2025 INTRODUCTION TO HISTOLOGY

BSMLS - 1M Francis Ian Salaver, RMT, MD

Histology  The smallest or the basic structural

and functional unit of the human
 the study of study of tissues, their body is the cell.
functions and their arrangement to  If the cell be group to the other
constitute an organ cells, as long as their functions
 Branch of anatomy = also known as are interrelated, they can form a
MICROSCOPIC ANATOMY tissue. If the tissues are group
together and their functions are
WHAT IS A TISSUE? also interrelated, they can make up
 Group of cells w/ interrelated an organ and an organ will always
functions belong to a certain human body
 More to a tissue than just being a system.
group of cells
URINARY BLADDER
 i.e., a neuron from the brain, a
hepatocyte from the liver,  One of the organs in the body, it
keratinocyte from skin STILL CANNOT belongs to the urinary system. Organs
BE CALLED A TISSUE (NEED TO HAVE are made up of tissue.
INTERRELATED FUCNTION)  X sign in the image (one of the
 The cells in the tissue need not to tissues that you can find in the
be in the same type of cell urinary bladder, composed of group
of cells because tissues are
 The important is their function must
actually group of cells.
be interrelated w/ each other.
 Tissues are made up of group of
 They don’t have to be in the same
cells w/ interrelated functions, if
size, same shape, same morphology as
tissues are grouped together, they
long as their functions are
can form an organ.
interrelated, then they can make up
a tissue. Four Basic Types of Tissue
 Fibers – hairlike structures All these types of tissues have group of
 Empty spaces that composed of cells, and if you’re going to combine
chemicals, empty spaces are called these four basic types of cells can form
GROUND SUBSTANCE an organ
 Group of cells specialized to carry  EPITHELIAL TISSUE (epithelium)
on a interrelated functions and  Nearest to the lumen (a luminous
their associated extracellular term referring to the channel
matrix within a tube such as a blood
 Cell + extracellular matrix vessel or to the cavity within a
(composed of fibers and ground hollow organ such as the
substance) intestine) of the stomach
 The ground substance and fiber are  Lining epithelium functions to
found outside the cell, they are provide covering, and functions
referred to as extracellular matrix. for absorption and secretion.
(i.e., lining epithelia of the
WHY STUDY CELLS IN A SUBJECT THAT IS large and small intestine.)
SUPPOSED TO STUDY TISSUES?  It is the epithelium that can
 Because tissues are made up of cell, provide protection against
you can never understand the stomach acid
characteristics off a tissue without  The cells that make up the
understanding the characteristics of epithelium tissue are tightly
its individual components of the packed (they do not tolerate
cell. spaces between
 The lining epithelium doesn’t
Organization in Human Body have their own blood supply
(AVASCULAR)

First Semester | Prelims

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 2025 INTRODUCTION TO HISTOLOGY
BSMLS - 1M Francis Ian Salaver, RMT, MD
 If these cells have a  Predominating cells are
blood vessel, then these fibroblast
cells are no longer  the bigger fiber is collagen
tightly packed, and they
fiber
cannot function for
 the thinner fibers are elastic
protection
fibers
 If the cells of the epithelium
are not getting enough oxygen, HOW CAN YOU FIT A LARGE HUMAN
then most likely the cells would ORGAN INTO A GLASS SLIDE?
die  in histopathology laboratory or
histopathology laboratory, we
HOW DO YOU THINK THE ORGANS IN THE BODY prepare the organ and make a
MAINTAIN THE CELLS OF THE LINING
thin cut section of the organ.
EPITHELIUM?
 the main purpose of preparing a
 The lining epithelium doesn’t have
thin section is that when
their own blood supply, but it is
placing this thin section on the
situated there because it’s the
epithelium that can provide protection top of the slide, the light
against HCl in the stomach coming from the light source of
 Connective tissue is highly the microscope can actually
vascularized meaning the connective traverse through the slide and
tissue has its own blood supply the specimen so that the light
 The epithelium is there to protect the can reach the eye of the
connective from the HCl observer.
 What will keep the cells of the
epithelium because they are getting Preparation of Tissues for Study
blood supply from the connective tissue  To study tissues, one must prepare
thin and translucent histological
 CONNECTIVE TISSUE sections or tissue slices that can
- Below the epithelium be studied with the aid of a
- Presence of the red-colored microscope.
capillary  fixation -> dehydration -> clearing
- Provides blood supply to the -> infiltration -> embedding
epithelium
Fixation
- What link to the epithelium to
the muscle? – it is because of Since cellular decomposition begins
the connective tissue, it tends immediately after the death of a
to connect the epithelium to the human/patient, tissues must be fixed to
other tissue in the organ. prevent alterations in their structure
 MUSCLE TISSUE through decomposition. (TO PRESERVE AND
- Below the connective tissue PREVENT CHANGES IN THE ORGAN)
- Incorporated to the organ to  If you have removed an organ from
provide movement. the body because you want to process
 NERVOUS TISSUE it, you have actually cut it from
- Somehow distributed between the its own blood supply, if no blood
connective tissue and muscle supply then most likely the cells of
tissue is the nervous tissue that organ will not receive oxygen
- something to feel/sensation, and the cells will start dying and
control, and information the organ will begin decomposing or
processing. decaying.
 The appendix should be immersed in
 Yellow cell – adipose cell or fat formalin and the main purpose of
cell which is not to allow the organ to
 Mast cell - appears to have a undergo decomposition or decay.
lot of dark granules

First Semester | Prelims

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 2025 INTRODUCTION TO HISTOLOGY
BSMLS - 1M Francis Ian Salaver, RMT, MD

 Neutrophils are antibacterial, the
white blood cells that fight off
bacteria.
 If you have abundance of these
neutrophils in your appendix it
would suggest that the appendix is
having a current infection. And
confirm diagnoses of having acute
appendicitis.
 The problem is; If the MedTech
forgot to fix the organ or if the
surgeon after removing the appendix
from the body of the patient forgot
to immerse the organ in the formalin
or in the fixative agent, then most
likely you will no longer appreciate
the presence of these neutrophils
because the appendix will undergo
the decomposition and decay.
PURPOSE OF THE FIXATION:
1. Avoid tissue destruction by digestive
enzymes (autolysis) or through
bacterial degradation
- (i.e., organ is small intestine,
where the enzymes from the pancreas
are drained onto, if the intestine
is removed and the enzyme is still
there, and the organ is not fixed
then these enzymes are
inactivated. The enzymes will start
digesting the intestine and you will
no longer be able to appreciate the
structure of the antecedent so
that’s what we mean by number one.
2. Terminate cell metabolism – glycolysis
without oxygen = lactic acid.
- The fixed the fixation step will
terminate cell metabolism
- Glycolysis is the conversion of
glucose to energy, if glycolysis
will happen without oxygen because
the organ was already cut off from
its blood supply, then the product
will be a lactic acid.
- When you have oxygen glucose will be
most likely converted to pyruvate,
and pyruvate will enter
mitochondria to complete the Krebs
cycle and the electron transport
chain and there would be generation
of atp molecules.
- In the absence of oxygen, the
glucose will be converted to lactic
acid or lactate. If the appendix
remove from the body and the cell
there will use up the glucose, the
remaining glucose in the appendix
and then instead of producing
energy, they are building up lactic
acid. The lactic acid will start
destroying the appendix the reason
why we must terminate cell
metabolism. We should not allow the
glycolysis to happen or else the
cells will start producing lactic
acid and the lactic acid will
destroy the appendix
- When the muscles contract they will
impinge on their blood vessels. The
muscles are contracting, the blood
vessels are impinged. Then most
likely the muscles are not getting
enough oxygen or blood supply, there
would be build up lactic acid in the
muscle. And the build-up of the
lactic acid now will now be felt as
the PAMAOL.
- If you’re going to cut a soft organ
in the body and it’s as soft as the
gelatin most likely the organ will
move while doing the cutting and
cannot make a perfect thin section.
- One of the purposes of fixation is
to harden, somehow harden the organ
not
so that it can facilitate now easy
cutting or easy sectioning of the
organ
3. Harden the tissue by cross-linking or
denaturing proteins.
- So, the MedTech can do the tissue
sectioning later
4. Kill pathogenic microorganisms such as
bacteria, fungi, and viruses.
- Some of the specimens remove
surgically from the body are
infected and to protect the people
who are working in the laboratory
especially in histopath laboratory
the fixation must kill the
pathogenic microorganisms
- The most common used fixative in the
laboratory is formalin.
the
Decalcification
 only done in specimens such as bone,
teeth, and calcified tissues
 Nitric acid
 For soft tissue we have harden them
a little so that we can make the
cutting. In cases of calcified

First Semester | Prelims

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2025 INTRODUCTION TO HISTOLOGY
BSMLS - 1M Francis Ian Salaver, RMT, MD
tissues such as bones and teeth we
need to decalcified them to somehow
soften them so that we can easily
cut them.
 The most common reagent for the
decalcification is nitric acid.
 Everything should start in fixation
and the second step will be
decalcification, but if the specimen
is a softest or soft tissue/organs
such as the lungs, the heart, we
first fixation but there’s no need
for decalcification.
 Decalcification is not only limited
to bones and teeth, it can also be
done to arteries especially arteries
with atherosclerotic plaque
formation (caused by the deposition
of the cholesterol on the walls or
the lumen of the artery. The
presence of the cholesterol will
actually trigger inflammation and
accompanying tissue destruction and
if the tissue destruction then there
would be deposition of the calcium.
And the area will somehow harden)
Dehydration
 done by successively bathing the
specimen in mixture of ethanol and
water from 70% to 100% percent%.
From a lower concentration of
alcohol to a higher concentration of
alcohol until such time you’re
already dipping or immersing the
specimen in 100 percent or absolute
alcohol.
 Alcohol (ethanol and water – because
dehydration starts with using lower
concentration of alcohol, so we can
50/70% diluted form of alcohol and
there’s a water in it) removes water
out of the tissue.
 Removal of water in the tissue
 From 70 we will transfer the tissue
to a higher concentration of alcohol
that could be 80 and 90, immersed in
a pure or 100 absolute alcohol.
 Why do we have to immerse the
specimen from a lower concentration
of alcohol to a higher concentration
of alcohol? – it actually takes
time, to preserve the morphology and
the appearance of the organ as it
was removed from the human body. In
First Semester | Prelims
dehydration we removed water in the
tissue and the water will be
replaced by alcohol. The tissue now
will contain alcohol.
Clearing
 Removal of the dehydrating agent by
immersing the specimen in the
solvent that the alcohol and
embedding medium is miscible
 Clearing agent are highly volatile
(becomes vapor once exposed to heat)
 Xylene and toluene
 Next to clearing is embedding and
embedding makes use of melted
paraffin that’s why this procedure
is usually done inside an oven set
at 52-60 degree Celsius, the melting
point of the paraffin.
 The clear fluid is the melted
paraffin. Once the tissue is dipped
into the paraffin the xylene or the
toluene will immediately become
vapors. Allowing now the melted
paraffin to infiltrate the tissue.
 In embedding we remove the clearing,
and we replace it with paraffin (the
essence of doing embedding)
 The paraffin now will be allowed to
cool down so that you will have the
formation of a black with the tissue
placed in the center
 The problem with using paraffin is
that a lot of medtechs are prone to
having BURN INJURIES
 In the lab, paraffin is replaced
with plastic resins (fluid at room
temp) once tried to place something
it will solidify immediately
- So there’s no need to do a
procedure where there’s an oven
(no chance of having burn
injuries)
- The problem with plastic resin
is that it is too expensive
(increased charges in biopsies)
Cutting and Sectioning
Cutting
 After the specimen is hardened, it
is trimmed into appropriately sized
blocks
 Cutting involves the use of a knife
to remove excess paraffin
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 2025 INTRODUCTION TO HISTOLOGY
BSMLS - 1M Francis Ian Salaver, RMT, MD

 We need to remove excess paraffin so iv. Absolute ethanol – 3 mins

that the block will fit into the v. 95% ethanol – 3 mins
block holder of the microtome - Immersed in 95% alcohol
Sectioning - To remove the tissue xylene,
 The block is then mounted in immerse the tissue in
microtome and cut with a steel knife decreasing concentration of
- As you rotate the drive wheel, alcohol
the block holder vi. 95% ethanol – 3 mins
- The tissue block will go up and vii. Distilled water – 30 secs. to
down and will come in contact 1 min
with the steel knife - To remove alcohol, immerse the
- And you will now have the tissue in distilled water
production of thin sections of - We will allow water to go back
the organ you want to study to the tissue
- Most of the stains that we are
 The main reason why you need to
utilizing in histopath lab or
place the tissue in the melted
histology lab are water soluble
paraffin is for us to have something
- So for the stains to impart
to hold the organ during the
color to the tissue, the tissue
sectioning
must contain
 Allow the thin sections to float on
the water, then scoop them out using Staining
the slide. The sections that we
 Since paraffin is colorless,
created using the microtome are
staining is a must
still transparent so even if you
have already placed them on top of  Application of color too the tissue
the slide and view them under the to highlight structures
microscope you won't be able to  Most commonly used stain:
appreciate the structures, the - Hemotoxylin – a basic dye thus,
composition, the cells w/in the it will stain acidic portions
tissue because the tissues are still of the cell.
transparent. - Stains blue=> PURPLE
 After sectioning, after placing the - Nucleus is acidic
section on the slide it is very - Stains nucleus and RNA –
important that we should perform the containing portion of
next tissue processing steep. Which cytoplasm
is STAINING - Eosin – acidic dye, thus is
- We need to impart color onto will stains the basic portions
the tissue so that you can see of the cell.
the structures under the - Stains pink =>RED
microscope - Cytoplasm is basic
- Cytoplasmic components and
Deparaffination and Rehydration collagen
- Remove the paraffin from the thin  These dyes are water-
sections soluble
i. Xylene – 5 mins.
- Remove paraffin using xylene  Tissues with negative charges/acid
- Thin section is now are readily stained with basic dyes
infiltrated w/ xylene – BASOPHILLIC
ii. Xylene – 5 minss. - NUCLEIC ACIDS = NUCLEUS
iii. Absolute ethanol – 3 mins  Tissues with positive charges are
- Remove xylene by using stained with acidic dyes –
absolute alcohol ACIDOPHILIC
- The tissue section at this - MITOCHONDRIA, COLLAGEN,
point will now infiltrated w/ CYTOPLASM
absolute alcohol
SPECIAL STAINS

First Semester | Prelims

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 2025 INTRODUCTION TO HISTOLOGY
BSMLS - 1M Francis Ian Salaver, RMT, MD

 Feulgen reaction – DNA Astrocytes a star shape, limits the entry

 Periodic acid Schiff – carbohydrates of substance from the capillary or blood
to the nervous system.
 Sudan black – lipids
 Silver stain – reticular fibers

 how long would it take for the

tissue to be fixed – dehydrated –
cleared – embedded – sectioned –
mounted – stained – read by the
pathologist.
 Philippine laboratory set-up the
tissue would require at least 48-72
hrs. tissue processing time. (2-4
days)

MOUNTING
- To preserve and support a
stained section for light,
microscopy, it is mounted on a
clear glass slide, and covered
w/ a thin glass coverslip
- Placing cut sections on a
slide w/ mounting media such
as glycerin or resins. (to
make the coverslip stick to
the specimen and glass slide)

FROZEN SECTIONS
- Rapid method
- Routinely done in hospital to
study specimen during surgery
- Lipids and enzymes are best
preserved in this method
 Fixation is done freezing compressed
carbon dioxide
 Sectioning is done thru cryostat, a
refrigerated compartment containing
microtome.

 Dehydration remove of water replace

alcohol
 Clearing remove alcohol replace w/
xylene. Clearing agents are highly
volatile.

In nervous tissue the cells are

categorized into neurons (create nerve
impulse) and neuroglia (microglia one of
the neuroglia in the nervous tissue, a
phagocytic cell eat up bacteria that kills
neurons, it support and protect the neuron
to keep it alive.

First Semester | Prelims

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