HISTOLOGY LAB
Microscopes REMINDERS
basic information
- Parfocal: you only need a slight adjustment between objectives
- 1 lens: simple
> 1 lens: compound
- Bright field: bright background and dark object
Dark field: dark background and light object
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parts of a microscope
Serves as a support for the
Base microscope; one hand is placed
here when carrying a microscope
Holds the ocular lenses, and
revolving nosepiece; also serves as
Arm a support for the microscope; the
other hand hold this part when
carrying the microscope
Ocular Lenses Has a magnification of 10x
OIO - 100x - white
HPO - 40x / 50x - blue
LPO - 10x - yellow
Objectives Scanner - 4x - red
Total Magnification = (objective)
(ocular)
Stage Where the slides are places
Stage Clips Holds the slides in place
Revolving Nosepiece Where objectives are attached
Coarse Adjustment Knob Found at the side of the microscope
and are used for refining the image
Fine Adjustment Knob seen in the microscope
Diaphragm Adjusts light passing through
Rheostat Adjusts light intensity
Concentrates light entering the lens
Condenser
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- Focusing (Order)
1. Scanner
2. Coarse Adjustment Knob
3. LPO
4. Coarse Adjustment Knob
5. HPO
6. Fine Adjustment Knob
- Storing
- Make sure the LPO is the one inside and OIO out
- Stage all the way down
- Condenser all the way up
- OIO is wiped if used
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types of microscopes
- Fluorescence Microscope
- Function
- Allows the visualization of naturally
fluorescent substances (fluorescent-
antibody (FA) technique, or
immunofluorescence) or those that
have been stained with a
fluorochrome to produce an image
- Stained using fluorochromes
- Acridine Orange
- Auramine - Rhodamine
- FITC: Fluorescein Isothiocyanate
- Methyl Green
- Principle
- When ultraviolet light hits an
object, it excites the electrons of
the object, and they give off light
in various shades of color.
- Application
- Most precise method of identifying specific proteins in
tissues
- Ex. Mycobacterium tuberculosis
- Interference Microscope
- Function
- Proides a 3D image showing very fine
structural detial by splitting the ligh
ray so that the beams pass through
different areas of the specimen
- Used to enhance the contrast in
unstained or transparent samples to
visualize living cells or tissues
- Cell additives: protoplasmic
- Principles
- Creates images using the difference between an
interfering beam unmodified by the specimen and an
identical beam that illuminates it
- Features
- 2 light beams separated by beam splitting prisms (shows
2D depth)
- Application
- Calculation of the mass of cellular components, provides
a 3D image of the specimen
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- Polarizing Microscope
- Function
- Used for stained linearly oriented
structures of living cells in tissue
culture or fixed stained preparation.
- It restricts the probing light to
preferred directions and orientations
making it possible to detect the
presence of orderly arranged fibrous
proteins or other arrays of long molecules.
- Used for anisotropic substances — usually
crystals (ex. uric acid, cholesterol)
- Principle
- A polarizer is used to intervene between the
light source and the sample which results in
the production of plane-polarized light. The
polarized light falls on a birefringent (or doubly
refracting) specimen that generates two wave
components perpendicular to each other. The waves
pass through the specimen in different phases. Then, they
are combined using constructive and destructive
interference, by an analyzer which leads to the
generation of high-contrast images.
- Birefringent: can rotate the light in a positive
(pyrophosphic substances) or negative (oric acid
crystals) way
- Features
- Modification: Installation of 2 filters
- Polaroid/Polarizing Filter – placed in the condenser
filter holder
- Analyzer Filter – placed in the head between the
objectives and the ocular
- Applications
- Mineral elements, Spindle fibers
- Confocal Microscope
- Function
- Used in measurement applications. They
provide
- A high-resolution image with all areas in
focus throughout the field of view. Also
enables non-contact non-destructive
measurement of three dimensional
shapes.
- The image is more focused thus, small
area is focused
- Principles
- Specialized form of standard
fluorescence microscopy that uses
particular optical components to
generate high-resolution images of material stained with
fluorescent probes.
- Features
- Uses fluorescence optics. Instead of illuminating the
whole sample at once, laser light is focused onto a
defined spot at a specific depth within the sample.
- Pinhole - to concentrate the image
- Filters other light not needed to visualize the object
- UV Microscope
- Function
- Ultraviolet microscopes can image
microscopic samples in the visible and the
ultraviolet region.
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- Principle
- The resolution of the UV microscope has
shorter wavelength than the other
microscopes; hence improving the resolution
beyond the diffraction limit of optical
microscopes using normal white light and
increasing the contrast of image.
- Features
- Quartz lenses and slides
- Uses ultraviolet radiation/light as the illumination.
- Bright Field Microscope
- Function
- The most common microscope use
- Used in histologic studies
- Note: Specimens should be transluscent,
fixed and stained to provide contrast and to
increase visualization
- Objects appear dark against a light
background illuminated by a condenser.
- It is where stained tissues are examined
with ordinary light passing through the
preparation
- Image is inverted of the actual image
- Principles
- An object is placed on the focus of the objective, forming
a virtual, inverted and magnified image of the object at
the least distance of vision from the eye.
- Features
- It has more than one lens and has own light source
- Uses subdued light when unstained specimens are used
- Phase Contrast Microscope
- Function
- Enhances visualization of
elements with low refractive.
- The phase contrast microscope
enables examination of unstained
cells and tissues and is especially
useful for living cells.
- A type of light microscopy that
intensifies contrasts of
transparent and colorless objects
by influencing the optical path of
light.
- Most useful in observing living
cells and examine cells in a
natural state. It shows components in a cell or bacteria.
- Principles
- Enhances contrasts of transparent and colorless objects
by influencing the optical path of light.
- Modifies refractive index or altering
- Features
- There is an annulus and phase plate that causes the
direct light to pass through and be retarded.
- Applications
- Living cells and tissues; extensively examine unstained
sections of plastic-embedded tissues (approx. 0.5um)
- Electron Microscope
- Uses electron beams
- High resolution and magnification
- Subcellular structures: organelles
- In microbiology: visualization and study of viruses
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 - SEM Tissue Processing
- Function
- Scans an electron beam CHECK OTHER REVIEWER NA LANG HEHE TY
across a specimen coated
with a thin layer of heavy Laboratory Safety
metal
- Principle - Precaution: treat all specimens as infectious
- Reflected and secondary electrons - Wear PPE
from the specimen are processed —————————————————————————————————————————
into a 3D structural image. waste containers
- Feature
- In contrast with TEM, the focused Yellow Infectious Anything that touches patient
electron beam does not pass through
the specimen; in SEM, the electron beam is moved Black Dry, non-infectious Paper, cartons
sequentially from point to point across its surface
- Surface image , 3D image Green Wet, non-infectious Food Wastes
- Black and white: heavy metal as stains
-TEM Red Sharps Biohazard symbol
-Function
-Sends an electromagnetically focused Orange Toxic Radioactive
beam of electrons at very high voltage
through ultrathin sections of tissue. —————————————————————————————————————————
-Principle hazards
-Tissue preparation for TEM involves - Physical: noise
adding heavy metal ions that - Biological: HIV, HEPA, blood-borne
associate at different electron - 5% Sodium Hypochlorite: standard disinfectant
densities with cell and tissue - 1:10 ratio is used in the lab
components, improving contrast in the - Disinfection: Nonliving (bleech)
resulting image. Antiseptic: Living (Alcohol)
-Higher magnification, 2D magnification - Ergonomic: repetitive motions, slouching, etc
-Internal image
-Black and white: Uranyl Acetate (superior
-
quality stain)
- Dark Field Microscope
- Function
- Used to enhance visualization of specimens that cannot
be seen easily with a bright-field
microscope.
- A bright-field microscope is easily
adapted for dark-field microscopy by
replacing the condenser with a dark-field
condenser that contains an opaque disk.
- It can be effectively used at high
magnifications to photograph living
bacteria, or at low magnifications to view
and photograph cells, tissues, and whole
mounts.
- Principles
- To view a specimen in dark field, an
opaque disc is placed underneath the
condenser lens, so that only light that is
scattered by objects on the slide can
reach the eye
- Applications
- For unstained specimens & Treponema pallidum
identification (STDs)

*OIO Oil - Cedar Wood Oil*

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 Visualization Techniques in Histology immunohistochemistry
- Analyte = antigen — from tissue
- Reagent = antibody
group staining - Monoclonal: reacts to one type of antigen (mice)
- Histologic Staining - Polyclonal: reacts to multiple types of antigen (rabbit)
- Direct Interaction of tissue and dye - Enzyme: ALP, Horseradish peroxidase
- Ex. H&E, methylene blue Fluorochrome: FITC
- Want to visualize tissue structure - Direct: Primary antibody directly on antigen
- Histochemical Staining Indirect: 1st antibody goes to antigen, 2nd forms immune
- Chemical substances found in the tissue complex (more sensitive)
- Ex. PAS (stain of glycogen), Perl’s Prussian Blue (stain of iron)
- Enzyme Histochemistry
- Demonstration of enzymes
- Heat labile: will be destroyed by heat
- Enzyme specimens need to be frozen sections when
processed
- Immunohistochemistry
- Antigen and antibody interaction —————————————————————————————————————————
- Binding of antigen and antibody, forming an
immunocomplex
hybridization techniques
- Uses antibodies as reagent - Pairing of single stranded nucleic acids to its complimentary NA
- Presence of tissue antigens can serve as tumor markers - FISH: Fluorescent In-Situ Hybridization
- Labels are attached to the antibodies - In-Situ : on site, directly on tissue
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- Single stranded NA binds with Probe NA
routine staining
- H&E
- Hematoxylin
- Stains the nucleus
- Basophilic stain
- Bluish color
- Eosin
- Stains the cytoplasm
- Acidophilic stain —————————————————————————————————————————
- Pinkish color
- Most commonly used H&E stain:Harris hematoxyllic & Eosin Y electrophoresis
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- Method of separating proteins into fragments
- Application of electrical current
autoradiography - Should have positive and negative electrodes
- Useful for newly produced macromolecules like DNA, RNA, and - Performed on an agarose gel (separating medium)
glycoprotein - Manner of separation is mitigation towards positive side
- To see structures you need to ass macrodetectors, usually - Proteins are placed on the negative side because they have
metals (particularly silver bromide) a naturally negative charge of 8.5
- Eventually emits radiation and appears as silver grains - Nearest anode = most anodal
————————————————————————————————————————— - Fastest to mitigate
cells and tissue cultures - Smallest in size
- Useful for culturing viruses like Chlamydia (bacteria), Rickettsia, - Farthest anode = least anodal
Cancer cells - Slowest to mitigate
- Commonality: All unculturable in artificial media (agar) and - Biggest in size
can only culture in living cells because they are intracellular - Less protein
- Primary cell culture: can only be used once or twice - Bands appear and are stained to visualize
- Immortal cell culture: can be used forever - Stains
- Ex. HeLa Cells, HEP-2 - Silver
- Visible results of viral growth on cell cultures - Ponceau
- Presence of cytoplasmic effects (CPE) — morphological - Coomassie
changes Brilliant Blue
- Multi-nucleation, aggregation, presence of cytoplasmic
inclusions (DNA: nucleus ; RNA: cytoplasm)
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gene splicing Sex Chromatin Test
- Plasmid: composed of extrachromosomal materials; outside
nucleus
- Get a portion of the gene — add resrtriction enzyme indications
- Cut a portion of the gene and place in another gene - The test is a simple method to identifying a person’s gender and
establish sexual identity in newborns and in adults
- The mouth has to be rinsed and washed before the test. A
spatula is used to gently scrape the inside of the person’s
cheek. This can be done by a health care provider or by self. A
smear of this BUCCAL SAMPLE is made on a slide, stained and
observed under the microscope.
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procedure
————————————————————————————————————————— 1. Get a Buccal Smear
pcr 2. Place smear on slide using plasma as the adhesive
- Polymerase Chain Reaction 3. Air dry
- Molecular Technique: we are after a particular gene 4. Fix for 30 mins in 95% alcohol
- Principle: Gene amplification 5. Stain with H % E
- Produces millions of copies of a gene 6. Air dry
- Process 7. View Barr Bodies on OIO
- Denaturation —————————————————————————————————————————
- Annealing physiology
- Extension / Elongation - A buccal smear is a painless test to detect BARR BODIES in a
sample of cells from inside the mouth (cheek)
- Oval structure near the nucleus on a neutrophil
- Only in GIRLS

BARR BODY
Lollipop shaped structure

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immunoblotting
- Uses nitocellulose paper
- Western blot: detection of proteins —————————————————————————————————————————
- Ex, Confirmation of HIV Status interpretation
- Detecting HIV antibodies: perform electrophoresis first - A person who is a biological female (46,XX) has a single BARR
then proceed to western blot BODY or sex chromatin while there is an absence of it in
- Detecting antibodies against HIV biological males (46,XY)
- Add detectors to detect presence of antibody
- If antibody is present bands will form (has HIV) Staining Blood Smear
no bands = no antibodies (no HIV)
procedure
1. Prepare blood smear & Air dry
2. Dip in:
- Solution 1: Absolute Methanol (fixative)
- Solution 2: Eosin
- Solution 3: Methylene Blue
- Solution 4: Buffer (water)
3. Blot dry with tissue paper
4. Label with pencil
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characteristics of a good blood smear
- Northern blot: detection of RNA - Tongue shape
- Southern blot: detection of DNA - Feathery Edge
————————————————————————————————————————— - Thick to thin transition
- Occupies 2/3 or 3/4 of the slide

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