University of San Agustin - COLLEGE OF PHARMACY AND MEDICAL TECHNOLOGY

Department of Medical Laboratory Science

MLS 9a: Parasitology Laboratory
MODULE 1: Microscopy

1.0 Overview a. Lens system which includes the oculars, objective and
ocular
PART 1: OVERVIEW OF DISCUSSION b. Illumination system includes the light source,
Microscope is an optical instrument that magnifies the condenser and field and iris diaphragm
size of the image of the object. c. Body which consists of a base, body tube and
●​ It is used to observe objects that are not visible nosepiece
with the unaided eye.
●​ It is probably the equipment that is the most
used (and misused) in the clinical laboratory.
●​ It is indispensable in many areas of the
laboratory, including urinalysis, hematology, and
microbiology.
●​ The microscope is a precision instrument and is
very important equipment thus, it must be kept
clean and in excellent condition, mechanically
and optically, at all times.
●​ The invention of this magnifying instrument was
credited to Galileo Galilei & Zacharias Janssen
and its use started with Anton Van Leuwenhoek.

Generally the microscopes are of 2 types: (a) simple

microscope which has a single biconvex magnifying lens
and (b) compound microscope which has 2 or more
lenses.
●​ The compound microscope is classified as to
light microscope (if it employs visible light as
source of illumination) or electron microscope The main framework of a compound microscope
(if the source of illumination is beams of consist of the mechanical parts:
electrons).
●​ This activity will refresh you on the use & care a. Base which provides support for the microscope. The
of a compound light microscope. base is large enough to support the entire structure and
let it stand by itself
PART 3: DISCUSSION b. Arm which serves as support structure wherein the
Microscope user will grip while handling and it holds also the
●​ one piece of equipment that is used for magnifying
magnification of images and allows visualization and adjustment system
of minute details of organisms or a certain c. Stage is the flat platform where the slide is placed for
microscopic structure. examination
d. Substage is located directly under the stage and holds
To fulfill its function a microscope must accomplish the condenser and diaphragm
three things. e. The mechanical stage with a X and Y movement
a. It must magnify the object permits movement of the stage while the Stage clips
b. It must resolve the details of the object holds the slide in place
c. It must make the details visible f. Coarse adjustment knob moves the stage up and
down for initial visualization of the specimen
COMPOUND MICROSCOPE g. Fine adjustment knob is a smaller knob which is
●​ A compound light microscope is a microscope used for fine adjustments in the distance of the specimen
with more than one lens and its light source. for detailed visualization which sharpens the image
●​ The most common type of microscopy which is h. Diopter adjustment knob found on the oculars can
classified under Bright field but essentially all be rotated and compensate for variations in vision
types of microscope have the same major between the operator’s eyes
systems. i. The rheostat is an adjustment part for regulating the
intensity of light.
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MLS 9a: Parasitology Laboratory
MODULE 1: Microscopy

The lens system and Magnification system are the TYPES OF COMPOUND MICROSCOPY
following parts and their functions:
a. BRIGHT FIELD MICROSCOPY
a. Nosepiece which is found at the end of the body tube ●​ This is generally used in compound
which hold the objectives microscopes, where light is either passed
b. Objectives are a set of lenses held by the nosepiece through, or reflected off, a specimen.
and are closest to the slide. Its purpose is to increase and ●​ Specimen is dark and contrasted by the
decrease magnification. Routinely there are 4 objectives surrounding bright viewing field.
that can be observed ●​ Illumination is not altered by devices that alter
■​ The scanner with magnification of 4 times (4x) its properties such as filters or polarizers.
■​ The Low power objective (LPO) with
magnification of 10 times (10x) b. DARK FIELD MICROSCOPY
■​ The High power objective (HPO) with ●​ This is a microscopic technique utilized to
magnification of 40 times (40x) observed unstained and transparent samples to
■​ The Oil Immersion Objective (OIO) with be visible, appearing bright against a dark
magnification of 100 times (100x) background.
c. Ocular or eyepieces of the microscope are located at ●​ It blocks the central light with a condenser so
the top of the body tube and is in contact with the eyes that oblique rays hit the object.
usually with 10 times magnification power (10x) ●​ Stereomicroscopes and Conventional compound
microscopes imply the principal elements.
The Illumination system are the following
c. PHASE CONTRAST MICROSCOPY
a. The light source located at the base of the microscope ●​ This Is an optical microscopy illumination
which provides the light technique in which small phase shifts in the light
b. Diaphragm controls the diameter of the light beam passing through a transparent specimen are
reaching the slide and adjusted for optimal illumination converted into amplitude or contrast changes in
c. Condenser at the substage focuses light on the the image
specimen and controls the light for uniform illumination ●​ . It enhances the contrast of transparent and
colorless objects. Usually for detailing live cell
TERMS IN MICROSCOPY or bacterial structures, wherein does not require
a. TOTAL MAGNIFICATION is the product of the staining.
magnifying power of the objective and eyepiece with
normal tube length of 160 mm. d. POLARIZED LIGHT MICROSCOPY
○​ Example with an ocular of 10x and Objective ●​ A type of microscopy which uses a
with 40x magnification and body tube of 160 contrast-enhancing technique which improves
mm its total magnification is 400x. the quality of the image obtained with
○​ If ever the microscope is fitted with a draw tube birefringent materials when compared to other
increasing the microscopes length. techniques.
○​ Total magnification is calculated as: ●​ Usually for anisotropic samples like crystals,
striated muscles and amyloid which there is
Objective X Eyepiece x (Working tube length/
birefringence on light.
Normal tube length) = Total Magnification

e. INTERFERENCE CONTRAST MICROSCOPY

●​ A technique which provides a three-dimensional
b. RESOLUTION is the ability of the lens to image showing very fine structural detail by
distinguish two small objects that are a specific distance splitting the light ray so that the beams pass
apart. through different areas of the specimen.
○​ The shorter the wavelength of light, the greater Two types:
the resolving power of the microscope ■​ Modulation contrast ( Hoffman )
c. PAR FOCAL is ability of the objectives to be ■​ Differential contrast (Nomarski)
approximately in focused even they are shifted
d. FOCAL LENGTH is the distance between the outer FLUORESCENCE MICROSCOPY
lens of the objective and the cover glass of the slide for ●​ type of microscopy which utilizes
examination. immunofluorescence.

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MLS 9a: Parasitology Laboratory
MODULE 1: Microscopy

●​ Fluorescence is the property by which atoms ○​ This is aggravated by leaving the

absorbed light of a particular wavelength and instrument uncovered, which can lead to
emits it at a longer wavelength. Allows viewing deterioration of image quality even
of naturally fluorescent substances or stained though the instrument may be
with fluorochrome or fluorophobe (fluorescent practically new.
dyes) ●​ Keep the microscope in a dry place. Do not
store the microscope in direct sunlight.
ELECTRON MICROSCOPY ○​ Sunlight can influence the quality of the
●​ uses beam of accelerated electrons as a source of specimen imaging.
illumination. ●​ Never touch the optical lens with bare
●​ It has a higher resolving power that can see the fingers.
ultrastructures of the specimen. ○​ Your body produces an oil that smudges
●​ Instead of visible light, a stream of electrons is the glass. This oil can even etch the
used which has a shorter wavelength. glass if left on too long.
It has two types. ○​ Use only LENS PAPER to clean the
a. TRANSMISSION ELECTRON MICROSCOPE glass. Toilet Paper, Kleenex, and paper
(TEM) shows a two dimensional view of the image. towels have fibers than can scratch the
b. SCANNING ELECTRON MICROSCOPE (SEM) lenses
shows surface of the object and seen in #-D. ●​ Dirt must be removed from the microscope
frame and mechanical parts by dabbing with
Care and Maintenance of the Compound Microscope a moist paper towel.
○​ Dirt that is non-adherent may be
The microscope is a delicate, precisely aligned removed from less delicate lens surfaces
instrument. Careless, incorrect operation and by gentle brushing with a clean camel
maintenance techniques not only result in unreliable and hair brush
poor quality images, but also cause productivity at the ●​ Clean smudges, fingerprints, oils, and other
microscope to suffer, and the instrument's useful lifetime contaminants from the lens with clean lens paper
to be greatly reduced. or a soft clean cloth moistened
●​ The top lens of the eyepiece should be polished
●​ Always support the microscope when to remove dust or finger marks, and the
carrying. microscope should be checked for critical
○​ It should be cradled - one hand holding illumination.
it by the arm, the other supporting the ●​ Optical components should not be immersed in
base. any solvent, and cleaning tissues should only be
○​ The best way to carry a microscope is in moistened, never saturated, with a cleaning
its case. solution. Remove oil when done with the Oil
●​ Never disassemble the microscope immersion Objective
○​ as doing so may cause electric shock or
damage to the microscope
●​ Keep the microscope covered when not in
use.
○​ Microscope life is extended when they
are covered during storage.
●​ Lint-free fabric covers are also available, and
provide an effective dust barrier that can
minimize the need for cleaning the microscope.
●​ Never store a microscope with the eyepiece
removed or uncovered,
○​ since dust drill collect in the body tube
and be very difficult to clean.
○​ Keep the body tube sealed at all times.
○​ A microscope that remains unused for a
lengthy period of time can accumulate
dust and debris from the air.