Lecture 1

Lecture Objectives
DLSU Biology
INTRODUCTION DLSU Biology At the end of the lecture, the student is expected to:
TO HISTOLOGY
Vitor - HISTOLO Vitor - HISTOLO 1. identify the different techniques for visualizing microscopic
specimens;

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2. discuss the various methods for histological preparation of
specimens; and
Rodel Jonathan S. VITOR II, DVM, MSc, PhD 3. understand the importance of stains in histological preparations.
Assistant Professor

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Department of Biology
College of Science
De La Salle University
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Etymology Fields of Anatomy

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Gross Anatomy Develepmental Anatomy Microscopic Anatomy

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histology Vitor - HISTOLO
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“study of microscopic anatomy of biological tissues”

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 Ultimate Goal of Histology
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• Understanding normal morphology of the cell, tissue, and organ
at levels not visible to the unaided eye including 3-dimensional

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relationships among biochemical constituents
Preparation
• Understanding the relationship between tissue structure and

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function
Tissues
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Study
• Establishing a basis for learning histopathology

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• Providing a basis for treating diseased and injured tissues

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Cell and Tissue Culture Smear Technique

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• Requires special instruments
and techniues
• Usually for blood and sperm

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• More expensive and time
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consuming

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• Specimens are difficult to
• May result to uneven thickness
and staining

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examine and cannot be stored
indefinitely

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 Nerve Teasing Technique Paraffin Technique
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Paraffin Technique Paraffin Technique

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 Paraffin Technique So Many Stains!
DLSU Biology DLSU Biology • Most tissue specimens are stained with hematoxylin and eosin.

Vitor - HISTOLO Vitor - HISTOLO • Most blood smears are stained with the Wright-Giemsa stain.

T3 2022-2023 T3 2022-2023 • Other stains (like silver, PAS, and acid-fast stains) can be used to
highlight special cells or features.

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Common Histological Stains and Common Histological Stains and

Reactions – Hematoxylin Reactions – Eosin
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Color Blue Color Pink

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Stains:
• Nucleus
Stains:
• Basic region of the
• Acidic region of the cytoplasm

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cytoplasm
• Cartilage matrix
• Collagen fibers

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 Common Histological Stains and Common Histological Stains and
Reactions – Hematoxylin and Eosin Reactions – Masson Trichrome
DLSU Biology DLSU Biology Dark Blue: nuclei

Vitor - HISTOLO Vitor - HISTOLO Red: muscle, keratin,

cytoplasm

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collagen

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Common Histological Stains and Common Histological Stains and

Reactions – Weigert’s Elastic Stain Reactions – Silver Stain
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Blue: elastic fibers Black: reticular fibers

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 Common Histological Stains and
Reactions – Iron Hematoxylin
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Black: striations of
muscle, nuclei,
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erythrocytes
Common Histological Stains and
Reactions – Periodic Acid-Schiff
Magenta: glycogen and
carbohydrate-rich
molecules

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Common Histological Stains and Advanced Visualization Procedures –

Reactions – Wright and Giemsa Stains Enzyme Histochemistry
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Pink: erythrocytes, • Method for localizing cellular
structures using a specific
eosinophil granules
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Purple: leukocyte
enzymatic activity present in
those structures
nuclei, basophil

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granules

Blue: cytoplasm of
Micrograph of cross sections of kidney tubules treated
histochemically to demonstrate alkaline phosphatases (with

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monocytes and
lymphocytes
maximum activity at an alkaline pH) showing strong activity
of this enzyme at the apical surfaces of the cells at the
lumens (L) of the tubules. (X200)

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 Advanced Visualization Procedures –
Methods of Immunohistochemistry
Immunohistochemistry
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• Identify and localize many
specific proteins, not just those

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with enzymatic activity that
can be demonstrated by
histochemistry

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A section of small intestine treated with an antibody
against the enzyme lysozyme. The secondary antibody
labeled with peroxidase was then applied and the localized
brown color produced histochemically with the peroxidase

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substrate 3,3'-diamino-azobenzidine (DAB). The method
demonstrates lysozyme-containing structures in scattered
macrophages and in the large clusters of cells. Nuclei were
counterstained with hematoxylin. X100.

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Advanced Visualization Procedures –

In Situ Hybridization
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• Implies the specific binding
between two single strands of

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nucleic acid
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ISH of this tissue section reveals that many cells contain the
human papilloma virus (HPV). The section was incubated with a
solution containing a digoxigenin-labeled complementary DNA
(cDNA) probe for the HPV DNA. The probe was then visualized

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by direct immunohistochemistry using peroxidase-labeled
antibodies against digoxigenin. This procedure stains brown
only those cells containing HPV. X400. H&E.

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 Methods of Light Microscopy –
Simple or Compound Microscope
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Interpretation
of Microscopic
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Sections

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Methods of Light Microscopy – Methods of Light Microscopy –

Phase Contrast Microscope Dark Field Microscope
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Enables examination of No direct light from the
unstained cells and light source is gathered

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tissues and is especially
useful for living cells.
by the objective lens

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 Methods of Light Microscopy – Methods of Light Microscopy –
Fluorescence Microscope Ultraviolet Microscope
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Use of the ability of Uses quartz lenses

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certain molecules to with an ultraviolet
fluoresce under light source
ultraviolet light

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Methods of Light Microscopy – Methods of Light Microscopy –

Confocal Microscope
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Combines components of
a light optical microscope
with a scanning system to
Polarizing Microscope
Uses the fact that highly ordered
molecules or arrays of molecules can

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dissect a specimen
optically
rotate the angle of the plane of
polarized light

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 Methods of Electron Microscopy Methods of Atomic Force Microscopy
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Methods of Virtual Microscopy

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 Histological Artifacts Histological Artifacts - Shrinkage
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improper processing
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• Structures or features in tissues resulting from poor handling or • Description: appearance of
spaces between portions of the

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• Interfere with normal histological examination by changing the
tissues

tissue appearance and hidign structures • Causes: type of chemical

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paraffin

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Histological Artifacts – Dark Precipitate Histological Artifacts – Folds

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• Description: crystals or DLSU Biology
granules that are not in focus
• Description: appear as dark
bands

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when tissue is in focus and
vice-versa
• Causes: failure of the tissue to
stretch (warm water bath is less

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• Causes: inadequate washing
dust or dirt which settled
before covered with the slip,
than 40°C)

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unfiltered or old stain

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 Histological Artifacts – Wrinkles Histological Artifacts – Scratches
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• Description: appear as tiny
corrugations running across
• Description: straight slashes or
ragged tears across section; if

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folded tissue Vitor - HISTOLO
the section; small creases of nick is big, scratches appear
broken

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• Causes: failure of the tissue to
stretch (warm water bath is less
than 40°C)
• Causes: nicks or dirt in cutting
edge of the knife (not sharp
enough)’

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Histological Artifacts – Chatter Histological Artifacts – Bubbles

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• Description: narrow parallel
bands, usually evenly spaced,
• Description: spaces and
bubble-like appearance

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across a tissue section
Vitor - HISTOLO • Causes: improper technique of
• Causes: knife vibrations which putting cover slip, too thin

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cause variations in thickness
(ripples)
mounting medium

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 Histological Artifacts – Pinching References
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• Description: jarred sections or
out of focus
Eroschenko, V. P. (2013). diFiore's Atlas of Histology with Functional
Correlations (12th ed.). Lippincott Williams & Wilkins.

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• Causes: few albumen (binder)
Gartner, L. P. (2017). Textbook of histology (4th ed.). Elsevier.

coating in the glass slide, dull

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Mescher, A. L. (2018). Junqueira's basic histology text & atlas (15th ed.).
knife is used, pinched too McGraw-Hill Education.
much with forceps
Ross, M. H., & Pawlina, W. (2015). Histology: A text and atlas with

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correlated, cell and molecular biology (7th ed.). Lippincott
Williams & Wilkins.

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