CYTOLOGY

HUMAN HISTOLOGY LABORATORY MIDTERM NOTES

➢ Study of plant and animal cell, its structure,
function, and chemistry.
Jay-Ann Abila 2nd Year – 3rd Semester
MED-244 Different Levels of organism
o Atoms
LESSON 1: INTRODUCTION TO HISTSOLOGY
o Molecules
o Cell
HISTOLOGY o Tissue
o Organs
➢ Came from the Greek word. o Organ system
o “Histos” means Tissue. o Organism
o “Logia” means knowledge.
➢ Histology is knowledge of tissue. ORGANOLOGY
➢ Study of normal tissue
➢ Study or structure and function of organs and
HISTOLOGIST organ system within the organism.
➢ Understanding the anatomy, organization,
➢ A health care professional that performs
development, and physiology of organs.
microscopy to study the normal structure of
the cells or tissue. ➢ By the normal physiological processes and
abnormalities of disease that affect organs.
HISTOPATHOLOGIST
DEVELOPING SKILLS IN MICROSCOPY
➢ A health care professional that performs
microscopy to study the abnormal structure
of the cells or tissue.

HISTOPATHOLOGY

➢ study of abnormal tissue

IMPORTANCE OF STUDYING THE APPERANCE OF

NORMAL TISSUE

➢ Identify and differentiate the appearance of ➢ Chondrocytes

normal and abnormal tissue in order to o cells that responsible for cartilage
determine whether the individual poses formation
specific illness. o found inside the cartilage.

MICROSCOPE

➢ The microscope is the most useful tool in

histology.

GOLD STANDARD

➢ Histologic diagnosis or microscopy is the gold

standard of clinical practice.
 LESSON 2: INTRODUCTION TO MICROSCOPY ➢ Limitation
o Absence of contrast between the
specimen and the surrounding
medium, which makes it difficult to
observe living cells.
➢ Specimens are usually nonviable, stained
preparations.
o The specimen can only be seen if its
stained.

2. DARKFIELD MICROSCOPE

➢ Similar to the
ordinary light
microscope.
➢ The condenser
system is
modified so that
the specimen is
not illuminated directly.
➢ The condenser directs the light obliquely
DIFFERENT TYPES OF MICROSCOPES (slanting or diagonal) so that the light is
deflected or scattered from the specimen.
1. Brightfield microscope ➢ The specimen appears bright against a dark
2. Darkfield microscope background.
3. Phase-contrast microscope ➢ Living species may be observed more readily.
4. Fluorescent microscope
5. Electron microscope
3. PHASE - CONTRAST MICROSCOPE

1. BRIGHTFIELD MICROSCOPE – Compound ➢ Observation of

microscope unstained
microorganisms is
➢ Contains 2 lens system for possible here.
magnifying specimen. ➢ Includes special
o the ocular lens in the objectives and
eye piece and the condensers that makes
objective lens visible cellular
located in the nose components that
piece differ slightly in their
➢ Specimen is illuminated by a refractive index.
beam of tungsten light. o Refraction – is
o It uses a tungsten halogen bulb. bending of light rays from one
▪ Temperature: (3200K) Warm medium to another.
white light. ➢ The image appears dark against a light
➢ Specimen appears dark against a bright background.
background.
4. FLUORESCENT MICROSCOPE ➢ The fluorescentportion of the dye becomes
visible against a black background.
➢ Used most frequently to visualize specimen
that are chemically tagged with a fluorescent
dye.
➢ The source of illumination is an
ultraviolet (UV) light.
o High-pressure mercury lamp or
hydrogen quarts lamp – light source.
➢ The ocular lens is fitted with a filter that
permits the longer ultraviolet radiations are
absorbed y the fluorescent label and the
energy is re-emitted I the form of a different
wavelength I the visible light range.
5. ELECTRON MICROSCOPE (EM)
➢ Fluorescent dyes absorbs at wavelength
between 230-350 nm and emit an orange,
➢ Provide a revolutionary method of
yellow or greenish light.
microscopy.
➢ Magnifies up to one million.
➢ Permits visualization of submicroscopic
cellular particles and viruses.
➢ The specimen is illuminated by a beam of
electrons rather than light.
➢ Focusing is carried out by electromagnets
instead of a set of optics.
➢ Components are sealed in a tube wherein a
complete vacuum is established.
➢ Specimens should be thinly prepared, fixed,
and dehydrated to allow the electron beams
to pass through freely.
o Fixed with formalin.
➢ A. Dyed with acridine orange.
o Dehydrated by alcohol.
o N – represents the nuclei of the cell
which emits yellow light. The cell is 2 types of Electron microscope
from the kidney tubule.
o R – the cell itself which emits orange 1. Transmission EM
light. o Requires
➢ B. Dyed with DAPI (4’,6-diamino-2- specimens that
phenylindole) are thinly
o DAPI binds the DNA prepared, fixed,
o Fluorescein-phalloidin binds the actin and dehydrated
filaments showing to allow the
▪ nuclei with blue fluorescence electron beam
and to pass through
▪ actin filaments stained green. freely.
➢ Used primarily for the detection of antigen- o As the electrons pass through the
antibody reactions. specimen, images are formed by
➢ Antibodies are conjugated with a fluorescent directing the electron onto
dye that becomes excited in the presence of photographic film.
UV light.
 ▪ This makes the internal 3. Base
cellular structures visible. o Bottom of the microscope and I used
▪ The wavelength of the for support.
electron beam is much
4. Mechanical stage
shorter compared to light
o Fixed platform with opening in the
resolution that is why it
center allows for the passage of light
allows a 1000-fold increase in
from an illuminating source below to
your resolution.
the lens system above the stage.
o The TEM permits resolution up to 3
o Provides a surface for the placement
nm.
of a slide with its specimen over the
o High resolution allows isolated
central opening.
particles magnified as much as
o Can be moved vertically or horizontally
400,000 times to be vied in detail.
by means of adjustment controls.
o The specimen (resin-embedded tissue)
o Stage clips – it hold the slide in place
should be thinly sliced into 40-90 nm
in the stage.
o Typically it can be magnified
(magnification) up to 120, 000 times. 5. Ocular or eyepiece lens
o Lens at the top of the microscope
2. Scanning EM o Usually 10x – 15x
o used for
visualizing 6. Illuminator or light source
surface o Positioned in the
characteristics base of the
rather than instrument.
intracellular o Built-in light source
structures. – provides direct illumination.
o A narrow beam of electrons scans o Mirror (one side flat and the other
back and forth, producing a 3- concave) – indirect illumination
dimensional image (3D) as the o Uses an external light source (lamp or
electrons are reflected off the sunlight) – placed in front of the
specimen’s surface. mirror to direct the light upward into
o The specimen will be dried out, then it the lens system
ill be sprayed it wih a very thin layer of ▪ Flat side – artificial light
heavy metal. ▪ Concave side – sunlight.
▪ Usally uses gold as a heavy 7. Condenser
metal. o Found directly under the stage and
▪ But the images are usuallt contains 2 sets of lenses that collect
black and white. and concentrate light passing upward.
o Illuminates the object the rays of light
that pass through the specimen
PARTS OF A COMPOUND BRIGHTFIELD MICROSCOPE obliquely as well as directly.

1. Body tube
o Connects the eye piece to the
objective lens.

2. Arm
o Supports the body tube and connects
it to the base
8. Iris diaphragm 12. High power objective (40x)
o Rotating disk under the mechanical o HPO – color blue
stage o Also called high- dry
o Can be adjusted the change the objective
intensity and size of the cone of light o Used for observing
that is projected upwards into the fine detail such as
slide. striations in skeletal
o Used to create contrast on cellular muscle, types of
components. nerve cells in the
o Tissues – open iris diaphragm more retina, etc.
o Cells or urinary sediments – close iris
13. Oil immersion objective (100x)
diaphragm for better contrast.
o OIO – Color white
9. Revolving nosepiece or turret o The longest
o Hold the objective
objective o Used for observing
lenses. details of the
o Allows easy individual cells
rotation of such as white
objective blood cells, red
lenses. blood cells, and immature cells.
o Must be used together with
10. Scanner objective (4x)
immersion oil.
o Color red
▪ Cedarwood oil
o Shortest objective
o Useful for 14. Immersion oil
getting an o Light that passes through the glass slide,
overview of the passes through air, then the lens – this
slide. light gets refracted
o For WHOLE o At high magnification, this refraction
ORGANS like a can blur the image.
section of the o To eliminate refraction between the
spinal cord, lung, slide and lens – replace the air gap with
digestive tract, etc. immersion oil.
o Safe to use since it cannot be lowered o The immersion oil has the same
to the point of contact with the slide. refractive index as glass
o Oil refractive index – 1.518
11. Low power objective (10x)
o Refreactive indec – ability to bend light
o LOP – color yellow
o The most useful lens for viewing slides
o Almost any feature
can be featured in
this objective
o Also safe to use
since it is cannot
be lowered to the
point of contacting
with the slide.
THEORETICAL PRINCIPLE OF MICROSCOPY MICROSCOPE CARE

1.Magnification Once the microscope is on the table

➢ Enlargement or magnification of a specimens ➢ remove all unnecessary materials (books,

the function of a 2-lens system. papers, purses, etc.)
➢ The ocular lens is found in the eyepiece. ➢ uncoil the microscope’s electric wire and plug
➢ The objective lens is situated in the revolving into the outlet.
nosepiece. ➢ clean all lens systems – ocular and objectives.
➢ The lenses are separated by the body tube. ➢ methanol or xylol (xylene) – lens cleanser.
➢ The objective lens is neared the specimen and
magnifies it.
After using the microscope
➢ The real image is projected up into the focal
plan and then magnified by the ocular lens to
➢ clean all the lenses with dry, clean lens paper
produce the final image.
with a drop or 2 of methanol.
➢ xylol can be used to clean the mechanical stage.
2.Resolving power or resolution ➢ place the objective back to LOP.
➢ center the mechanical stage.
➢ Unlimited enlargement is not possible by ➢ coil the electric wire are the body tube.
merely increasing the magnifying power of ➢ return the microscope.
the lenses or by using additional lenses.
➢ Because lenses are limited by a property called
RESOLVING POWER.
➢ Resolving power
o It is the ability of lens to shoe 2
adjacent objects as discrete entities.
o It is dependent on the wavelength of
light used and the numerical aperture.
- Numerical aperture –
characteristics of each lens
imprinted on the objectives.

3.Numerical aperture

➢ Function of a diameter of objective lens in

relation to its focal length.
➢ it is doubled by use of the substage
condenser.