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Experiment No.

1:
Aim: Preparation of a slide of given plant or bacterial sample and visualize it under under bright field, dark
field and phase contrast microscope.
Requirements: Bacterial culture, slides, crystal violet dye, iodine, ethanol, saffranin, microscope, distilled
water, inoculating loop.
Theory:-
Bright field Microscopy: is used to detect the specimen that possess colour or have other properties that
affects the amount of light that passes through it. Many biological samples lack these characteristics and thus
must be stained with dyes to observe under bright field. Sample illumination is through transmitted (i.e.,
illuminated from below and observed from above) white light and contrast in the sample is caused by
absorbance of some of the transmitted light in dense areas of the sample.
Dark field microscopy:- is used for observing live and unstained biological samples such as smear from a
tissue culture or individual water borne single celled organisms. In dark field microscope the condenser is
designed to form a hollow cone of light. The objective lens sits in the dark hollow of this cone although the
light travels around and past the objective lens, no rays enter it. When a sample is placed on the stage, the
light at the apex of the cone strikes it. The image is made only by those rays scattered by the sample and
captured in the objective lens. The image appears bright against dark background.
Phase contrast microscopy : used for observing cells that are intact and still living for example for viewing
unstained cells growing in tissue culture, bacteria, cellular organelles and other small entities. This
microscopy improves contrast without sectioning and staining by exploiting differences in the thickness and
refractive index of various regions of the specimen being examined. The phase change produced in the light
coming out of various regions of specimen is amplified by using phase plate. Based on the pattern of
diffracted and undiffracted light an image is formed with highly contrasting bright and dark areas against an
evenly illuminated background. The phase-contrast microscope was designed to exaggerate contrast without
sacrificing resolving power.
Procedure:
Gram staining of bacterial samples
1. Clean the slides properly. Put a drop of distilled water on the slide and properly mix the bacterial culture.

2. Heat fix this smear for 2-3 minutes and air dry the sample.

3. Add a drop of crystal violet to the smear and keep for 60 s.

4. Wash it with water, add iodine and keep for 30 s.

5. Wash the slide with ethanol, add saffranin and keep for 60 s.

6. Wash off with running water and air dry.

7. Observe it under bright microscope.

Observations:
1. Observe the stained slide under bright field microscope
2. Observe the unstained live bacterial cells under dark field and phase contrast microscope.

3. Observe the difference in images under all three microscopic techniques, and took the photograph.

Precautions:
Hold a microscope firmly by the stand, only. Never grab it by the eyepiece holder, for example.

Since bulbs are expensive, and have a limited life, turn the illuminator off when you are done.

Always make sure the stage and lenses are clean before putting away the microscope.

NEVER use anything but good quality lens tissue on any optical surface, with appropriate lens cleaner or
distilled water; oganic solvents may separate or damage the lens elements or coatings.

For dark field microscope, use a clean slide to avoid visualizing dust particles.

Focus smoothly; don't try to speed through the focusing process or force anything.