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Microscopic Report

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Date:
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Question 1

Why is Kohler Illumination utilized?

Kohler Illumination is a specimen illumination method used for reflected and transmitted light
optical microscopy. The method is used to produce an extremely homogenous illumination of the
specimen to ensure that the image of the source of illumination does not become visible in the
image formed. The incident light is properly aligned and the image of the illumination source is
perfectly defocused, allowing the best imaging possible in a field of light that is evenly
dispersed. This enables collection of more imaging data. The contrast between areas of interest is
increased, generating a sharper final image with high overall resolution. It is the modern
technique for specimen illumination in modern light microscopy. Other reasons why this method
is used is because it eliminates glare and reflections in the optical system, and specimen heating
is also reduced. Excessive light reflections affect quality of image contrast while heating of
specimens is problematic especially when viewing living specimens (Hibbs, 2004).

Question 2

Why would you stain specimens prior to examining under a microscope?

Cells and most elements that make up a specimen are colorless, except a few natural pigments,
e.g. melanin. In order to obtain a detailed structural information using a microscope, staining of
the specimen is essential. This is made possible by the fact that staining increases contrast by
changing the color of the specimen under observation, allowing for clearer imaging. Different
staining techniques are employed in biology and medicine to define particular microorganisms,
or highlight structures of interest in biological tissues so as to have a clear view under a
microscope. Stains can be used in the examination and defining bulk tissues, for example
connective tissue or muscle fibers, or examining cell populations, e.g. for classification of
different blood cells, or cell organelles (Karp, 2009). The detailed information provided through
specialized staining techniques is important in histopathology to obtain a full differential
diagnosis.

Staining technique is not limited to biological tissues, but it can also be used to examine
morphology of materials such as the lamellar structures in semi-crystalline polymers.
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Question 3

What structures do haematoxylin and eosin stain and with what colours?

Haematoxylin and eosin (H&E) stain is a combination of these two dyes routinely and
universally used as a point of start in providing essential structural information. Hematoxylin is a
positive/basic violet or dark blue stain that binds to substances that are basophilic. Eosin is a
negative/acidic pink or red stain that binds to substances that are acidophilic (Crocker & Burnett,
2005). Using this method, eosin stains the cytoplasm, proteins and other extra-cellular
components with shades of pink, orange or red, while haematoxylin stains the cell nucleus, and
other objects such as calcified material and keratohyalin with a deep blue-purple color.
Generally, oesinophilic structures are composed of extracellular or intracellular protein.
Examples of such structures include the Mallory bodies and Lewy bodies, and the red blood cells
which are intensely stained with red color. The cytoplasm is largely eosinophilic (Bancroft &
Gamble, 2008). In micrscopy, we observe:

 Cytoplasm as red
 Nuclei as deep blue/purple
 Muscles as dark red
 Basophils as purple/red
 Collagen as pale pink
 Erythrocytes as cherry red
 Mitochondria as pale pink

It is possible to diagnose many conditions in histopathology by examining an H&E only.

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Question 4:

Why might you use microscope co-ordinates?

Microscope coordinates are important in relocating an organism or artifact on the slide. Each
feature on a specimen is located on a unique X-Y coordinate that can be used to identify a
particular feature on the slide. This is particularly important to point out for referral or share your
findings with people who are interested. The coordinates are normally recorded on the X (from
left to right) axis and the Y axis (from top to bottom). It enables the observer to make notes and
record positions on the specimen for future references. To move to a particular reference point,
the X-Y coordinates are simply entered on the microscope. Coordinate assignment is also
important in the case of specimen identification and validation, and taxon types, especially where
it will require future researchers to locate a particular specimen as referenced in the protologue,
and specimens from materials – specifically, vouchered or type specimens (Paddock, 1999).

Question 5:

What different epithelial cells might you observe?

Epithelium form part of the main tissues in the body. It is usually made up of millions of
epithelial cells that are arranged in tubules or sheets attached to the underlying membrane
(Krause, 2004). There are four different epithelial cells that can be observed:

 Squamous epithelial cells – These are flattened cells that are mostly found lining on
surfaces that need a thin surface for molecular transfer, such as air sacs found in the
lungs, or a smooth fluid flow such as the blood vessels.
 Cuboidal epithelial cells – These are single layered cube-shaped cells that are typically
found in substance secreting or absorbing tissues, such as the glands (e.g. the thyroid
gland), ducts and kidney tubules (Krause, 2004).
 Columnar epithelial cells – These cells are long, thin and columnar-shaped arranged in a
single layer. They are found lining in areas that secrete mucus such as the intestine,
stomach, and in gall bladder and the uterus.
 Ciliated columnar cells – These cells usually have their apical surface covered with cilia
– tiny little hairs. They are useful in pushing mucus or other particles for smooth flow
(Krause, 2004).
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Question 6:

What different types of epithelial cells might you observe from your cheek cells?

The human cheek cell is typically thin, flat and irregular in shape and has a large nucleus with
the DNA. It is made up of a tissue called basal mucosa that lines the inside walls of the mouth.
This tissue is composed of squamous epithelial cells whose function is to continually secrete
mucus that play an important role of keeping a moist environment inside the mouth. In
association with salivary glands, the cheek epithelial cells provide enough supply of moisture for
enzymes to function well. The moisture assists in softening food, swallowing and starts the first
steps of food digestion. The epithelial cells are relatively permeable to selectively allow certain
particles or molecules in and out of the cell (King, et al., 2001).

These cells can be obtained through mouth rinsing or a simple swab. Though simple, these cells
contain the whole genetic makeup of an individual. Thus, they are used in investigations
involving DNA identification, e.g. in establishing paternity (King, et al., 2001).

Question 7:

Can you determine from which part of the body the epithelial cells came from, simply by
looking at it through the microscope?

It is possible to determine the part of the body from which the epithelial cells observed through
the microscope. Different epithelial cells have different apical shapes that vary with the part of
the body from where they are obtained. There are four main shapes of epithelial cells: squamous,
columnar, cuboidal and ciliated columnar. Squamous epithelial cells are thin and flat-shaped and
are normally found in blood vessels of air sacs in the lungs. Columnar epithelial cells form long,
thin and tiny columns and are normally found in mucus-secreting places such as the stomach,
intestines, and on the linings of the gall bladder and in the uterus. Cuboidal epithelial cells have
a cubical cross section and are typically obtained in tissues such as glands and the kidney.
Ciliated columnar cells are characterized by small hairs known cilia covered on their outer face.
Other cell shapes used to identify epithelial cells depends on whether they are simple stratified.
Stratified cells are commonly found in areas that are subjected to wear and tear, e.g. the skin
(Solomon, 2015).
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Question 8:

Are there any other techniques which might identify from which part of the body the
epithelial cell originated?
There are other techniques available used to identify the part of the body from which epithelial
cells have originated. These are discussed below:

 The Lugol’s Iodine Staining technique – This technique involves using iodine in the
detection of extracellular glycogen. It is believed that some areas have higher
concentration of epithelial cells than other areas of the body (Wise, 2002).
 Dane’s, staining technique – This technique uses histological staining to distinguish
between buccal, vaginal and skin epithelial cells. The samples are stained and analyzed
quantitatively by examining the patterns or colour, and the morphology of the cells.
Under a microscope, epithelial cells from different areas of the body will have different
staining patterns and morphology.
 Immunohistochemical staining techniques – This technique involves staining a sample
of epithelial cells with antibodies to identify certain proteins that make up a specific cell.
It is widely used to identify vaginal epithelial cells. It has increased the ability to identify
other categories of cells under a microscope (Wise, 2002).
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Question 9:

Why might identifying the origin of the epithelial cell be useful in forensic science?

There are several forensic cases that may require the identification of the type of epithelial cells
from which certain DNA originated. This would provide significant probative evidence. Many
types of human body fluids may be retrieved from a crime scene and can be potentially analyzed
and become useful in identifying the perpetrator of the criminal act. For example, when
investigating exhibits retrieved from a sexual assault scene, forensic science target to obtain
evidence that will confirm or refute allegations of occurrence of a sexual intercourse that is non-
consensual (James, et al., 2005). Evidence may be unfolded as trace evidence or stains from the
body fluid of the suspect, the victim or the scene of crime. Saliva is typically composed of
enzymes, water, mucus, various electrolytes, and epithelial cells originating from the cheeks. It
contains the same proteins as those found in blood and urine, thus, it is one ideal body fluid for
DNA profiling.
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References

Bancroft, J. D. & Gamble, M., 2008. Theory and Practice of Histological Techniques.
s.l.:Elsevier Health Sciences.

Crocker, J. & Burnett, D., 2005. The Science of Laboratory Diagnosis. 2nd ed. s.l.:John Wiley &
Sons.

Hibbs, A. R., 2004. Confocal Microscopy for Biologists. s.l.:Springer Science.

James, S. H., Nordby, J. J., Bell, S. & Nordby, J. J., 2005. Forensic Science: An Introduction to
Scientific and Investigative Techniques. s.l.:CRC Press.

Karp, G., 2009. Cell and Molecular Biology: Concepts and Experiments. 6th ed. s.l.:John Wiley
& Sons.

King, T., Reiss, M. & Roberts, M., 2001. Practical Advanced Biology. s.l.:Nelson Thornes.

Krause, W. J., 2004. The Art of Examining and Interpreting Histologic Preparations: A
Laboratory Manual and Study Guide for Histology. s.l.:Universal-Publishers.

Paddock, S. W., 1999. Confocal Microscopy: Methods and Protocols. s.l.:Springer Science .

Solomon, E. P., 2015. Introduction to Human Anatomy and Physiology. s.l.:Elsevier Health
Sciences.

Wise, C., 2002. Epithelial Cell Culture Protocols. s.l.:Springer Science & Business Media.