic organic solvent crystalline. During the end of the cycle,
the specimen is immersed for an hour or more in pots of molten paraffin wax the end product is
tissue impregnated with molten wax. The next step is blocking the specimen in solid wax, carrier
is transferred the tissue check and bedding stations and the cassettes immersed in the wax
reservoir so the warmth keeps the wax molten. The tissue is removed and orientated as required
in a metal mold which has been filled with molten wax. The base of the cassette of the specimen
ID on its side forms a lid for the mold, the mold is topped up with extra wax and placed onto the
adjacent cold plate. The wax solidifies and the paraffin block with the specimen is removed from
the metal mold, the tissue sample is now ready for the next step section cutting. Sections are cut
using a rotary microtome, the tissue block is clamped into the micro time. The knife is inserted
and clamped into the knife holder, the knife stage is eased towards the block. When final
adjustments have been made the stage is locked in place and the dial set at the required thickness
routinely 7 microns, by rotating the handle the block is advanced by 7 microns and lowered
down pass the knife to produce a section. These sections of wax and tissue form a ribbon that we
lifted off the knife and laid down in strips, due to compression the tissue sections are slightly
wrinkled but most folds can be removed by floating up the ribbon or individual sections on a
warm water bar sections are collected up onto a microscope slide and dried in a 40-degree oven
overnight. The wax within the tissue sections is dissolved away by the clearing agent, the slides
are then rehydrated since the solvents are not miscible with the dyes. This stage is simply a series
of alcohol washes of decreasing strengths down to distilled water, the dewaxing and rehydrating
steps are referred to as bringing the sections to water. The sections are now ready to be stained,
hematoxylin and eosin are routinely used to stain histological sections. Hematoxylin stains many
nuclei are blue to black color, fearsome stains cytoplasm and intercellular components pink.
Sections are over stained in here schema toxin solutions and then they are placed in a wick
ammonium solution to produce a deep blue color, over standing with hematoxylin results in
many tissue components being colored blue. The diet is then selectively removed until the nuclei
can be seen clearly, this is checked with the microscope. This process is known as
differentiation, the slides are then stained eosin. To make the section permanent it is dehydrated
in absolute alcohol and then cleared in the clearing agent that is miscible with PIX the clearing
agent mix section translucent PIX is a synthetic matting medium and acts as a clear glue-like
substance that adheres the coverslip to the same section. Droplet P IX is placed on the cover slip
on the slide with the stained section is lowered onto it, P IX will harden after the slide is placed
in a 40 degree oven overnight. The mountain to P IX has the same refractive index as glass, this
means that in the light microscope black rays are not further distorted as they pass through the
section then the mountain and up through the coverslip. In summary tissue processing involves
fixation where the tissue is preserved, processing where water is removed and the tissue is
impregnated with wax section cutting where a thin section is cut from the paraffin-embedded
tissue and finally staining with hematoxylin and eosin to give the a stain in the section.
MODULE 2
Topic 1:
ACTIVITY 1: Use 3 adjectives that best describe proper methods of tissue preparation
1. Time Consuming
2. Perplexed
3. Delicate
ACTIVITY 2:
Reflection:
Tissue Processing Steps for Frozen Samples
Based on the video that I have watched, which is frozen tissue sectioning. Grossing is
defined as the process in which pathologic specimens are examined and trimmed to proper size
and best part is selected for further microscopic examination. To obtain diagnostic information
cryomicrotome may be required during the performance of surgical procedures for rapid
diagnosis tissue is snap frozen in a fluid called OCT and sectioned in a cryostat sections are
picked up with a glass slide and stained.
Topic 2:
ACTIVITY 1:
1.During your second year in this program, you took up Histology course and you were asked
to view prepared tissue slides. Why do you think the structures of these tissues are still
preserved?
During our second year in medical technology program, we found out that the fixation
process could preserve tissue's shape and integrity fully. Fixation is the most crucial step in
preparing the tissue for observation. Fixation involves two steps which is destroying the tissue to
end its natural life processes and stabilizing the tissue's structure called preservation.
Topic 3:
ACTIVITY 1:
The bones in our body are made up of Calcium, what do you think will happen to our
bones if Calcium is removed?
For our bones to be dense and strong, our body needs calcium. Low bone density can lead to
fragile and weak bones. Even when there is no obvious damage, these fragile bones are more likely to
break. Vitamin D facilitates calcium absorption. Bones can't help a person move if there isn't calcium in
them, they won't even be able to function correctly.
ACTIVITY 2:
Give the importance of Decalcification
Decalcification’s role is to remove calcium salts from mineralized tissue while keeping organic
components intact. Calcium-rich bones are very difficult to analyze, scientists then use bone
decalcification to prepare specimens for research.

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Bellerry C.

FIXATION DEHYDRATIO CLEARING INFILTRATIO

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