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EXAMENUL MICROSCOPIC

Principiul microscopului optic: lumina provenita din lampa trece prin


condensor si apoi prin proba. Lumina care trece prin si pe linga proba fara a
fi deviata este numita lumina directa. Lumina de fond ce trece pe langa
specimen este de asemenea lumina directa. O parte din lumina ce cade pe
specimen este deviata prin refractie sau difractie si este numita lumina
difractata. Aceasta lumina este defazata cu 180 grade. Aceasta lumina
difractata poate interfera cu lumina directa. Imaginea este preluata de
obiectiv, marita de ocular si proiectata pe retina.

What has happened is that the direct or undeviated light is projected by
the objective and spread evenly across the entire image plane at the
diaphragm of the eyepiece. The light diffracted by the specimen is brought
to focus at various localized places on the same image plane, as illustrated
in Figure 2; and there the diffracted light causes destructive interference,
and reduces intensity resulting in more or less dark areas. These patterns of
light and dark are what we recognize as an image of the specimen. Since our
eyes are sensitive to variations in brightness, the image then becomes a
more or less faithful reconstitution of the original specimen
Lentilele microscopului optic sunt reprezentate de oculare i obiective. lens
and an objective lens (see figure 2.2). The ocular lens allows comfortable
viewing of the specimen from a distance. It also has some magnification
capability, usually 10 times (10) or 20 times (20). The purpose of the
objective lens, which is located near the specimen, is to provide image
magnification and image clarity. Most teaching microscopes have three
objective lenses with different powers of magnification (usually 10, 45,
and 100). Total magnification is obtained by multiplying the magnification
of the ocular lens by the magnification of the objective lens. Thus, when
using a 10 ocular lens with a 45 objective lens, the total magnification of
the specimen image is 450 diameters. the technique of using lens immersion
oil in place of water as a medium for transmission of light rays from the
specimen to the lens of the oil immersion objective. Oil with a density more
akin to the microscope lens than that of water helps to decrease the loss of
transmitted light, which, in turn, increases image clarity.
In cases where an objective is used for transillumination, image brightness
decreases rapidly as the magnification increases in a series of objectives
having identical correction.
Principiu
Treponema pallidum
Microscopie cu fond intunecat (500X) Escherichia coli
1. Use both hands to transport the microscope. Keep upright. If inverted, oculars
may fall out.
2. Do not touch lenses with your hands. Use lens paper instead. Use of other
cleaning materials such as handkerchiefs and Kleenex tissues is discouraged
because they may scratch the lens.
3. Do not force any of the various microscope adjustment knobs. If you
experience problems making adjustments, consult your instructor.
4. Do not remove objective or ocular lenses for cleaning, or exchange them with
different microscopes.
5. For routine cleaning of the oil immersion objective lens, it is necessary only to
wipe off excess oil with a piece of dry lens paper. Any special cleaning should
be done under the guidance of the instructor.
6. Before storing the microscope, make certain that the ocular lens is also clean.
Frequently, sweat deposits from your eyes, which are acidic, can etch the
glass. The presence of other foreign particles can be determined by rotating
the ocular lens manually as you look through the microscope. The presence of
a pattern that rotates is evidence of dirt. Clean the upper and lower surfaces
of the ocular with lens paper moistened with a drop of distilled water. If dirt
persists, consult your instructor. Any dirt remaining after cleaning with a
suitable solvent indicates either a scratched lens surface or the presence of dirt
on the inside surface of the lens.
7. 7. A blast of air from an air syringe may be effective in removing any
remaining dust particles from the lenses.
Wet mounts are easier to prepare but dry out more rapidly due to contact
between the coverslip and air on all four sides. The drying out process can
sometimes create false motility positives.
Drying out can be reduced by ringing the coverslip edges with petroleum
jelly.
Other disadvantages are the inability at times to see the microorganism
because it is not sufficiently different in refractive index from the suspending
fluid (this can sometimes be resolved by reducing the light intensity).
It is not particularly useful for observing thick preparations such as hay
infusions.
bright-field microscopy is used with wet mounts to observe bacterial motility
and form.
In observing bacterial motility, it is important to distinguish true motility
from Brownian movement, a form of movement caused by molecules in the
liquid striking a solid object, in this instance the bacterial cell, causing it to
vibrate back and forth. If the bacterial cell is truly motile, you will observe its
directional movement from point A to point B, providing the cells are not in
the resting stage of the growth curve.
Measurement of cell viability with methylene blue may also be skewed. When
resting stage cells are used (Kleyn et al., 1962) they, although viable, are often
unable to reduce the dye to a colorless form. Thus, it is preferable to observe
cells from the early logarithmic stage of the growth curve (see figure
10.1).
The cells of choiceyeastare sufficiently large for ease of observation with
bright-field microscopy when using the high dry objective. Unstained cells
from the same stage of the growth curve will also be observed for viability by
using dark-field microscopy
Thus, you will be able to compare viability results for the two methods with
one another. Hopefully they will vary no more than 10%one accepted
standard of error for biological material.
Procedure for wet mount
1. Prepare clean microscope slides and clean
coverslips by washing them in a mild detergent
solution, rinsing with distilled water, and then
drying them with a clean towel. Examine
visually for clarity.
2. Suspend your broth culture of S. epidermidis by
gentle tapping on the outside of the culture tube.
Hold the tube firmly between thumb and index
finger and tap near the bottom of the test tube
with your finger until the contents mix.
3. Remove the test tube cover and with a Pasteur
pipet, finger pipette approx. 0.1 ml of the broth
culture.
4. Transfer a drop of this suspension to the surface of
a slide.
Note: The drop must be of suitable size; if it is too
small, it will not fill the space between the
coverslip and the slide; if it is too large, some of
the drop will pass outside the coverslip, which
could smear the front lens of the microscope
objective. If such occurs, prepare a fresh wet
mount.
Discard the Pasteur pipet in the designated container.
Procedure for wet mount
1. Prepare clean microscope slides and clean coverslips by washing them in a mild
detergent solution, rinsing with distilled water, and then drying them with a
clean towel. Examine visually for clarity.
2. Suspend your broth culture of S. epidermidis by gentle tapping on the outside of
the culture tube. Hold the tube firmly between thumb and index finger and tap
near the bottom of the test tube with your finger until the contents mix.
3. Remove the test tube cover and with a Pasteur pipet, finger pipette approx. 0.1 ml of
the broth culture.
4. Transfer a drop of this suspension to the surface of a slide.
Note: The drop must be of suitable size; if it is too small, it will not fill the space
between the coverslip and the slide; if it is too large, some of the drop will pass
outside the coverslip, which could smear the front lens of the microscope
objective. If such occurs, prepare a fresh wet mount.
Discard the Pasteur pipet in the designated container.
Prepararea frotiului
bacterian pentru coloraii
uzuale
Prepararea frotiului
bacterian pentru coloraii
negative
Prepararea frotiului bacterian pentru
coloraii uzuale
Prepararea frotiului bacterian
pentru coloraii negative
Coloraia simpl
Morfologie bacterian
Endospori. Examen n contrast de faz
Capsula bacterian
(col. Tu India)
Capsula bacterian
(col. Cristal violet)