Basics of a Compound

microscope
Types of microscope
A. Compound (simple) microscope (routinely used in
medical laboratories)
B. Phase contrast microscope
C. Dark field microscope
D. Fluorescence microscope
Phase contrast
Based upon the conversion of slight differences in
refractive index within the specimen into differences
in amplitude or briightness
Dark field
Dependent upon black background against which
objects that scatter light are seen
In dark field microscope, the light enters a special
condenser, which has a central blacked out area or a
condenser fitted with dark field stop so that the light
cannot pass directly through it to enter the objective.
Instead, the light is reflected to pass through the outer
edge of the condenser lens at a wide angle. The only
light entering the eye comes from the microorganisms
themselves, with no light entering the eye directly from
the light source.
Fluorescent
In Fluorescence microscopy, ultra - violet light, which
has a very short wavelength and is not visible to the
eye, is use to illuminate organisms, cells, or particles,
which have been previously stained with fluorescing
dyes called fluorochromes.
These dyes are able to transform the invisible short
wavelength ultra - violet light in to longer wavelength
visible light. The fluorescent stained organisms, cells,
or particles can be seen glowing (fluorescing) against a
dark background.
Electron
Light Beam is substituted by a beam of electrons
Compound Microscope
Microscope is an important device that enables us to
visualize minute objects (animate and inanimate) that
cannot be seen by our naked eye.
Major parts of microscope
A. Frame work of the microscope
This includes:
- An arm (stand): - The basic frame of the microscope to
which the base, body and stage are attached.
- A stage: - the table of the microscope where the slide or
specimen is placed.
- A foot, or base: - is the rectangular part up on which
the whole instruments rest.
B. Focusing system
• Coarse and fine focusing adjustments-
1. Course adjustment:- The course focusing adjustment is
controlled by a pair of large knobs positioned one on each side of
the body. Rotations of these knobs move the tube with its lenses,
or in some microscope the stage, up or down fairly rapidly.
2. Fine adjustment: - While low power objectives can be focused by
the course adjustment, high power objectives require a fine
adjustment.
3. Condenser adjustments:- The condenser is focused usually by
rotating a knob to one side of it. This moves the condenser up or
down. The condenser aperture is adjusted by the iris diaphragm,
which is found just below the condenser. The principal purpose of
the condenser is to condense the light required for visualization.
C. Magnification system
1. Objectives: -
Objectives are components that magnify the image of the
specimen to form the primary image. For most routine
laboratory work, 10x, 40x, and 100x (oil immersion) objectives
are adequate.
2. Eyepiece
Eyepiece is the upper optical component that further magnifies
the primary image and brings the light rays to a focus at the
eye point. It consists of two lenses mounted at the correct
distance. It is available in a range of magnifications usually of
4x, 6x, 7x, 10x, 15x and sometimes as high as 20x.
Based on their number of eyepiece, microscopes can
be classified as monocular and binocular microscopes.
D. Illumination system
• Condenser and iris
- Condenser is a large lens with an iris diaphragm.
- The condenser lens receives a beam from the light source and passes
it into the objective.
- The iris is a mechanical device mounted underneath the
- Condenser and controls the amount of light entering the condenser.
• Mirror
- Mirror is situated below the condenser and iris.
- It reflects the beam of light from the light source up wards through
the iris into the condenser. The mirror is used to reflect ray or
electrical light. Some microscopes have a built in light source.
• Sources of illumination
Light source: Electric light
An ordinary 60-watt pearl electric bulb placed about
18 inches from the microscope is sufficient for most
routine work. Quartz halogen (quartz iodine) and
other high intensity lamps are available and are very
good light sources because they give excellent white
illumination and do not blacken like ordinary
tungsten lamps. Many microscopes are now provided
with correctly aligned built-in sources of illumination,
which use tungsten or quartz halogen lamps operating
on 6,8or 12 volts through variable transforms.
Light filters
Light filters are used in the microscope to:
- Reduce the intensity of light;
- Increase contrast and resolution;
- Adjust the color balance of the light to give the best
visual effect;
- Provide monochromic light;
- Absorb light;
- Transmit light of selected wavelength; and
- Protect the eye from injury caused by ultra-violet light.
Objective Eyepiece Total
Magnification Magnification Magnification
10x 10x 100x
20x 10x 200x
40x 10x 400x
100x 10x 1000x
Oil immersion objective
Working principle of an oil immersion objective
When a beam of light passes from air into glass it is
bent and when it passes back from glass to air it is bent
back again to its original direction. This has effect on
oil immersion objective and affects the NA of the
objective and consequently its resolving power. The
bending effect on the objective can be avoided by
replacing the air between the specimen and the lens
with oil, which has the same optical properties as
glass.
Using the microscope
A microscope must always be used with gentleness; care and the
following should be noted.
1. Place the microscope on a firm bench so that it does not
vibrate.
• Make sure that it is not be exposed to direct sun light.
• The user must be seated at the correct height for the
convenient use of the microscope.
2. Select the appropriate source of light.
3. Place the specimen on the stage, making sure that the
underside of the slide is completely dry.
4. Select the objective to be used.
• It is better to begin examination with 10x objective.
• The 10x objective can be used for adjusting the illumination
and for searching the specimen before using a high power lens.
5. Bring the objective as close as possible to the slide
preparation and while viewing in the eye piece slowly move
the objective up ward with the coarse adjustment until the
image comes into view and is sharply focused.
6. Adjust the light source until the illumination of image is at
its brightest.
7. Focus the condenser. To do this, open fully the iris of the
condenser. Using the condenser adjustment knob, focus
the condenser on the details of the light source.
8. Adjust the aperture (opening) of the condenser iris
according to the specimen being examined.
The wider the condenser aperture, the brighter will be
the specimen and the smaller will be the details, which
can be resolved.
• The smaller the aperture, the greater will be the
contrast.
9. Examine the specimen by systematically moving the
slide with the mechanical stage.
10. For a higher magnification, swing the 40x objective
into place.
• Focus the 40x objective, using the fin adjustment.
• If for any reason the image is not visible, lower the
objective until it is nearly but not quite touching the
specimen.
• Then looking through the eyepiece, focus up wards
with the fine adjustment until the image comes into
view.
Any Streptococcus?