histo assignment 3

8/11/2019 Histo Assignment 3

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Carl G. Buscato

Bio 158

1.

What are the different stages of Histological Preparation? Provide pictures of protocols of histological

preparation.

a.

Tissue Fixation

Fixation confers chemical stability on tissue, hardens tissues for sectioning, and most

importantly, halts autolysis and bacterial degradation. Chemical fixatives preserve tissues by

denaturing proteins through coagulation, cross-linking, or both. Changes to the molecular form

mean that fixation is often therefore a compromise between retention and preservation. Fixation

also alters penetration and antigen exposure.

b.

Tissue Processing

The three main procedures on preparation for tissues embedding are dehydration, clearing

and infiltration.

Dehydration

Removal of fixative and water from the tissue and replacing it with dehydrating fluid

such as isopropyl alcohol.

Clearing

Replacement of dehydrating fluid with organic solvent like xylene or any that is miscible

with both the dehydrating fluid and embedding medium.

Infiltration

The tissue is placed in melted embedding medium like paraffin until it becomes

completely infiltrated by this substance.

c. Embedding

After the tissue is infiltrated, the tissue becomes surrounded by a large block of molten

embedding agent creating a block. Once the block solidifies it produces a support matrix that

allows very thin sectioning.

d. Sectioning

Hardened tissues are cut into very thin sections, 3-5 microns, using a microtome. Once

cut, the tissue ribbons are carefully transferred to a warm water bath. Here they are allowed to

float on the surface and can then be scooped up onto a slide placed under the water level. Slidesare dried uprightly at 37C to gently melt the excess embedding agent, leaving the tissues section

intact.

e. Staining

Staining enables transparent and colorless cells to be viewed. Histochemical stains are

used to provide contrast to tissues sections, making tissue structures more visible and easier to


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evaluate. Following staining, a cover slip is mounted over the tissue specimen on the slide, using

optical grade glue, to help protect the specimen.

Summary Overview:

Pictures of Protocols of Histological Preparation

Fresh Sample

A fresh, unfixed specimen after surgical removal. To

prevent degeneration or drying-out the specimen should

be fixed as soon as possible.

A. Tissue fixation

A surgical specimen fixing in formalin and ready for

grossing. Note that there is a generous volume of

fixative compared to the size of the specimens. Fixation

is a crucial step in preparing specimens for microscopic

examination. Its objective is to prevent decay and

preserve cells and tissues in a life-like state.

Sectioning Staining


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B. Tissue Processing

A tissue processor being loaded with a basket of cassettes

containing tissue specimens for processing. Where large

batches of specimens are processed for paraffin section

preparation automated instruments called tissue processors

are used. These instruments allow the specimens to be

infiltrated with a sequence of different solvents finishing in

molten paraffin wax. The specimens are in an aqueous

environment to start with (water-based) and must be passed

through multiple changes of dehydrating and clearing solvents

(typically ethanol and xylene) before they can be placed in

molten wax (which is hydrophobic and immiscible with

water).

C. Embedding

An embedding center helps the operator by integrating a cold

plate, a hot plate and a controlled flow of molten paraffin wax.

Stomach specimen being placed and oriented in an embedding

mold on the hot plate of an embedding center.

A block ready for microtomy. A stomach specimen has been

processed and embedded ready for section preparation using a

microtome. The pale blue cassette was used to contain the

specimen during processing and now forms part of the block.

It will be firmly clamped in the specimen holder of the

microtome and sections will be cut from the face of the block.


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D. Tissue Sectioning

A ribbon of sections being cut from a paraffin block using a

rotary microtome. Note that the sections which are 4m thick

(4/1000 of a millimetre), show little distortion or disruption.

A paraffin section being mounted on a microscope slide afterbeing floated out on warm water to flatten it.

E. Staining

A rack of paraffin sections being loaded onto an automated

stainer for H&E staining. This instrument will stain the

sections and the adjacent automated glass coverslipper will

apply glass coverslips to the surface of the sections to preserve

them and provide optimal optical conditions for microscopy.

The H&E stain. This is a microscopic image (micrograph) ofa paraffin section of the wall of a human appendix taken using

brightfield microscopy. Cell nuclei are stained blue while

smooth muscle, collagen, and other components are stained in

shades of pink. The large clear spaces belong to fat cells, the

fat having been dissolved out during processing.


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2. How do you keep cells/tissues preserved for a long time of study?

Tissues/cells are preserved by means of preparing prepared slides on which fixation process is

applied to prevent autolysis (enzyme digestion) and putrefaction (bacterial attack). Fixation process uses

chemical fixatives such as formalin, aldehydes and acetone to preserve the tissues for long-time purposes.

These chemicals bind to cells to release of lysosomic enzymes and to the receptors of microorganisms to

prevent them from attacking the cells.However, larger specimens, such as tissues of an entire organ like brain, special preservation

techniques are done such as cryophilization and other freezing methods and addition of chemical

preservatives such as formalin and ethanol.

3. Why is there a need to go into prepared slides?

Prepared slides are essential tools for instruction and education in histology and serves as

standard basis for normal structure-function of a tissue. Its offers convenience in instruction and

education because it is readily made and can be viewed right away on the microscope as needed. It also

serves as a standard basis as to what is normal on pathological context. Prepared slides for study aside

from that it can be preserved for a long period, it gives a clear image of cellular/histological structures

that relates and explains their function under normal conditions.

In medical perspective, there is a need for knowledge on histological procedure especially on

conducting biopsy. For instance, an organ of a patient with tumor must undergo biopsy in order to

determine whether it is malignant or not that is to provide proper medical attention to the patient

regarding the tumor.

4. Provide an explanation of magnification using scale bars on tissues.

Magnification using scale bar is the ratio between the size of the tissue on the image and on its

actual size as viewed on microscope. The micron bar serves as the ruler (measuring device) as to how

long a micron is on the actual image. For example, a micron bar representing 0.1m, measures 10 mm on

the image and the entire image is 100 mm wide thus, under microscope, the actual size of the image is1m.

The length of the micron bar drawn is dependent on the magnification of image and on its

resolution of the picture when it is printed. For example, if the magnification of a tissue on journal or in

picture is 125,000X, one 0.1m micron bar should be 12.5 mm long so that its ratio will be proportionate

to the actual image of tissue seen on microscope.


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5. Give 5 micrographs of tissues coming from the three organs namely the heart, skin and pancreas.

A. Heart

Slide 1: Intercalated discs

Intercalated discs are located at adjacent ends of myocytes at irregular intervals. It contains three

types of cell to cell junction namely gap junctions, desmosomes and fascia adherens. Intercalated discs

enable ionic communication and continuity between adjacent cardiac fibers. They are located at adjacent

cardiac fibers to help multiple cardiac muscle cells contract rapidly as a unit just as in synctium.

Slide 2: Subepicardial Adipose Cells

Adipose cells are mainly found in subepicardial layer along with blood vessels, however on islands of

mature adipose cells there is no fascial structure that divides them from the myocardium. Aipocytesprimarily stores lipids that supplies energy for cardiac cells for spontaneous activity. They are located

nearby the myocardium, region of cardiac fibers to supply adequate energy to these fibers for cardiac

rhythm whenever they need it right away.

1 mm

Adipose cells

Myocardium

Subepicardial layer


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Slide 3: Sinoatrial Node

Sinoatrial node is located at the right atrium of the heart, it surrounds an artery so that it will

receive rich blood supply for passing the signals to the AV node. SA node is the pacemaker of the heart it

is where the muscular contraction begins, fibers in SA node depolarize and repolarize the fastest in the

heart, it sets the pace for heartbeat.

Slide 4: Cardiac Fibers

Cardiac muscle fibers are located at the myocardium, deep of the epicardium and surface of the

endocardium. Cardiac fibers are the conducting tissues of the heart, they are branched that helps to

prevent them from separating apart. The nuclei are centrally located compared to peripherally locatednucleus of the skeletal muscles. The cross-striations are visible representing the bands, sarcomere which

is the simplest contracting unit.

250 m

Sinoatrial nodal arterySinoatrial node

30m

nuclei

endomysium

fibrocytes

branching muscle

fiber


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Slide 5: Purkinje Fibers

Purkinje fibers are located in the inner ventricular walls of the heart. They are muscle derivatives

from cardiac fibers but its main function is not contractility but conductivity. They are larger and thicker

than cardiac fibers. They deliver stimuli in gap junction throughout the myocardium.

B. Skin

Slide 1: Thin Light Skin

Skin is composed of the epidermis, dermis and hypodermis. Arrow marks the stratum basale, thelowest layer of the epidermis. Below this is the dermis. Note the lymphocyte infiltration in the dermis.

30m

100m


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Slide 2: Thin Dark Skin

The thin layer of the epidermis distinguishes thin skin. The dark pigment in the stratum basale isthe main factor in determining the color of skin.

Slide 3: Thin Dark Skin

The melanin pigment, a derivative of tyrosine, is produced by melanocytes and stored in

melanosomes. Some are transferred to epidermal cells so the two cells are indistinguishable with lightmicroscopy.

250 m

250 m


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Slide 4: Sweat Gland

Sweat glands, like hair follicles, are found only in the dermis of thin skin. Notice that the sweat

duct stains darker than the gland.

Slide 5: Hair Follicle

The hair follicle is rooted in the dermis of thin skin. Notice that this is the same magnification as

the previous slide.

250 m

250 m


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C. Pancreas

Slide 1: Islet of Langerhans

While the islets may get all the attention, notice how much more exocrine tissue is present. Theexocrine tissue releases several digestive enzymes into the duodenum.

Slide 2: Pancreatic Acinar Glands

Compound acinar glands are those in which the secretory units are acinar in form and drain into abranched duct system. The pancreas shown in this micrograph consists of numerous acini, each of whichdrains into a minute duct. These minute ducts, which are just discernible in the centre of some acini, drain

into a system of branched excretory ducts of increasing diameter which are lined by simple cuboidalepithelium.

1mm

30 m

Excretory ducts


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Slide 3: Pancreas

The pancreas is a lobulated gland covered by a thin collagenous capsule which extends as delicate

septa between the lobules. The exocrine component of the pancreas consists of closely packed secretoryacini which drain into a highly branched duct system. Most of the secretion drains into the main

pancreatic duct, which joins the common bile duct to drain into the duodenum via the ampulla of Vater. In

most people, a small accessory pancreatic duct drains into the duodenum more proximally. Interlobularducts can be seen in this micrograph. Their surrounding supporting tissue reinforces the septal framework.

The endocrine tissue of the pancreas forms islets of Langerhans of various sizes scattered throughout theexocrine tissue. Occasional adipocytes are scattered throughout the parenchyma. These are scanty in

young adults but are seen in increasing numbers in older people, reflecting the natural atrophy of thegland with age.

Slide 4: PancreasThe pancreas is most readily identified by the numerous Islets of Langerhans. Recall that the

pancreas serves both endocrine and exocrine functions.

1mm

Islet of

Langerhans

Adipocytes

Interlobular

ducts

1mm

Islets of

Langerhans


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Slide 5: Pancreas

In this photomicrograph of a thin section, an intercalated duct can be seen beginning within a

pancreatic acinus. The cells forming the duct within the acinus are the centroacinar cells. The eosinophiliczymogen granules are clearly seen in the apical cytoplasm of the parenchymal cells

1 m

histo assignment 3

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