Introduction to Histology

MLS 407 | Human Histology

Prelim | 1st Semester

Histology
| is the study of tissues, their functions, and their
arrangement to constitute an organ

Tissue
|Group of cells with interrelated functions

Neuron = brain x = group of cells

Hepatocyte = liver
Keratinocyte = skin

Cells do not have to be of the same shape,

morphology as long as their function is
interrelated they can make up a tissue

Histology
 Branch of anatomy = also known as
Microscopic anatomy
 Is the study of tissues, their functions and
their arrangement to constitute an organ.

Why study cells in a subject that is supposed to 4 Basic Types of Tissue

study Tissues? 1. Epithelial Tissue
You never understand the characteristic of the 2. Connective Tissue
tissue without understanding the characteristic of 3. Muscle Tissue
its individual components, the cell. 4. Nervous Tissue

Organization in Human Body

1. Cells – smallest or basic structural and
functional unit of the human body
2. Tissues
3. Organs
4. System
 Epithelium is avascular (no blood supply)

Nervous tissues – between connective and

muscle tissue

Epithelial tissue to provide (covering)

protection, and functions for absorption and
secretion (Yellow star)
Connective Tissue provides blood supply to
Lining epithelium of the stomach is situated epithelium and links it to other tissues
nearest to its lumen Highly vascularize

Stomach ‘s function to produce hydrochloric

acid, epithelial serves as protection

Muscle provides movement of tissue

Nervous tissue provides sensation, control and

information processing

The cells in the epithelium is tightly packed thus

there is no space for blood vessels
What is a tissue?

Predominating cells = fibroblast

Bigger fibers = collagen fibers
 Group of cells specialized to carry on a Thinner fibers = elastic fibers
interrelated functions and their
associated extracellular matrix Histology Laboratory
 Tissue = Cells + extracellular matrix
 Tissue = group of cells + (fibers +
ground substance)

Extracellular Matric
 The extracellular matrix is composed of
many kinds of
o (1) Ground substance and
o (2) Fibers
 TISSUE PROCESSING STEPS
The main purpose of preparing a thin section wo 1. Fixation
that we can place it on top of the slide. By this, 2. Decalcification
the light coming from the light source can 3. Dehydration
traverse through the slide and the specimen 4. Clearing
5. Embedding
Appendix 6. Cutting and sectioning
7. Staining
8. Mounting

1. Fixation
To preserve organ and prevent changes in the
organ
Preparation of Tissues for Study Since cellular decomposition begins
To study tissues, one must prepare thin and immediately after the death of a human/patient,
translucent histological sections or tissue slices tissues must be fixed to prevent alterations in
that can be studied with the aid of a microscope. their structure through decomposition. (TO
PRESERVE)
 C. Hardens the tissue by cross-linking or
denaturing proteins
D. Kill pathogenic microorganisms such as
bacteria, fungi and viruses
a. Commonly used fixative is
formalin

2. Decalcification
 Are only done to organs of the body that
are calcified, such as bone and teeth
 Only done in specimens such as bone
and calcified tissues
 Nitric acid – commonly used reagent for
calcification

( R) Scanner View
(L) High Power Magnification
-present of multiple neutrophils

Neutrophils – are antibacterial (white blood cells

that fight of bacteria)
Abundance of neutrophil in the appendix
suggest that the appendix is having an infection

Fixation Purposes
A. Avoid tissue destruction by digestive
enzymes (autolysis) or through bacterial
degradation.
B. Terminate cell metabolism
a. Glycolysis without oxygen =
lactic acid Decalcification can also be done to arteries
Latic acid will destroy the especially arteries with atherosclerotic plaque
sample formation (deposition of the cholesterol on the
walls of the arteries or lumen)

3. Dehydration
  Removal of water 5. Embedding
 Done by successively bathing the  After the clearing procedure, the tissue
specimen in mixture of ethanol and is placed in a melted paraffin in an oven
water from 70% to 100%. (increasing set at 52-60 degree Celsius.
concentration of alcohol)  The heat causes the clearing agent to
 Alcohol removes water out of tissue evaporate so that the tissue will be filled
 Starting from lower concentration of up with the paraffin.
alcohol to higher concentration of  Remove the clearing and replace it with
alcohol will produce minimal tissue paraffin
distortion

 The tissue and paraffin will harden after

removal from oven
o Plastic resins – makes use of
plastic solution which hardens
tissue by cross-linking polymers
4. Clearing o Eliminates the need to use oven
 Removal of the dehydrating agent by and paraffin; little tissue
immersing the specimen in the solvent distortion
that the alcohol and embedding medium
is miscible
 Clearing agent are highly volatile
(becomes vapor once exposed to heat)
 Xylene and Toluene
6. Cutting and Sectioning
 After the specimen is hardened, it is Sectioning
trimmed into appropriately sized blocks.
o Cutting is removal of excess
paraffin
 The block is then mounted in microtome
and cut with a steel knife.
o Sectioning is done with the aid
of microtome

Microtome The sections will be placed in a water bath and

will be scoop out with a slide
The sample is still transparent

Sections are transparent

  An alkaline/basic dye will be attracted to
portions that are acidic
Eosin
 Stains pink to red
 Is an acidic dye, thus will stain the
basic/alkaline portions of the cell

Before Staining
Deparaffinization and Rehydration

1. Xylene – 5 mins
2. Xylene – 5 mins
3. Absolute ethanol – 3 mins
4. Absolute ethanol – 3 mins Hematoxylin stains the nucleus because DNA
5. 95% ethanol – 3 mins are present in the nucleus and DNA are acid
6. 95% ethanol – 3 mins
7. Distilled water (30 seconds to 1 min)  Hematoxylin and Eosin – Most
commonly used stain in histology
Rehydration is done, because most od the stains  Hematoxylin- basic dye; usually stains
that are utilized in histopath laboratory or nucleus and RNA-containing portion of
histology laboratory are water soluble. So for cytoplasm
stains to impart color to the tissue, it must  Eosin-acidic dye – usually cytoplasmic
contain water components and collagen

7. Staining Tissues with negative charges/acids are readily

 Since paraffin is colorless, staining is a stained with basic dyes – BASOPHILIC
must. o Nucleic acids = nucleus
 Application of color to the tissue to o Acidic = basophilic
highlight structures Tissues with positive charges are stained with
 Most commonly used stain: acidic dyes – ACIDOPHILIC
Hematoxylin and Eosin o Mitochondria, collagen,
o These dyes are water soluble cytoplasm
o Basic = acidophilic

Special Staining
 Feulgen reaction – DNA
 Periodic acid schiff – Carbohydrates
 Sudan Black – Lipids
 Silver stain – reticular fibers
Hematoxylin
 Stains blue to purple 48- 72 hours tissue processing hours
 Is a basic dye thus, it will stain acidic
potion of the cell 8. Mounting
  Placing cut sections on a slide with  Sectioning is done thru cryostat, a
adhesives such as pinene or acrylic refrigerated compartment containing
resins microtome
 Cover slip is places on top of the stained
specimen and its main purpose is to
preserve the specimen, to prevent
washing off of the stain, to prevent
contamination, and provide support.
 Before the glass slide is placed on top of
the stained specimen, we place a
mounting media such as glycerin or
resin to make the coverslip stick to the
glass slide
Cryostat

Frozen Section
 Method is rapid
 Routinely done in hospital to study
specimens during surgery
 Lipids and enzymes are best preserved
in this method

Resected Colon Mass

Frozen Sections
 Fixation is done rapid freezing using
compressed carbon dioxide
Cytoplasm 1
MLS 407 | Human Histology
Prelim | 1st Semester
Composed of three basic parts:
PART OF A CELL  (1)Cell membrane
 (2) Nucleus
 (3)Cytoplasm
o Solid portion = Organelles
o Fluid portion = Cytosol

Cytoplasm = organelles plus cytosol plus

inclusion bodies

Inclusions
Cell Membrane – boundary or the border of the  Not a functional organelle
human cell  May or may not be present in the cell
Cytoplasm – space inside the cell  Most are stored nutrients or cell
Nucleus – largest organelle | most of the time product
situated in the center  Can be present in the cytoplasm and at
Nucleolus – inside nucleus some point might disappear because
Organelles – solid structure in the cell | are majority of it are stored nutrient
functional structures of the cell  Lipofuscin, lipid, glycogen, etc.
Cytosol – fluid where the organelles are
suspended
Cytoplasm = organelles plus cytosol

Lysosome- organelle that contains digestive

enzymes | is involved in intercellular digestion

The cells in the body can break down fats, lipid

and carbohydrates and the digestion of theses
fats, lipids and carbohydrates takes place in out
lysosome.
Lysosome does not only digest carbohydrates,
lipids, and fats.

Presence of injury can cause some organelles

1. Nucleolus inside the cell to become damaged and with the
2. Nucleus damage organelles are no longer functioning.
3. The cell will be discarded and will be
transported to lysosome. The lysosome will
digest the organelles. With this, lysosome does
CELL not only digest lipid, fats, and carbohydrates but
also digest damaged organelles. The
accumulated waste products of the digestion will Lipofuscins tend to accumulate in cells that are
eventually become lipofuscin. not actively undergoing mitosis.

Inclusion body: LIPOFUSCIN When you have a stroke, neurons cannot be

 Yellow-brown pigment composed of replace.
residues of lysosomal digestion When you have an injury in your akin the
 Accumulates through the years fibroblast and keratinocytes will multiply in
 “wear and tear” pigment = cellular number just to replace the damaged tissue.
senescence or aging
Neurons has limited capacity to multiply and the
keratinocytes and fibroblast in the skin have
unlimited capacity to multiply. So, keratinocytes
and fibroblast most likely to lose their lipofuscin
because they undergo mitosis while neurons
does not go under mitosis, so it has the chance to
accumulate lipofuscin. Therefore, we can
conclude that lipofuscin is best demonstrated in
organs or cells that are not anymore actively
undergoing mitosis.
Rich in lipofuscin
Present in postmitotic cells such as nerve,
skeletal muscles and cardiac muscles

Studies have shown that neurons cannot

regenerate but in recent researches suggest that
neurons can regenerate. It is just to slow that
they cannot replace the damage neurons in time,
so mostly likely a patient with stroke will die
even before the neurons in can regenerate
themselves.
Maroon circle = lipofuscin
Cell A is older than cell b because cell A has
higher amount of lipofuscin.

People who had acute myocardial infraction or

heart attack are advised not to any more engaged
in strenuous activities because they already have
lost some of their cardiac muscles. Strenuous
activities will most likely to kill the remaining
healthy cardiac muscles.
If the cell undergo mitosis, the two-daughter cell
will no longer have the lipofuscin. Lipofuscin in Brain
 acetyl coenzyme A will participate in the kreb
cycle and the product of the kreb cycle will
proceed to the electron transport chain with 6
nadh and 2 fadh2
6 nadh and 2 fadh2 will transported to the inner
mitochondrial membrane for electron transport
chain

Lipofuscin in Cardiac muscle

Insulin will facilitate the storage of excess
glucose in the muscle and liver
Glycogen – storage form of glucose

Inclusion bodies: Glycogen

 Storage form of glucose
 Stored in skeletal muscles and liver
 NOT STAINED with H and E
 Appears like clear areas
 Requires special stain such as periodic
acid Schiff (red in color)

Glucose to ATP
As glucose enter the cell, it undergoes glycolysis
in the cytoplasm. The product of the glycolysis
is two pyruvate molecule.
Pyruvate -> mitochondria = acetyl coenzyme A
 Glycogen in Vagina

The presence of the glycogen in the cytoplasm

of the cells of the lining epithelium of the vagina
is actually acted upon are the normal flora of the
Period acid Schiff vagina. Meaning they are normally present in
there and we call them as Lactobacillus
acidophilus.

Lactobacillus acidophilus – convert glycogen

into lactic acid.

The lactic acid makes the environment of the

inside of the vagina acidic, the acidity will not
promote the growth of microorganisms thereby
Glycogen in Vagina protecting the vagina from infection. That is
Vagina – lined with stratified squamous why semen has to be alkaline or else the sperm
epithelium cells will be killed by the lactic acid of the
vagina.

Inclusion bodies: Hemosiderin

 Indigestible residue (breakdown
product) of hemoglobin
 Can be normally found in the red pulp
of the spleen
A. Human skin  Often results from bleeding when red
B. Lining epithelium of the vagina cells and hemoglobin are degraded by
macrophages
Vagina (Stratified squamous non-keratenized)
 Alveolar macrophage if there is
hemorrhages in the alveoli
 Appears as brown granules
  Can be found in areas in the body cells have to be flexible for them to squeeze
where there is hemorrhage or bleeding themselves into the sinus.

Spleen is the graveyard for red blood cells When rbc is damaged or old it loses its
If the rbc in the cells are already more than 120 flexibility, what will happen will it will be
days old or damage they are destroyed in the trapped or stranded in this sinuses and when
spleen particularly in the red pulp of the spleen. they are trapped or stranded they will be eaten
It is expected that hemoglobin will also be by macrophages which are present on the splenic
degraded there cords.

Splenic cords – are composed of macrophages

and are waiting for red blood cells to be trapped.
Since the rbc are eaten up by the macrophages
they will be destroyed hemoglobin will be
destroyed hemoglobin will be degraded and now
have the formation of hemosiderin.

Spleen is compose of:

(1) White pulp – rich in lymphocytes or
white blood cells
 Nodular pattern
(2) Red pulp – where damage red blood
cells are destroyed

Splenic sinuses – are spaces within the red path

where the red blood cells will flow. These
sinuses are too small that the rbc have to blood
People with mycobacterium tuberculosis would
have destruction of the walls of their alveoli
which could cause of bleeding.
The presence of rbc in the alveoli can affect the
gas exchange and would have difficulty in
breathing. So, there is need to remove the rbc in
the alveolar space to restore the normal gas
exchange function of the lungs.

The photo suggest that the pathologist the idea

that previously there was a massive bleeding or
hemorrhage in this alveolar space

Inclusion Bodies: Lipids

 Adipose cells (common site for the fats
to be deposited )
 Accumulates through time in adipocytes
(Yellow (R)) Alveolar Macrophages – try to
displacing all organelles and nucleus
clear up the rbc in the alveolar space by
into periphery
digesting them.
 Extracted by solvents during tissue
RBC -> hemoglobin -> hemosiderin preparation, thus appears “hole in the
cytoplasm”

Clear cytoplasm – are adipose cells | fats that

have been accumulated through time | it may
appear empty but previously they dad fats inside
them
During the tissue processing especially in the
clearing and the dehydration step, the alcohol
and the clearing step will dissolve the fat leaving
behind an empty space or a hole

Lined by stratifies squamous keratinized

epithelium

Fat is steadily increasing while the nucleus is

displaced towards the periphery up until the fat
will become big that it has already displaced the
organelles and the nucleus in the perphery Cell Membrane
 Outermost boundary of the cell
 Regulates passage of molecules in and
out of the cell.
 Bilipid layer

BILIPID = 2 layers of Phospholipids

Melanin
 found on the basal layer of the epidermis
 responsible for the brown color of the
skin (present in the base)
Cell Membrane
Composition
1. Phospholipid – most abundant

How are the phospholipids arranged in the

bilipid layer?

2. Cholesterol

3.
4. Proteins

5. Carbohydrates - outside

Phospholipids
 Amphipathic - substance that has both
hydrophilic and hydrophobic portions
2 layers of Phospholipids : Head and Tails

Since cytoplasm is rich in water, hydrophilic

potions or the head are the ones facing the
water.

Do you know that Phospholipids tend to

migrate from one place to the other?
Head = Phosphate = Hydrophilic = water loving
Tail = 2 fatty acids = HYDROPHOBIC Glucose transporter

Phospholipid
1) polar/ hydrophilic/ water-attracting
phosphate head group and;
2) non-polar/ hydrophobic/ water-repelling
fatty acids
If insulin is present, it will bind to the insulin
receptors (protein) that will respond by causing
the blue colored glucose transporter to open.
Making a way for glucose molecule to enter the
cell. That’s why it is known that glucose will
never enter a human cell without the presence of
insulin. The absence of insulin will cause the
 Cholesterol has hydrophobic ring and
glucose to remain in the blood making the
hydrophilic hydroxyl group
patient diabetic.
 Hydrophobic ring - Hydro cyclopentane
Glucose transporter phenanthrene ring
 Hydrophilic – terminal(end) portion of the
cholesterol
 Circle - Hydroxyl group (OH) - hydrophilic
 Ring – four-fused ring, per-
hydrocyclopentanol ring
 Inserted among closely packed
phospholipids restricting their movements
What if the membrane is highly fluid? and thus modulating fluidity and movement
There will be a tendency that the phospholipids of membrane components.
will drag along with them the components that
are found beside them.
Glucose receptor
Change of position of the glucose transporter
will not allow the glucose molecule to enter the
cell. Making the glucose to stay in blood will
result to hyperglycemia.
Cholesterol – keeps phospholipid stay in place

Lipid raft
 Localized region within the plasma
membrane that contains high levels of
Cholesterol cholesterol and variety of peripheral
and integral proteins.
 Restrict the movement of the
 Area is limited in fluidity thus restricting
phospholipids, making the glucose
movement of the proteins which may
transporter to stay in place.
be involved in cell signaling processes
 Amphipathic
 Abundant in proteins, but called lipid
 Cholesterol has hydrophobic ring and
raft because it is also rich in cholesterol
hydrophilic hydroxyl group
that restrict the proteins to move
around
Blue – protein components of the cell membrane
Yellow – cholesterol structure

Notice that the presence of cholesterol in lipid

raft

 Signal transduction in lipid rafts occurs

more rapidly since proteins are found in
proximity to each other

Receptor are composed of several proteins. For

the receptor to be form the proteins should not
be moving apart from each other. That’s why Proteins
lipid raft is important.

 •Can be divided into:

Lipid Rafts as Signaling platforms
o (1) Integral (transmembrane)
 G-coupled protein composed of 7
proteins
transmembrane proteins and alpha,
 Directly incorporated
beta and gamma protein subunits
into the cell membrane
 Ligands bind to the G coupled receptor
 Channels, carriers and
which later cause the activation of the
transporters and even
alpha, beta and gamma subunits
as receptor
 o (2) Peripheral proteins
 Exhibits looser Peripheral proteins – ankyrin and actin, and
association with one of spectrin
the membranes
 Responsible for the Spectrin makes ankyrin and actin interact with
shape of the cell and each other
transport of the Ankyrin and actin will pull each other using
substances within the spectrin
cell
Hereditary spherocytosis = absence of spectrin
(people with no gene spectrin)
Will develop anemia
They cannot maintain the biconcave shape of
the rbc

Th biconcave shape helps the rbc to squeeze

through small capillaries

Red lines = cell membrane

Blue circles = peripheral proteins
Peripheral proteins – interact with each other
and they tend to pull each other. As they pull
Freeze Fracture model of the cell membrane
each other they cause the cell membrane to be
pulled towards the center. This is the reason why Developed to study the integral proteins
the red blood cells have boconcave shape present in the cell membrane

Th cell is freeze and hammer.

The hammer fracture the cell. Most of the time
the fracture is created by the hydrophobic
portion of the cell membrane (fatty lipid).
Separating the membrane into two;
(1) Extracellular layer – outer layer part of the
cell
(2) Cytoplasmic layer – close to the cytoplasm
Viewed under electron microscope

When examined they saw protrusions. The

protrusions are actually caused by the presence
of the integral proteins
Every protrusion on the extracellular layer of the
membrane there is a corresponding depression
on the cytoplasmic layer of the cell membrane Glycolipids – carbohydrates that is attached to
and vice versa. lipids
Using the freeze fracture method prove the Glycoprotein – carbohydrates that is attached to
presence of the integral or transmembrane protein
protein.
Functions:
1. Cell adhesion
2. Cell-to-cell interaction

Carbohydrates
Can be Glycolipids or Glycoproteins
 Generally found outside the cell Blood flowing through its lumen
membrane normally/majority of the white blood cells are
circulating in the central portion of the blood
 Its function have something to do with
while the majority of the red blood vessels are
the interaction of the cell to its
circulating in the periphery because the rbc have
environment
to share oxygen to the cells and they have to
 May act as receptors that participate in pick up carbon dioxide. For their function not to
cell adhesion, and cell-to-cell be interfered majority of the white blood cells
interaction should be positioned in the central portion of the
blood.

If there be any infection, the white blood cells

will have to go the margin or border of the blood
vessels and they have to go to the area of
infection in the body.
 Green – bacteria
Endothelial cells – lining epithelium of the blood Bacterium is digested by the lysosome
vessel | presenting the wall of the blood vessels
Neutrophil – polymorphonuclear leukocytes | Carbohydrate – where the bacteria in the cell is
has to go to the from the central portion of the loaded | structure of that bacterium was
blood towards the periphery displayed on the surface of the macrophages

Glycolipids and Glycoproteins – helps the

neutrophil to attach the wbc to the periphery of
the vessel
- Can help in cell adhesion (example is
chemotaxis)

Bacteria are phagocytosed by the macrophage

Carbohydrate can mediate cell-to-cell interaction

- The t cells that was activated by the
macrophage using the carbohydrate that
will interact with the b cell
- The b cell will eventually transform to
plasma cell
- The plasma cell will produce antibodies
 1) Simple passive transport (diffusion)
2) Facilitated (with the aid of channels or
carrier)
From higher to lower concentration directly
across the cell membrane
No need to energy consumption because the
molecules will move on their own

Facilitated Diffusion

Out of 4 bacteria only 1 was killed by the

macrophage. The 3 bacteria left will be killed by
the antibodies produced.

Channel – if the protein does not change its

shape

Since one of the functions of the cell membrane

is to regulate the entry and exit of the
substances in and out if the cell. It is also Carrier – changes the shape of the protein
important to discuss how these substances are
transported.

Passive Transport
Active Transport
 Requires energy consumption
 Pumps transport molecules from lower
to higher concentration (against
concentration gradient)
 ATP has to power the protein to
transport the molecule from lower
concentration to higher concentration
 The protein is now called a pump

 Requires energy?
 Oxygen and carbon dioxide diffusing
across the membrane without the need
of carrier via ________________?
Simple passive transport
 Transport of molecules is against
concentration gradient?
 Transport of molecule is from higher to
lower concentration and requires
carrier?