ORIGINAL COMMUNICATION

Anatomy Journal of Africa. 2023. Vol 12 (1): 2252-2262

PREPARATION OF HISTOLOGY SLIDES AND

PHOTOMICROGRAPHS: INDISPENSABLE TECHNIQUES IN
ANATOMIC EDUCATION.
Ubi Essien Isaac1, Emmanuella Oyo-Ita2, Nancy Paulina Igwe3, Eze Lazarus Ije3
1
Department of Human Anatomy, Faculty of Basic Medical Sciences, Cross River University of
Technology, Nigeria
2
Department of Biochemistry, Faculty of Science, Cross River University of Technology, Calabar,
Nigeria
3
Department of Anatomy, College of Medical Sciences, Alex Ekwueme Federal University, Ndufu-
Alike, Ikwo, Nigeria
Corresponding Author: Dr Ubi E. Isaac, E-mail: [email protected]. ORCID: 0000-0003-
4577-6278

ABSTRACT
The relevance of histology slides in microscopy cannot be overemphasized. However, a major
challenge to many scientists in the field stems from paucity of published data to nearly lack of
access to a comprehensive technical guide to prepare tissue samples as needed for practical
demonstrations, reviews or diagnostic purposes. The current research project was carried out to
produce histology glass slides and photomicrographs, as well as to document the step-by-step
procedures for model by concerned practitioners and scientists. The standard procedures for
paraffin method of histological preparation were employed. This involves sample fixation,
dehydration, clearing, impregnation, embedding, section-cutting, staining and mounting. Harris
hematoxylin and eosin were used for section staining. Tissue specimens were sourced from
laboratory rats (Rattus norvegicus). Other materials used included reagents- formalin, alcohol,
xylene, distyrene plasticizer xylene (DPX) as well as instruments/equipment- embedding mould,
water bath, oven and rotary microtome. Photomicrographs were produced using digital camera
attached to light microscope that is connected to a computer interface. 500 pieces set of slides
comprising 20 different organ specimens was produced as samples, with each specimen mounted
on 25 glass slides of either longitudinal or transverse sections. 40 digital images taken at low and
high magnifications were also generated from the various specimens and subsequently stored on
a disk. This project occupies a special place in practice due to the clarity about the techniques
and the good quality of the slide series obtained which both contribute to knowledge.
Keywords: tissue processing, embedding, microtome, photomicrographs, microscopy
DOI: https://dx.doi.org/10.4314/aja.v12i1.1
INTRODUCTION
Histology slides sometimes referred to as photomicrograph or simply micrograph
microscope glass slides are objects usually (often generated from the glass slide
prepared to demonstrate or explore the produced) is a photograph taken through a
microscopic structure of cells, tissues and microscope to show a magnified or digital
organs as well as to understand how the image of a specimen or an object. Thus, it is
structure relates to function. These objects a graphic reproduction of the image of an
are often biological specimens secured or object formed by a microscope (Connett,
held on thin flat pieces of glass and most 2017). The technique or process by which
times stained to highlight various structural images of specimens are captured or
features, for visualization and evaluation photographed through a microscope is
with the aid of a microscope. A known as photomicrography.

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The availability of a collection of histological Over the years, major revisions within the
glass slides and photomicrographs for review medical teaching curricula of many
is essential in the study and practice of institutions have placed severe constraints
anatomy and other microscope-based upon the time allocated to the teaching of
disciplines including pathology and molecular traditional laboratory courses in the
biology. It is imperative for scholars to know anatomical disciplines (Fitzharris, 1998;
how to prepare tissue samples as well as Hightower et al., 1999; Cotter, 2001).
apply the knowledge to interpret cells and Despite the wealth of real and virtual imaging
tissues through a microscope, rather than capabilities available to students in some
reproducing the information found in institutions in developed countries, the most
histology textbooks. However, due to the important resource to stimulating learning
technical know-how required and/or the remains the one-on-one or small group
difficulty often faced in preparing or interactions of students with faculty
obtaining good quality slides as well as the (Heidger, 2002). Hence, that approach helps
high cost of purchasing ready-made slide to maintain the teaching of histology by
collections for teaching and demonstration, using microscope glass slides and
histology is often taught today without photomicrographs. However, a common
laboratories, and a histology atlas is problem facing many anatomists and other
frequently used as a replacement. This is concerned laboratory scientists is the lack of
unfortunate because according to Sorenson a comprehensive practical guide to produce
and Brelje (2005), no matter how good the histology slides whenever needed. Moreover,
few images in a text book or histology atlas the world of science is fast advancing beyond
are, they cannot replace the experience of theoretical knowledge. Hence, this research
viewing a specimen through a microscope. project has been designed to rekindle
This traditional method of histology which is scientific or practical interest in traditional
centred on microscopy allows the observer methods of histopathology as well as to
an exceedingly close view of minute or very encourage efficiency or mastery of slides and
small structures at a scale convenient for photomicrographs production for the
examination and analysis (Ford and purpose of visualizing the real microstructure
Shannon, 2021). of cells, tissues and organs.

MATERIALS AND METHODS

Various materials in the form of reagents, The following step-by-step procedures
instruments, tools and/or equipment were constitute techniques for preparing
used and these included formalin for tissue histological tissue specimens as modified
fixation, alcohol or ethanol for dehydration, from the methods of Slaoui and Fiette
xylene for clearing tissue, paraffin wax for (2011):
impregnation and embedding, DPX mountant
for keeping specimen in place and protecting Tissue Sampling
from accidental contact, glycerin for making This process involves the collection of tissue
section adhesive, dyes for staining, either by biopsy, surgical excision or
processing cassettes, embedding mould, postmortem dissection. Access to human
water bath, frosted slides, staining racks and tissue is not always easy or practical, and is
dishes, cover slips, rotary microtome, electric complicated by ethics and legislation over
hot plate, hot air oven, plastic sample both data and tissue samples (Thomas,
bottles, dissecting set, digital camera and 2014). For this research project, laboratory
light microscope. rats were euthanized through cervical
dislocation to enable dissection and

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extraction of various organs including the identity of the sample-specimen as it is

different brain portions (cerebral cortex, passed through subsequent steps).
cerebellum, hippocampus), heart, pancreas,
liver, spleen, stomach, kidney, testis, seminal Dehydration
vesicle, prostate gland, ovary, breast and This procedure is carried out to remove
uterus. However, it is important to note that water from the tissue and involves the
whatever method of euthanasia chosen in immersion of samples in ascending grades of
any case (i.e., depending on the research alcohol. See end note 2 for guide to prepare
purpose) must follow scientifically accepted alcohol solution
procedures. I. Immerse in 50% alcohol for 45 minutes;
afterwards use forceps to transfer sample
Tissue Fixation to the next grade of alcohol.
This procedure is necessary to preserve the II. 70% alcohol for 45 minutes
tissue from decay or to keep it in a near life- III. 90% alcohol for 45 minutes
like state as possible while also making it IV. 100% (absolute) alcohol for 45 minutes
firm. Start fixation process immediately after V. Absolute alcohol (second change) for 30
the removal of selected organs to prevent minutes
decomposition caused by microbial activity. VI. Absolute alcohol (third change) for 30
I. Trim tissue/organs to reach the adequate minutes
size for rapid fixation
Clearing
II. Label sample bottles or vials indicating
This step is necessary to remove alcohol
sample name and collection date
from the tissue prior to paraffin embedding,
III. Fill the bottles with fixative though not to
using an agent that is miscible both in alcohol
the brim but ensure level up to two third of
and paraffin. This treatment most often
the vial
involves immersion of sample in xylene
IV. Put the organs in separate vials with
which also increases the refractive ability of
histological fixative such as 10% formalin
the tissue by giving it a transparent
(if tissue is very delicate, use Bouin’s fluid).
appearance.
See endnote 1 for information on how to
I. Immerse in xylene for 1 hour
prepare fixative.
II. Repeat the process in two changes of
V. Leave the tissue to fix depending on the
xylene for 30 minutes each.
appropriate duration or the type of fixative
used and the temperature condition Some other agents may be used for clearing
(formalin: 10-24 hours; Bouin’s fluid: 4-8 but the time would vary as tissues would
hours, at room temperature) need to be kept for a longer period. These
VI. Next trim the tissue to obtain 3 – 5mm thick include:
sample size that is compatible with
subsequent histology steps as well as to get a. Chloroform: widely used for its hardening
the proper orientation. effect; ideal for hard and delicate tissue
VII. Place the sample in a tissue processing b. Toulene: tissues can be kept in this agent
cassette together with the appropriate label for a longer period as it causes less
and then cover it properly. (When making shrinkage
labels during this step, use pieces of paper c. Cedar wood oil: preferred for delicate
from plane sheet and make inscription with tissue due to its low penetrating rate.
pencil. Do not use ink. Also, ensure the
inscription on the label shifts toward the
right half of the piece of paper leaving the
left half space blank. This helps to retain

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Impregnation I. Insert the appropriate knife in the holder or

This process is carried out to infiltrate the change disposable blade if necessary and
tissue sample with molten paraffin wax to tie or screw properly. Ensure to tie the knife
replace clearing agent and provide support. clamp screw securely
I. Use glass containers or aluminum jars to II. Apply ice to the surface of the block for a
melt wax in hot air oven at a temperature few seconds and wipe off the water from
of 60 – 670C. (Do not exceed this the block surface. This is done to aid
temperature to avoid tissue frying and sections cut easily
shrinkage) III. Place or clamp the paraffin block in the
II. Transfer the tissue while enclosed in the microtome by pulling the medial arm
processing cassette to molten wax for 20 forward, in a position away from the knife
minutes. or blade to avoid accidental cut through the
III. Repeat this process with another two tissue.
changes in molten paraffin wax for 20 IV. Adjust the microtome stage so as to move
minutes each to completely remove the the paraffin quadrate close to the
clearing agent. microtome plate
V. Unlock the microtome wheel with the
Note: Ensure not to allow the wax to solidify appropriate button.
when transferring the tissue from one VI. Next place both hands on either side of the
change to the other; keep the wax liquefied finger guard and pull it down as to reveal
by adding molten wax at regular intervals. the microtome plate. (Always lock the
wheel back into place when not sectioning).
Embedding
VII. Use the section thickness adjustment knob
This enables the careful positioning of the
to set initially to approximately 15µ
tissue inside a metal base mold or mould
(microns) so as to trim any excess wax and
filled with melted paraffin.
thereby expose a suitable area of tissue for
I. Pour into the mold an adequate amount of
sectioning.
molten wax heated at 650C.
VIII. After exposing a suitable area of tissue, set
II. Use warm forceps to place the tissue in the
the section thickness to 5µ. (The
mold and adjust it appropriately to orient
appropriate thickness level for routine
tissue surface toward the base of the
purposes is between 3 and 5 µ).
mould.
IX. Move the tissue surface of the block parallel
III. Next, submerge the mould into cold water
to the edge of the knife to obtain straight
at 200C or place on a cooling surface such
ribbon of sections.
as the cold plate to avoid wax
X. Turn the wheel to begin to cut into the
crystallization.
paraffin specimens and continue turning
IV. Allow the blocks for a little time to harden
until a desired section is obtained, making
and then remove them from the mould
a ribbon that contains multiple sections of
V. Trim or cut the paraffin blocks into suitable
the tissue.
sizes using a surgical blade or small knife.
XI. Use twizers or small forceps to collect the
VI. Next, melt the top surface of paraffin block
paraffin ribbons from the microtome and
with a heated knife and then mount it on a
then place on the slide that has been
wooden block to firmly attach.
smeared with adhesive.
Sectioning XII. Gently pour 30% alcohol upon one end of
This is a process in which the tissue is cut the slide and tilt it obliquely to enable the
from the paraffin block with a microtome. solution to spread over the ribbons and
When ready to section a paraffin embedded thus facilitate their flattening out during the
organ or tissue: subsequent step.

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XIII. Next, place the ribbon to float on a water X. Rinse in tap water twice for 5 seconds each.
bath regulated at 450C so as to flatten or XI. Next, immerse sample in hematoxylin
straighten the ribbon and also remove any neutral solution for 15 minutes (see end
shrinkage. note 4a).
XIV. Use a blade or forceps to split the ribbon of XII. Dip in 1% acid alcohol to differentiate or
sections floating on water to obtain get rid of excess stain (see end note 5, on
individual or groups of sections as desired. how to prepare the acid alcohol).
XV. Prior to picking up tissue sections that float XIII. Rinse in water twice for 5 seconds each.
on water to mount on a blank slide, first XIV. Next, immerse in eosin for 5 minutes.
apply some adhesive on the frosted surface XV. Dip in water to rinse for 2 seconds.
of the slide, as noted in XI above (see end
note 3 for a guide to prepare adhesive). Mounting and placing coverslips
XVI. When picking up the section from water, This involves covering the tissue section in
immerse the adhesive-smeared slide an ideal resin medium such as DPX or
vertically to about three-fourths the length Canada balsam to keep specimen in place
of the slide to bring the section in contact and protect it from accident contact.
with it. I. Use a dispenser rod to apply 1 or 2 drops
XVII. Lift the slide vertically from the water to of DPX on the slide while being placed on a
find the section flattened on to the slide level surface.
XVIII. Blot sections lightly with blotting paper to II. Place the cover slip gently from one side of
remove excess water and to increase the droplet to avoid any air bubble inside it.
contact between slide and section, or keep III. Put slides in a warmer, or just expose them
the slide in upright position for several to normal room air to dry for at least 6
minutes to drain. hours and then cut excess medium from
the edges of cover slip with a razor blade.
Staining
This involves a series of protocol aimed at Photomicrography
colouring specimens to highlight important This is a process by which images of
features of the tissue as well as to enhance specimens of interest are captured using a
the tissue contrast. The following steps are camera attached to a microscope that is
required for Hematoxylin and Eosin (H/E) connected to computer interface.
method. Photomicrographs of histological tissue can
I. Prepare each dye or stain separately and be taken using Motic image plus 2.0 ML – the
put in a staining dish (see end note 4a and model 35 moticam camera
b, for guide to prepare H/E stock solutions I. Fix the camera to the eye-piece of the
respectively). photomicroscope to view the tissue through
II. Arrange the glass slides in a staining rack. the various objectives of the
III. Immerse the tissue sample in xylene for 10 photomicroscope.
minutes. II. Connect a lap-top as computer interface to
IV. Rinse the sample in fresh xylene for 5 the photomicroscope to aid viewing.
seconds. III. Observe the image of the tissue viewed on
V. Rinse in absolute (100%) alcohol for 10 the lap-top screen.
seconds. IV. Capture the image and store for evaluation
VI. Rinse in another absolute alcohol for 5 or studies as desired.
seconds. V. For each sample of tissue under study take
VII. Rinse in 90% alcohol for 5 seconds. photomicrograph at low power objective
VIII. Rinse in 70% alcohol for 5 seconds. x10 and at high power objective x40 of the
IX. Rinse in 50% alcohol for 5 seconds areas for study.

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RESULTS
The following histology slides and
photomicrographs are sample products of
the research project:

Figure 3: Photomicrograph of sample of

kidney x400
Figure 1: Photo showing stained microscope
glass slides ready for photomicrography

Figure 2: Photomicrograph of sample of Figure 4: Photomicrograph of kidney sample

cerebellum transverse section x400 (high x100 (low magnification)
magnification)

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Figure 8: Photomicrograph of sample of

Figure 5: Photomicrograph of sample of prostate gland x400
testis (x400)

Figure 9: Photomicrograph of sample of

pancreas (x100)

Figure 6: Photomicrograph of ovarian sample

(x400)

Figure 7: Photomicrograph showing

longitudinal section of lung sample (x400) Figure 10: Photomicrograph of sample of
epidydimis (x400)

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Figure 11: Photomicrograph showing

longitudinal section of stomach sample
(x100).

DISCUSSION

As presented in figure 1, glass microscope become readily available, the stained glass
slides were produced by paraffin method of slide has for centuries been the mainstay and
histological tissue preparation. The various its examination will remain the “gold
tissue specimens or samples were secured or standard” (Tsai et al., 2007). Since optical
mounted on the slides. This arrangement instruments were first used in the early 17th
enables a slide-mounted specimen to be century to visualize biological material at the
quickly inserted in the microscope for microscopic scale, the morphological study of
examination and thereafter removed for cells and tissues has largely been driven by
storage in the appropriate slide box. The technological advances, such as the
traditional glass slide with the aid of a development of high quality light
microscope serves to give the observer an microscopes, new procedure for preparing
extremely close view of very small structures biological specimens for visual analysis and
at a scale convenient for proper evaluation. novel microscope equipment and approaches
The origin of the concept was pieces of ivory (Hortsch, 2013).
or bone, containing specimens held between
disks of transparent mica that would slide For many years, the teaching of
into the gap between the stage and the microanatomy has relied on banks of light
objective of a microscope). These “sliders” microscopes and boxes of histological glass
were popular in Victorian England until the slides. This gave students an opportunity to
Royal Microscopical Society introduced the learn how to operate a light microscope.
standardized glass microscope slide However, this approach suffered from a
(Connett, 2017). great variability in the quality of the slide
material, even as good quality histology glass
Apart from enabling close attention to slides are often difficult to obtain or
structural details when viewed under the prohibitively expensive. In addition, many
microscope, images of specimens are tissue and organ preparations are difficult to
captured or photographed at either low or come by. These problems and the upkeep of
high magnifications as shown in figures 2-11. many light microscopes are a continuous
Despite the variation in format and content expense, especially during times of
between institutions offering anatomical diminishing financial support for higher
sciences and/or other microscope-based education (Drake et al., 2009; Horsch, 2013).
courses, light microscopy is still the most Even in settings where virtual microscopy
important tool for practical teaching and tends to be replacing light microscopes and
photomicrographs remain the mainstay of glass slides for the teaching of histology,
self-study material (Marshall et al, 2004). there are limitations as access to a single
Even as digitized images and virtual slides virtual microscopy website relies on a fast,

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stable internet connection and its contents histopathology and cytopathology.

may not align with different curricular and Microscopy plays an important part in
course structures (Logan et al, 2009). haematology, microbiology and more
broadly in the areas of biology, zoology, and
Over the last ten years, many schools and botany because all these disciplines involve
universities in some countries have moved the examination of specimens under the
away from the use of real microscopes and microscope (Rolls, 2021). The availability of
have adapted a novel, electronic way to high quality digital consumer cameras at
expose student to the structure of cells and relatively low prices has made photography
tissues at the microscopic scale (Coleman, with the microscope significantly easier than
2009; Drake et al., 2009). Although this with traditional film, together with recent
emerging modality known as virtual developments in software aimed at amateur
microscopy is perceived as a useful tool for digital images, which now offers the
learning, the hybrid or traditional approach photomicrographer capabilities for images
still remains the most preferred for histology that were not possible only a few years ago
learning (Telang et al, 2016). According to (Quekett, 2021).
Heidger (2002) the contention is that,
despite the wealth of real and virtual imaging CONCLUSION
capabilities available to students of histology, The output of this research project is
the most important resource to stimulating relevant in scientific education, due to the
learning remains the one-on-one or small ease of reproducibility and replication of the
group interaction of students with faculty, as techniques as well as the use of its products
this approach helps to maintain the teaching for teaching and practical demonstrations.
of histology by traditional means.
ENDNOTES
There are many reasons to examine human
1. Preparation of 10% formalin: requires 1
cells and tissues under the microscope.
part of stock formalin (i.e formaldehyde
Medical and biological research is
solution) with 9 parts water. For example
underpinned by knowledge of the normal
when using a 100 mL calibrated cylinder,
structure and function of cells and tissues,
simply measure 10 mLs of formalin and then
and the organs or structures that they
add 90 mLs of water.
makeup. In the normal healthy state (as
2. Dehydrating stock solution constitutes
visible in the photomicrographs in figures 2-
ethanol and water. To get a particular
11), the cells and other tissue elements are
concentration, apply the formula V1=C2V2÷C1
arranged in regular, recognizable patterns.
where V1 and V2 represent initial and final
However, in the event of changes induced by
volume respectively while C1 and C2 stand for
a wide range of chemical and physical
the initial and final concentration. Example,
influences, these are reflected by alterations
to prepare 60% ethanol in a final volume of
in the structure at the microscopic level, and
100 mL: V1=?, V2=100mL, C1=100%, C2
many diseases are characterized by typical
=60% V1=C2V2÷C1.
structural and chemical abnormalities that
differ from the normal state. Therefore (60%)(100mL)÷100% =60 mL. A
quick way is to divide 60 by 100% and
Without a sound knowledge of normal
multiply by 100mL (which is the total volume
histomorphology as obtained in the present
of container). This yields a 60mL measure of
research, it is impossible to identify these
ethanol, while the remaining 40 mLs is of
changes and link them to particular diseases
water.
that should form the basis of important
specializations of modern medicine such as

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3. Section adhesive preparation: mix 50% to cool. Afterwards the stain can readily be
glycerin with 50% albumin (fresh egg white) used.
to make this.
However, if the progressive method is
4. Stains chosen, about 4 ml of glacial acid per 100 ml
of solution should be added and filtered
4a. Hematoxylin preparation: Either of two before use.
methods can be used. One of these is the
progressive method which requires the 4b. Eosin preparation: i. dissolve 1 g of the
addition of excess acid or salts in the solution dye in 80 ml of distilled water. ii. mix the
to increase the accuracy of nuclear staining. solution with 320 ml of 95% alcohol. iii. Add
The other method known as regressive 0.4 ml or a few drops of glacial acetic acid.
staining requires a neutral hematoxylin
solution to over stain the tissues; afterwards 5. Preparation of 1% acid alcohol when using
wash the slides by dipping in acid to remove a total volume of 100 mL, requires the
excess stain and then rinse in water to addition of 99 mL of 70% ethanol to 1mL of
neutralize the acid wash. hydrochloric acid (HCL).

I. Dissolve 5 g of hematoxylin in 50 ml of ACKNOWLEDGEMENT

absolute alcohol.
II. Dissolve 100 g of potassium alum in 1000 The Authors acknowledge funding support
ml of distilled water by heating. from Tertiary Education Trust Fund
III. Mix the two solutions obtained in the (TETFund) of Nigeria, by means of
preceding steps i and ii and then heat as institution-based research grant, with
fast as possible so as to boil within one reference number:
minute while stirring at frequent intervals. TETF/DR&D/CE/UNI/CAL/RG/2021/VOL.1
IV. Next, remove from heat and add 2.5 g of
CONFLICTS OF INTEREST
mercuric oxide slowly.
V. Heat again to boil slowly until it turns dark The authors affirmed that there are not any
purple, conflicts of interests
VI. Remove from heat and immediately thrust
the kettle or flask into cold water and allow

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