RWQ 1330B MICROBIAL WATER QUALITY

WEEK SIX
TOPIC FIVE LECTURE NOTES
MICROSCOPY
TRAINER: EVANS AMUNZE
Introduction
• This is the technical field of using microscopes to view
objects that are too small to be seen by the naked eye.
• Magnification is the ability to make small objects
seem larger, such as making a microscopic organism
visible.
• Resolution is the ability to distinguish two objects
from each other.
Objectives
By the end of this topic, the trainee should be able to;
a) Define the terminologies used in microscopy
b) Discuss the types of microscopes
c) Explain the differences between light and electron
microscopes
d) Identify the parts of a light microscope and give their
functions
e) Explain how to care, handle and maintain a light
microscope
Definition of terms
Definition of terms Cont…
• Microscopy: This is the technical field of using microscopes to view objects
that are too small to be seen by the naked eye.
• Magnification: Is the ability to make small objects seem larger, such as
making a microscopic organism visible.
• Resolution: Is the ability to distinguish two objects from each other. It is the
clarity of an image
• Limit of Resolution (resolving power): Actual measurement of how far apart
two points must be for the microscope to view them as being separate.
• Numerical Aperture: A Measure of a lens's ability to "capture" light coming
from the specimen and use it to make the image.
Definition of terms Cont…
• Par focal Lenses: Lenses that is oriented in such a way that the specimen
remains in focus as the objectives are rotated into place.
• Depth of Field: It is the thickness of the specimen which can be seen when
in focus. As you increase magnification the depth of field decreases.
• Working Distance: The space between the front mount (bottom) of the
objective lens and the top of the cover slip of the microscope slide is the
working distance. The working distance decreases as the objective lens
magnification increases.
• Total Magnification: The total magnification of the lens system is the
product of the magnification of the ocular lens by the magnification of the
objective lens being used (ocular X objective).
Types of Microscopes
There are two major types of microscopes namely:
1. Light microscope
a) Simple light microscope
b) Compound light microscope
2. Electron microscope
a) Scanning electron microscope
b) Transmission electron microscope
Advantages and Disadvantages of a Light
Microscope
Advantages of a light microscope
• Cheap to purchase and operate
• Small and portable
• Unaffected by magnetic fields
• Natural colour of the material can be observed
• Living as well as dead specimen can be viewed
• Preparation is relatively quick and simple
Disadvantages of a light microscope
• The depth of the field is restricted
• Preparation may distort specimen
• Magnifies objects up to 1500 times
Advantages and Disadvantages of an Electron
Microscope
Advantages of an electron microscope
• Have a higher resolution up to 500000 times
• It is possible to view a greater depth of the field
Disadvantages of electron microscope
• Are extremely expensive
• Sample preparation is more complicated
• Sample must be dead hence not possible to observe living specimen
• Require more training and experience to operate
• Space requirements are high i.e. a whole room
Parts
Parts and Functions:
Functions of the Parts of a Light Microscope
• Ocular Lenses: These eyepieces further magnify the image formed by the objective lens. It
does not improve resolution. They make up an adjustable binocular system. The
magnification of the oculars is 10X. They are equipped with a pointer inside one of the
lenses which makes it easier to point out specific structures or determine the real
dimensions of the specimen you are observing.
• Arm: Connects to the base and supports the microscope head. It is also used to carry the
microscope.
• Light Intensity knob: Light on/off switch and intensity knob. It controls the intensity of the
light coming from the bulb.
• Fine Focus Adjustment Knob: It controls precise focusing of the object. Only the fine
adjustment knob should be used with the high magnification lenses - high power and the
oil immersion objective lenses. Moving the fine adjustment knob also helps you to
determine the third dimension (depth) of the specimen you are studying.
• Coarse Focus Adjustment Knob: This is used for rapid (or coarse) focusing on the specimen
when using the scanning objective lens and the low power lens. This course focusing knob
is rotated until the specimen is roughly in focus and then left alone.
Functions of the Parts of a Light Microscope
Cont…
• Mechanical Stage Adjustment Knobs: Two knobs that are located on the side of the stage
and are used to move the slide around to locate your specimen; one knob moves the slide
from side to side and the other moves the slide forward and backward. The mechanical
stage permits precision movements of your slide, without you touching the slide yourself.
• Base: The bottom of the microscope used for support.
• Lamp: Provides a steady light source and illumination of the specimen.
• Condenser Lens: Connected to the iris diaphragm and located just under the stage. It
contains a system of lenses that directs light from the lamp through the slide specimen.
• Iris Diaphragm: Located on the condenser. The lever of the iris diaphragm is used to adjust
the amount of light striking the specimen.
• Stage: Holds the slide to be observed. The center of the stage has an aperture or hole
through which light passes to illuminate the specimen on the slide.
• Stage Clips: They fix into position your slide on the stage.
Functions of the Parts of a Light Microscope
Cont…
• Objective Lenses: The pattern of light formed by the specimen is focused into a real
image by the objective lenses. When the proper illumination is provided the
resolving power of a microscope depends on the quality of its objective lenses.
There are four of them with magnifications of;
a) 4X - Scanning objective lens
b) 10X – Low power objective lens
c) 40X – High power objective lens, and
d) 100X – Oil immersion objectives lens
• Revolving Nosepiece: The four objective lenses are mounted on this rotating turret
or nosepiece. Rotating this turret allows you to switch between the objective lenses
as you need them.
Specialized Types of Light Microscopy
A. Bright-field Microscopy: Produces an image made from light that is transmitted
through a specimen.
• The specimen restricts light transmission and appears shadowy against a bright
background - where light enters the microscope unimpeded. Because most
biological specimens are transparent, the contrast between the specimen and the
background can be improved with the application of stains to the specimen.
• The price of the improved contrast is that the staining process usually kills cells.
This is especially true of bacterial-staining protocol.
B. Dark-field Microscopy: A special condenser is used so that only the light reflected
off the specimen enters the objective.
• The appearance is of a brightly lit specimen against a dark background, and often
with better resolution than that of the bright-field microscope.
Specialized Types of Light Microscopy Cont…
C. Phase Contract Microscopy: It uses special optical components to exploit subtle
differences in the refractive indices of water and cytoplasmic components to produce
contrast.
• Light waves that are in phase reinforce one another, and their total intensity (because of
the summed amplitudes) increases. Light waves that are out of phase by exactly ½
wavelength cancel each other and result in no intensity – darkness. Wavelengths that are
out of phase by any amount will produce some degree of cancellation and result in
brightness that is less than maximum but more than darkness. Thus, contrast is provided
by differences in light intensity that result from differences in refractive indices in parts of
the specimen that put light waves more or less out of phase.
• Specimen appears as various shades of darks against a bright background.
D. Fluorescence Microscopy: It uses a fluorescent dye that emits fluorescence when
illuminated with ultra-violet radiation.
• In some cases, specimens posses naturally fluorescing chemicals and no dye is needed..
Calculating Total Magnification
The total magnification of the lens system is the
product of the magnification of the ocular lens by the
magnification of the objective lens being used (ocular
X objective).

Magnification = Ocular lens magnification X Objective

lens magnification
Examples:
1. Name the 4 types of objective lens that are on the microscope and what is their objective
magnification and total magnification?
Answer:
• Scanning Lens - 4X, 40X
• Low Power Lens - 10X, 100X
• High Power Lens - 40X, 400X
• Oil Immersion - 100X, 1000X
2. Name the four types of Light Microscopy
Answer:
• Bright-field
• Dark-field
• Fluorescence
• Phase Contrast Microscopy
Examples Cont…
4. What begins Image Formation?
Answer:
• It begins with light coming from an internal or an external light source.
5. What are the benefits of using oil immersion?
Answer:
• Using immersion oil between the specimen and the oil objection lens minimized the light
loss as well as increased its numerical aperture and, in return, makes its limit of resolution
smaller. The result is better resolution. Used in conjunction with cytological stains is used to
identify microbes from patient specimens or the environment.
6. What does "D" equal to in light microscopy?
Answer:
• It is the minimum distance at which two points can be resolved - the actual limit of
resolution.
Examples Cont…
7. Where is lens paper used in microscopy?
Answer:
• Used for gently cleaning the condenser and objective
lenses.
8. What is the relationship between resolution and the limit
of resolution (resolving power)
Answer:
• Resolution improves as the limit of resolution (resolving
power) is made smaller.
Differences between Light and Electron
Microscope
Light Microscope Electron Microscope

Beam of light is source of energy Beam of electrons is source of energy

Microscopic slide is used to place the specimen Electromagnetic plate is used to place the specimen

Specimen is dead or alive Specimen must be dead

Magnification is low Magnification high

Lower maintenance costs Maintenance costs are very high

Initial purchase and operation cost are relatively lower Initial purchase and operation costs are quite high
 Care, Handling and Maintenance of a
Microscope
Tips for Microscope Care
Note: Proper care and maintenance of your microscope can extend its life by many years.
Tip 1: Handle with care
• Most microscope problems occur as a result of improper handling. When carrying your
microscope, hold it by the base and the metal support arm. Do not pick it up by the stage,
as this can cause misalignment. When transporting it, use a microscope bag.
Tip 2: Keep lenses clear of slides
• When using your microscope and adjusting the focus you will need to lower the objective
lens down as far as it will go. However, you should never allow the lens to touch the slide
you are looking at. Dirty lenses can be difficult to clean.
Tip3: Clean after using immersion oil
• If using immersion oil, always ensure the objectives are cleaned immediately after use.
Objective, eyepieces and condenser may be removed for cleaning. Use only lens paper and
lens cleaner. Do not use solvents.
 Care, Handling and Maintenance of a
Microscope Cont…
Tip 4: Cover when not in use
• All microscopes are sold with dust covers. Always keep your microscope covered when not in use
even if the microscope is stored in a cabinet. Eye tubes also need to be kept free of dust so do not
store a microscope without the eyepieces. If the microscope eyepieces must be removed, cover the
tubes with caps or a plastic bag with a rubber band around the eye tube.
Tip 5: Look after the bulb
• After using the microscope, turn off the illuminator and wait for it to cool for several minutes
before putting it away. By allowing the bulb to cool you will extend its life. When turning the
microscope on and off, use the switch not the power point. Do not switch the microscope on while
using full light intensity. Never touch the bulb with your fingers as the body oils can burn into the
bulb and reduce its life. Use a tissue. Keep a store of replacement bulbs and always use the correct
bulb.
Tip 6: Store in a clean, dry place
• Make sure you do not store your microscope in an area that has corrosive chemical fumes that can
destroy lenses or metal parts or beside solutions that may leak. Salt air and pervasive damp can
also cause damage over time. Make sure your cabinet is ventilated.
Care, Handling and Maintenance of a
Microscope Cont…
Tip 7: Only use special lens paper or wipes for cleaning the lenses
• Microscope lenses can easily be scratched and should be treated with great care.
Use an aspirator to remove dust.
• Sticky residue can be removed with lens paper moistened with distilled water or
lens cleaning solution and rubbed gently using a circular motion.
• Never use sharp instruments or anything abrasive on the microscope lenses.
Tip 8: Keep your User's manual and wrenches in a safe place
• Each microscope should come with a user's manual and specialist wrenches as
required. Always consult the User's Manual before making any adjustments to your
microscope and use the wrenches provided.
• Never over-tighten or use force when performing any maintenance on your
microscope, or use inappropriate tools. This can damage the parts.
Care, Handling and Maintenance of a
Microscope Cont…
Tip 9: Perform an annual maintenance check
• On an annual basis moving parts on the microscope should be
cleaned and lubricated. Clean grease and dirt from sliding surfaces
using a clean cloth.
• Apply a very thin layer of lithium-based grease to the sliding
surfaces. Do not grease the teeth of the rack and pinion gears.
Inspect the power cords and plugs for safety and stock up on a
supply of replacement bulbs.
Tip 10: Have your microscope serviced professionally
• A rule of thumb for frequency of servicing is every 200 hours of use
or every 3 years, whichever comes first.
Types of Sophisticated Light Microscopes
Type Brief Description

Bright-field microscope A typical microscope that uses transmitted light to observe targets at high magnification.

Polarizing microscope A microscope that uses different light transmission characteristics of materials, such as crystalline structures, to produce an image.

Phase contrast
A microscope that visualizes minute surface irregularities by using light interference. It is commonly used to observe living cells
microscope
without staining them. It is used to increase contrast when viewing transparent specimen.

Differential interference This microscope, similar to the phase contrast, is used to observe minute surface irregularities but at a higher resolution. However, the
contrast use of polarized light limits the variety of observable specimen containers. It gives an image with a 3D quality however the image is
microscope optical rather than morphological.
Fluorescence
A biological microscope that observes fluorescence emitted by samples by using special light sources such as mercury lamps. When
microscope
combined with additional equipment, brightfield microscopes can also perform fluorescence imaging.
 THE END

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