2. Importance of the Microscope
• Important for hematology, microbiology, TB, and
malaria testing
• Compound microscope used in bacteriology,
biology, and medicine to examine minute objects
such as bacteria, other unicellular organisms, and
plant and animal cells and tissue
• Advances in fluorochrome stains and monoclonal
antibody techniques caused growth in use of
fluorescence microscopy in both biomedical
analysis and cell biology
3. ADVANTAGES
Brightfield microscopy is very simple to use with
fewer adjustments needed to be made to view
specimens.
Some specimens can be viewed without staining and
the optics used in the bright-field technique don’t alter
the color of the specimen.
It is adaptable with new technology and optional
pieces of equipment can be implemented with bright-
field illumination to give versatility in the tasks it can
perform.
.
4. DISADVANTAGES
Certain disadvantages are inherent in any
optical imaging technique.
By using an aperture diaphragm for contrast,
past a certain point, greater contrast adds
distortion. However, employing an iris
diaphragm will help compensate for this
problem.
Bright-field microscopy can’t be used to observe
living specimens of bacteria, although when
using fixed specimens, bacteria have an
optimum viewing magnification of 1000x.
5. DISADVANTAGES
Bright-field microscopy has very low contrast
and most cells absolutely have to be stained to
be seen; staining may introduce extraneous
details into the specimen that should not be
present.
Also, the user will need to be knowledgeable in
proper staining techniques.
Lastly, this method requires a strong light source
for high magnification applications and intense
lighting can produce heat that will damage
specimens or kill living microorganisms.
6. TERMINOLOGIES
Resolution
Ability to distinguish (resolve) two close-
together points as separate.
Contrast
Differences in intensity between two objects,
or between an object and background
Important in determining resolution
Staining increases contrast
9. DESCRIPTION
Brightfield microscopy is the most
elementary form of microscope illumination
techniques and is generally used with
compound microscopes.
The name "brightfield" is derived from the
fact that the specimen is dark and contrasted
by the surrounding bright viewing field.
Simple light microscopes are sometimes
referred to as brightfield microscopes.
10. WHEN TO USE BRIGHT FIELD MICROSCOPY
Bright field microscopy is best suited to
viewing stained or naturally pigmented
specimens such as
stained prepared slides of tissue sections or
living photosynthetic organisms.
It is useless for living specimens of bacteria,
and inferior for non-photosynthetic protists or
metazoans, or unstained cell suspensions or
tissue sections.
11. OBSERVED USING BRIGHT-FIELD MICROSCOPY,
AND APPROPRIATE MAGNIFICATIONS
Prepared slides, stained - bacteria (1000x), thick
tissue sections (100x, 400x), thin sections with
condensed chromosomes or specially stained
organelles (1000x), large protists or metazoans
(100x).
Smears, stained - blood (400x, 1000x), negative
stained bacteria (400x, 1000x).
Living preparations (wet mounts, unstained) - pond
water (40x, 100x, 400x), living protists or metazoans
(40x, 100x, 400x occasionally), algae and other
microscopic plant material (40x, 100x, 400x). Smaller
specimens will be difficult to observe without
distortion, especially if they have no pigmentation
13. STEPS
Mount the specimen on the stage
Optimize the lighting
Adjust the condenser
Think about what you are looking for
Focus, locate, and center the specimen
Adjust eyepiece separation, focus
Select an objective lens for viewing
Adjust illumination for the selected objective
lens
15. In bright-field microscopy a specimen is
placed on the stage of the microscope
and incandescent light from the
microscope’s light source is aimed at a
lens beneath the specimen. This lens is
called a condenser.
The condenser usually contains an
aperture diaphragm to control and focus
light on the specimen; light passes through
the specimen and then is collected by an
objective lens situated in a turret above
the stage.
16. The objective magnifies the light and
transmits it to an oracular lens or eyepiece
and into the user’s eyes. Some of the light is
absorbed by stains, pigmentation, or dense
areas of the sample and this contrast allows
you to see the specimen.
For good results with this microscopic
technique, the microscope should have a
light source that can provide intense
illumination necessary at high magnifications
and lower light levels for lower magnifications
19. Power switch
Light intensity control
Condenser
Stage
Objectives
Eyepiece lens
Stage motion
control knobs
Course and fine
adjustments knobs
Field diaphragm ring
Aperture diaphragm
20. Care and Use of the Bright
Field Microscope
Malaria parasite
Mycobacterium tuberculosis
21. CARE AND MAINTENANCE OF THE
MICROSCOPE
Good preventive maintenance and care includes:
Regular cleaning of oculars and objectives
Avoid damaging oculars and other optics with eye
make-up or other debris
Careful handling to avoid abrupt motions
Protect from direct sunlight, high temperature,
humidity, dust and vibration
Use appropriate materials to clean the lenses
Cover when not in use with vinyl or plastic dust cover
22. CLEANING THE MICROSCOPE
Routine Cleaning Supplies:
Commercial lens tissue for optics
Caution: Do not use paper towels or
other rough paper products
Cotton swabs with wooden shaft
(optics)
70% isopropyl alcohol
Dilute methanol is satisfactory
Mild detergent and soft cloth for stage
and base of microscope
23. CLEANING OCULARS AND OBJECTIVES
Unplug the microscope
Wash hands
Remove dust from optical glass
surfaces
Carefully remove eyepieces,
objectives, condenser, and filters–one
at a time
Excessive rubbing can cause damage
to iridescent coating on lens
Clean and replace as completed
Do Not take eyepiece or objectives
apart
24. Unplug microscope and allow bulb to
cool
Carefully place microscope on its
side
Open bulb house; use tissue to
remove bulb
Use tissue (to avoid fingerprints) to
pick up new bulb
Insert new bulb and close bulb
house
Replacing Microscope Bulb
25. Plug in microscope and turn on illuminator.
Rotate nosepiece to lock 10X objective in place
Place smear on stage and center it under the
10X objective
Open the field diaphragm all the way and close
condenser diaphragm all the way
Move up (rack up) stage to its highest position
Adjust the oculars for interpupillary distance so
that only one circle of light is seen
Rack up condenser as high as possible
Setting the Koehler
Illumination
26. • Close field diaphragm half way and focus smear at
10X
• Close field diaphragm until diameter of illuminated
image is smaller than the field of view
• Lower condenser with positioning knob until you have
a sharp, focused image of the edges of the field
diaphragm
• Adjust condenser using centering screws so that the
circle of light is centered in field
• Open field diaphragm until illuminated image is just
larger than the field of view. If more light is needed,
use the transformer.
• Koehler illumination is now set. It is important not to
move the condenser up or down or change the field
diaphragm.
Setting the Koehler Illumination
(continued)
27. OPERATION OF THE MICROSCOPE –
EXAMINING SMEARS
Put smear on stage and center it under the 10X
objective
Adjust intensity of the light to a comfortable level
with the transformer
Open condenser diaphragm about 70% to
achieve a good balance of resolution and
contrast
Adjust oculars for interpupillary distance so that
when looking with both eyes only one circle of
light is seen
28. Examining Smears (continued)
• Adjust sharpness of image by moving
adjustment ring on adjustable ocular
• Once 10X focus is achieved, rotate nosepiece
so that the 40X objective is in place
• Readjust the intensity of light to a comfortable
level using the transformer
• Use the fine adjustment knob to focus up and
down through the different planes of the field
29. Microscope Problems –
Troubleshooting 1
Problem: Black Field
Possible Causes:
• Microscope not plugged in
• Power not available at outlet
• Illuminator not turned on
• Bulb burned out
• Objective not clicked into place
• Condenser too low with
diaphragms closed
30. Microscope Problems –
Troubleshooting 2
Problem: Field only partially
illuminated
Possible Causes:
• Objective not clicked into position
• Condenser not centered correctly
• Condenser too low
• Field diaphragms closed too much
31. Problem: Difficulty focusing with
10X
objective
Possible Causes:
• Wrong objective in place
• Objective not screwed into place
• Not in correct plane of focus
Microscope Problems –
Troubleshooting 3
32. Microscope Problems –
Troubleshooting 4
Problem: Difficulty focusing with 40X
objective
Possible Causes:
• Not in correct plane of focus
• Not initially focused at 10X
33. Microscope Problems –
Troubleshooting 5
Problem: Blurry image at 10X or
40X
Possible Causes:
• Dirty objective
• Dirty slide
• Dirty coverslip
Problem: Ground glass
appearance
Possible Causes:
• Condenser too high
34. REFERENCES:
http://www.microscopemaster.com/brightfield-microscopy.html
Advanced Light Microscopy vol. 1 Principles and Basic Properties
by Maksymilian Pluta, Elsevier (1988)
Advanced Light Microscopy vol. 2 Specialised Methods by
Maksymilian Pluta, Elsevier (1989)
Introduction to Light Microscopy by S. Bradbury, B. Bracegirdle,
BIOS Scientific Publishers (1998)
Microbiology: Principles and Explorations by Jacquelyn G. Black,
John Wiley & Sons, Inc. (2005)
Microscopy and Imaging Literature
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/microscopy
.html
