LP 1 - Examenul Microscopic Refacut

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EXAMENUL MICROSCOPIC

Transcript of LP 1 - Examenul Microscopic Refacut

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EXAMENUL MICROSCOPIC

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• 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

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•Lentilele microscopului optic sunt reprezentate de oculare și 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.

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Principiu

Treponema pallidumMicroscopie cu fond intunecat (500X)Escherichia coli

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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.

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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 choice—yeast—are 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.

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Procedure for wet mount1. 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.

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Procedure for wet mount1. 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 colorații uzuale Prepararea frotiului

bacterian pentru colorații negative

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Prepararea frotiului bacterian pentru colorații uzuale

Prepararea frotiului bacterian pentru colorații

negative

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Colorația simplă Morfologie bacteriană

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Endospori. Examen în contrast de fază

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Capsula bacteriană

(col. Tuş India)

Capsula bacteriană

(col. Cristal violet)

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