Blesile S. Mantaring, MD Cell YL5: 2.

0 07/01/2013

CELL
Histology Lecture
OUTLINE  Eyepiece
I. Histology III. The Cell  Binocular (used with both eyes)
II. Studying Tissues and A. Cellular  Adjust distance between your eyes comfortably
Cells Organization  Magnification of 10x
A. Parts of the Light B. Cellular  Course and Fine Adjustment
Microscope Components
 Course adjustment: Scanning and low powered
B. Measurement IV. Review Questions
objective lens
C. Slide Preparation V. References and
 Fine adjustment: High powered and oil immersion
D. Common Additional Readings
objective lens
Histologic Stains VI. Quotation
 Objective Lenses
 Always start with the lowest objective first
(Scanning lens)
I. HISTOLOGY
 Focus specimen with increasing magnification
 Magnification: How much bigger a sample
 Encompasses the study of cells, extracellular appears than it is in real life
components, and their organization into tissues, organs,
organ systems Lens Color Magnification
 Makes use of: Ocular Lens - 10x
 Light microscope
Scanning Objective Red 4x
 Makes use of visible light to view a specimen.
Light first passes through or is absorbed by the Low Power Objective Yellow 10x
specimen. The remaining light is refracted or High Power Objective Blue 40x
bent by the lenses magnifying the image Oil Immersion Objective
White 97x-100x
(Solomon et al, 2008). (no need to adjust)
 Electron microscope
 Utilizes a beam of energized electrons which  Stage
passes through or is bounced off by the  Make sure that slide is adjusted and secured on the
specimen. The remaining electrons are stage clip
focused by electromagnets to create an image  Light adjustment
(Solomon et al, 2008).  Adjust for light intensity
 Scanning EM: Electrons pass on the
 Diaphragm
surface
 Regulates the amount of light transmitted unto the
 Transmission EM: Passes through the
stage
specimen
 Base and Arm
 Where to hold and support the microscope
 There is a knob on the arm to rotate the head

B. M EASUREMENT

Units of Measurement
All images above are samples of Candida albicans viewed
using different microscopes System International Unit Symbol Value
(A) Scanning EM; (B) Transmission EM; (C) Light Microscope Micron (micrometer) μ (μm) .001 mm; 10 -6 m
Millimicron (nanometer) m (nm) .001 um; 10 -9 m
II. STUDYING TISSUES AND CELLS Angstrom Å 0.1 nm; 10 -10 m

A. PARTS OF THE LIGHT M ICROSCOPE Estimating the Size of Structures

 Cells differ in size and shape, thus it is necessary to

estimate sizes
 Basis of comparison
 HPO = 0.4mm field of vision
 RBC = 7.74 micrometers

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 Blesile S. Mantaring, MD Cell YL5: 2.0 07/01/2013

C. SLIDE PREPARATION  Ex: Kidney, renal tubules

D. COMMON HISTOLOGIC STAINS

Process Method Purpose
 Periodic acid-Schiff
Fixation Bathe in fixative Terminates cellular  magenta: glycogen & carbohydrates rich molecules
e.g. formalin processes,
(formaldehyde) or Preserves cellular
Glutaraldehyde architecture / tissue
morphology,
Prevents autolysis of
organelles,
Protects against
bacteria

Dehydration Bathe in To remove water from

progressively the cell
increasing
concentrations of
Periodic acid schiff stain colors the mucin secretion
ethyl alcohol (ex.
10-60%) (heavily glycosylated) of goblet cells purple
(Image taken from http://histology.leeds.ac.uk/what-is-
*H20/ alcohol are histology/histological_stains.php)
imiscible in
paraffin  Wright and Giemsa stain (Differential staining for blood
cells)
Clearing Bathe in lipid- To remove alcohol and  Pink: erythrocytes, eosinophil granules
solvent eg lipids in preparation for  Purple: leukocyte nuclei, basophil granules
benzene or xylene next step (water and  Blue: cytoplasm of monocytes and lymphocytes
(miscible in alcohol are not miscible
paraffin) with paraffin; xylene is
only miscible with
paraffin); tissue
becomes transparent

Embedding Bake specimen in To get a rigid/fim

melted paraffin specimen for sectioning
wax (58-60oC) into thin slices, NB;
different media used for
electron microscope
because it requires a Wright stain colors the nuclei white blood cells
thinner slice dark violet (in this case eosinophil & NK cell)
and Giemsa stains colors red blood cells pink
(Image taken from http://imagebank.hematology.org/AssetDetail.
Sectioning Use microtome Sections tissue thinly (3-
aspx?AssetID=2719&AssetType=Asset)
8um for LM & .02-.1um
for EM)
 Orcein elastic stain
 Brown: elastic fiber
Staining Use appropriate To distinguish different
stain structures

 Staining
 Hematoxylin
 Basic dye (basophilic; reacts with acidic
components)
 Blue: Nucleus (DNA, RNA); acidic regions of
cytoplasm; cartilage matrix
 Stains the DNA and RNA due to their
acidic natue
 Eosin
 Acidic dye (acidophilic; reacts with basic Orcein stain colors the Tunica Media of
components) arteries brown showing the elastic fibers
 Pink: basic regions of cytoplasm collagen (Image taken from http://millette.med.sc.edu/Lab%205%20
fibers pages/C5%20image%20page.htm)
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 Blesile S. Mantaring, MD Cell YL5: 2.0 07/01/2013

 Silver stain III. THE CELL

 Black: reticular fibers
A. CELLULAR ORGANIZATION

Organ
Cell Tissue Organ
System

 The cell is the basic structural and functional unit of all

organisms
 Cells have different shapes and sizes according to their
different functions

(Image taken from http://www.lab.anhb.uwa.edu.au
/mb140/core pages/connective/Images/liv042re.jpg) 1.
 Iron hematoxylin stain
2.
 Black: striations of muscles, nuclei, erythrocytes
B. CELLULAR COMPONENTS
Nucleus
 Contains DNA that encodes the instructions for
synthesis of all proteins and enzymes of the cell
Cytoplasm
 Contains organelles and inclusions suspended in a
fluid medium called the cytosol
 Principal site of metabolic functions of the cell

Organelles Inclusions
- From the word organ, - Metabolically inert
meaning - Accumulation of cell
important/indispensible products
to the cell (losing them, - Dispensable
the cell dies) - Not bound by a
- Metabolically active membrane
- Internal cellular organs
carrying out specific
(Image taken from http://biology.clc.uc.edu/fankhauser/Labs/ essential functions
Anatomy_&_Physiology/A&P201/Muscle_Histology/cardiac_ - Bound by a membrane
muscle_100x_PB082240labeled.JPG)
3. Cell Membrane
 Massom’s trichrome  Also called plasmalemma or plasma membrane
 Light blue: collagen fibers  Only seen under EM
 Dark blue: nuclei  Trilaminar/unit structure due to phosphilipid bilayer
 Red: muscle, cytoplasm  Composed of phospholipids & proteins

(Image taken form http://www.polysciences.com/SiteData/images

/masson/c852c7ae73be82e8f9afcab1dba0861d/masson.jpg)
(Image taken From http://www.invertebratesclassification.com
/2012/protists-characteristics-1)

 Phospholipids in Plasma Membrane

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 Two components of lipid bilayer
 Hydrophilic head portion: Outer and inner
surface of cell
 Hydrophobic tail portion: Middle portion
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 Blesile S. Mantaring, MD Cell YL5: 2.0 07/01/2013

 Responsible for cellular form and permeability  Adenine : Thymine

properties  Cytidine : Guanine
 Proteins in Plasma Membrane  Proteins
 Kinds of protein  Folded polypeptides composed of specific
 Intrinsic: Spans the entire width of plasma sequence of amino acids
membrane  Functions:
 Extrinsic: Found only on inner or outer surface  Structural component of cells
of plasmalemma  Enzymes catalyzing metabolic reactions
 Act as carrier proteins, channel proteins, or  Secretory products
receptors  Triplet or codon: Sequence of three nucleotides that
 Carrier proteins: Transports a substance form encodes a specific amino acid
the external environment intracellularly  Gene: Sequence of triplets providing the
 Channel proteins (transient pore/channel): information for the synthesis of a particular protein
Allows communication between external &  RNA
internal environment of the cell; allows  DNA remains in the nucleus because it is too big to
molecules to go in & out pass through the nuclear pore
 Receptor: binding domain for substances such  Types of RNA
as hormones (recognizes certain substances  Messenger RNA (mRNA): a single strand RNA
resulting to change of cell function) molecule transcribed from template DNA within
 Glycocalyx the nucleus with the help of RNA polymerase II
 Carbyhydrate chains attached to proteins and/or  Transfer RNA (tRNA): a triloop cloverleaf
phospholipid molecules in outer leaflet shaped single RNA strand that delivers amino
 Forms a coat on outer surface of the cell acids to the translation site. The loop on the tip
 Under EM: Mat of delicate branching of the clover leaf contains a group of three
polysaccharide filaments bases called the anti-codon which complement
 Functions: the codon in the mRNA
 Cell recognition
 Intercellular adhesion
 Mechanical & chemical protection for cell
membrane

4. Nucleus
 Largest organelle of the cell
 Contains the genetic material of the organism
 Components:
 Chromatin
 Nucleolus: contains mRNA, therefore present
in active cells (i.e. constantly producing
protein) (Image taken from http://www.emc.maricopa.edu/faculty/
 Nuclear envelope farabee/biobk/biobookprotsyn.html)
 Nucleoplasm
 Site of transcription (then mRNA is transported out  Ribosomal RNA (rRNA): an RNA and protein
to cytoplasm) complex that catalyzes the attachment of each
 Translation
new amino acid onto the growing peptide chain
 3 consecutive nucleotides  1 codon  1
amino acid  sequence of amino acids  (The ribosome itself)
gene
RNA is similar to DNA except
Protein synthesis in brief  Has one strand instead of two
 Has uracil instead of thymine
DNA transcription in nucleus produces mRNA  mRNA  Has ribose instead of deoxyribose
transported to cytoplasm  translation occurs tRNA
carries amino acid while rRNA reads mRNA  3
consecutive nucleotides = codon which codes for one  Chromatin
specific amino acid  sequence of amino acids form a  Clumps of basophilic material seen in the nucleus
protein  Consists of DNA and associated proteins
 Nucleosome
 “beads on a string”
 DNA  Consists of a core of eight histone molecules with
 Two polypepetide chains containing nucleotide two loops of DNA wrapped around the octomer
subunits
 Nucleotides, Sugar (deoxyribose), Phosphate Base
 Specific pairing of the bases
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 Blesile S. Mantaring, MD Cell YL5: 2.0 07/01/2013

edu/weblinks/images/nucpore6.jpg)
Organization of a Eukaryotic chromosome (6-fold
 Nuclear pore
shortening)
 avenue of communication between nucleoplasm
1. DNA double helix (2nm in diameter) and cytoplasm
2. Nucleosome Beads = complex of 8 histone molecules  not uniformly distributed
(10nm in diameter)  closed by a pore diaphragm
3. Solenoid complex (30nm in diameter)  Heterochromatin lies adjacent to the inner
4. Extended chromatin/chromatin loops (300nm fibers) membrane
5. Condensed chromatin (700nm in diameter)  Nuclear Pore Complex
6. Condensed Chromosome (1400nm in diameter)  Non-membranous structures associated with
nuclear pore
 Components:
 8 particles attached to the inner and outer ring
 8 radially arranged spokes projecting inward
 Principal barrier to movement of particles > 10 nm
 Nucleolus
 Consists mainly of RNA and protein
 Site of ribosomal RNA synthesis
 Stains intensely with hematoxylin
 Size and number varies (depending on activity of
the cell)
 Can be more than one when active
5. Endoplasmic Reticulum
 Extensive system of membrane bounded canaliculi
 Consists of a series of flattened sacs called
cisternae arranged in parallel array
 The more active, the bluer stain (hematoxylin)
 Two kinds:
(Image taken from http://www.med.unc.edu/~bstrahl/research.html)
 Rough ER
 Continuous with nucleus
 Chromosomes  Outer surface of membranes contain
 Discrete bodies containing condensed and tightly ribosomes
coiled chromatin  Functions:
 Found in dividing cells (a) Synthesis of protein for export from
 46 chromosomes in humans organized as 23 pairs the cell
(b) Synthesis of integral protein of the
Heterochromatin Euchromatin plasma membrane
- Dark dense ‘clumps’ - Active chromatin (c) Abundant in protein secreting cells
adjacent to nuclear - Chromatin stretched  Contribute to cytoplasmic basophilia
membrane out so that genetic
- Inactive chromatin in a information of DNA
Are you following?
condensed state can be read and
 Nerve cells of the CNS are constantly active &
hence more basophilic transcribed
in appearance produce a lot of protein
- Coiled  Contains a lot of ribosomes
 Contains a lot of mRNA
 Reacts readily with hematoxylin
 Nuclear envelope
 Cytoplasm is deep blue ie Basophilic Stippling
 Two parallel membranes separated by a
perinuclear cisterna
 Ribosomes attached to outer membrane  Smooth ER
 Absence of ribosomes in the membranes
 Functions
(a) Involved in the synthesis of fatty
acids and other lipids
(b) Plays a role in the detoxification and
metabolism of lipid-soluble exogenous
drugs and alcohol
(c) Abundant in steroid secreting
endocrine glands

(Image taken from http://mvc.bioweb.dcccd.

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 Blesile S. Mantaring, MD Cell YL5: 2.0 07/01/2013

Are you following?  more intricate cristae (contains enzymes for

oxidative phosphorylation)
 The liver plays an important role in
detoxification  Can’t be visualized under light microscope
 we can expect a biopsy of a liver sampled from  Principal biochemical activity: oxidative
alcoholics to have increased smooth phosphorylation
endoplasmic reticulum  Oxidation of nutrients like glucose  energy as
ATP  ATP broken down for energy
 Ribosomes  Covered by two membranes
 Complex structures composed of ribonucleic acid  Smooth outer membrane
and proteins  Cristae mitochondrialis (inner membrane
 Composed of a large and small subunit thrown into folds, increasing its surface area)
 May occur free in cytoplasm or attached to rough  Contains a high content of enzymes involved in
ER oxidative phosphorylation to generate ATP
 Free ribosomes  Two spaces:
 10 or more ribosomes maybe associated  Intermembrane space (between inner & outer
with m-RNA (polysomes or polyribosomes) membrane)
 Involved in synthesis of proteins to be  Intercristal space (area within inner membrane)
used in the cytosol
 Ribosomes attached to ER
 Involved in synthesis of proteins for export
or for membrane system
6. Golgi Apparatus
 Abundant in cells that produce a lot of secretion
 Does not stain hence produces a negative image
(no nucleic acid). Usually presents as an empty
space in plasma cells
 Functions
 Packaging: Forms secretory granules
Cis-phase Trans-phase
- Convex side - Concave side http://fusionanomaly.net/mitochondria.jpg
- Outer - Inner
- Immature - Mature
 Concentrating: Addition of oligosaccharides in Are you following?
glycoproteins
 Sorting: Product is “addressed” to where it is A cell that is continuously active requires a lot of energy
supposed to go to & needs to produce more energy  increase cristae to
 Membrane synthesis and membrane recycling increase the surface area for oxidative phosphorylation

8. Lysosome
 Round, electron dense bodies
 Assembled by fusion of vesicles coming from the
Golgi complex and endosomes
 Digestive organelles of the cell containing hydrolytic
enzymes to digest foreign bacteria
 Vary in number
 No consistent form or staining properties
 Important in programmed cell death/apoptosis
 i.e. Breast cells undergoing apoptosis to return
to normal size and shape after breastfeeding

(Image taken form http://www.ezeelearning.com/htmlmindmaps/

Class%20-%209/Biology/ClassIX-cell-unit%20of%20life-
797/golgi%20apparatus.jpg)
7. Mitochondria
 Provides energy for the biosynthetic and motor
activities of the cell
 usually rod-shaped but can sometimes be rounded
 Number depends on the size and energy needs of
the cell
 Increases in an active cell

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 Blesile S. Mantaring, MD Cell YL5: 2.0 07/01/2013

http://course1.winona.edu/sberg/IMAGES/lysosome.jpg  Provides means for movement of cellular

9. Peroxisome or Microbodies
 Function similar to smooth ER
 Small spherical bodies
 Contain oxidative enzymes and catalase
 Involved in detoxification of alcohol, uric acid and
toxic drugs
 Oxidation of long chain fatty acids is a source of
metabolic energy → H2O2
 Numerous in liver & kidney cells
 Difficult to identify even under electron microscope
10. Centrosome
 Not membrane bound
 Recognized by containing centrioles
 Granular area within the cytoplasm containing a
pair of centrioles
 Acts as the organizing center for the growth of
microtubules of the cytoskeleton
11. Centriole
 Paired, short, rod-like bodies found near the
nucleus in non-dividing cells
 Arranged at right angle with each other therefore,
under microscope, one should appear longitudinally
cut while the other appears cross-sectionally cut
 Consists of nine triplets of microtubules
 Provides basal bodies which is necessary for the
formation of the flagella and cilia
 Source of mitotic spindles and tail of the
sperm.
organelles
 Provides movement of specialized structures
such as cilia and flagella
 Responsible for contractility of muscle
 Microfilament
 Composed of fine strands of the protein actin
 Often grouped as bundles under the plasmalemma
 Gives the peripheral region of the cytoplasm a firm
gel-like consistency
 Interact with myosin for muscular contraction
 Microtubules
 Largest structural element of the cytoskeleton
 Dynamic structure of the cytoskeleton
 Assemble in a new pattern to meet the
cytoskeletal requirement so f change in cell
shape
 Arises from the centrosome
 Important role in directing movements of organelles
and vesicles
 Serve as tracks along which vesicles are
moved from one region of the cytoplasm to the
other
 Functions:
 Serves as tracks providing means transport of
vesicles and organelles
 Cell elongation and movement
 Maintenance of cell shape
 Beating of cilia and flagella
 Movement of chromosomes during mitosis

 Intermediate Filaments
 Intermediate in size between microfilaments and
microtubule
 Stable supporting function of the cytoskeleton
 Keratin filaments
 Main structural component of skin cells
(keratinocytes)
(A) Bull’s sperm
 Axons of nerve cells (neurofilaments)
(B) Electron micrograph of the cross-section through a
sea-urchin sperm showing the nine doublet
IV. REVIEW QUESTIONS
microtubules surrounding the central pair of singlet
microtubules 1. Which of the following organelle is responsible for
(Images taken from http://www.springerimages.com/Images/ the detoxification of toxic drugs and alcohol that
RSS/1-10.1007_s12195-008-0004-z-4) enters the cell?
a. Smooth Endoplasmic Reticulum
12. Cytoplasmic Inclusions b. Golgi Apparatus
a. Glycogen – visible through PAS stain c. Lysosome
b. Lipid – signet ring appearance (signet nucleus; ring d. Mitochondria
is the empty lipid place) 2. Which of the following is used to stain reticular
 Emptied during clearing step fibers black?
c. Proteins – Pigment a. Wright and Giemsa Stain
i. Lipofuscin b. Silver Stain
 Residual granules/bodies in a cell c. Massom’s Trichome
 The older the cell (e.g. nerver & cardiac d. Orcein Stain
cells), the more lipofuscin granules there 3. What is the center of organization of microtubules?
are a. Actin
 Seen with H&E stain b. Centrosome
13. Structural Elements of the Cytoskeleton c. Centrioles
 Functions: d. Peroxisome
 Provides structural support for the plasma
membrane and cellular organelles
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 Blesile S. Mantaring, MD Cell YL5: 2.0 07/01/2013

4. It is possible to identify the boundaries of the

nuclear membrane based on the boundary between
the nucleus and cytoplasm.
a. True
b. False
5. Transcription occurs in the _________, while
translation occurs in the __________.
a. Nucleus; Golgi Apparatus
b. Cytoplasm; Nucleus
c. Cytoplasm; Endoplasmic Reticulum
d. Nucleus; Cytoplasm
6. Using a Light Microscope, you can only see which
of the following?
a. Nucleus
b. Heterochromatin
c. Cytoplasm
d. All of the above

Answers: A, B, B, A, D, D

V. REFERENCES AND ADDITIONAL READING

 Solomon EP, Berg LR, Martin DW. 2008. Biology. 8th
ed. California: Thomson Higher Education Corp.

VI. RELEVANT/IMPORTANT QUOTATION

Everything you'll ever need to know is within you; the secrets

of the universe are imprinted on the cells of your body.
– Dan Millman

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