3.

0 MATERIALS
 Cultures (from previous experiment)
 Inoculating loops
 Slides
 Gram staining reagents in dropping bottles (crystal violet, safranin, malachite green)

4.0 PROCEDURE
a. Slide preparation for living cultures/ pond water specimens
1. A small drop of specimen was placed on the centre of the slide.
2. A clean cover slip was placed with carefully over the drop, avoid bubbles.
3. The preparation was sealed with clear nail polish if no fluid was escaped from under
the edges of the cover slip. The polish was allowed to dry thoroughly before
examined preparation under the microscope.
b. Slide observation
1. The microscope was placed on the bench squarely in front of you.
2. The specimen slide in the clips was placed on the mechanical stage. The slide was
moved to center the specimen over the opening in the stage directly over the light
source.
3. The microscope stage up was raised as far as it will go. The low-power lens was
rotated into position.
4. The eyepieces was adjusted to your own personal measurements.
5. The substage condenser was adjusted to achieve optimal focus.
6. The light source was adjusted routinely by means of the light source and/or
iris diaphragm for optimum illumination for each new slide and for each change in
magnification.
7. After the observation was completed under low power, the observation was continued
with high power lens, a drop of immersion oil was placed in the center of slide if
necessary ( for oil immersion lens only).
8. The observation was recorded and the magnifications was noted.
9. This procedure was repeated with all available specimens.
10. All lenses with lens paper was cleaned and the microscope was placed back to its
cabinet on completion of the laboratory exercises.
c. Simple stain
1. The smears with crystal violet was covered 10 seconds.
2. The smears with crystal violet was rinsed with water.
3. The slide was completely dried before put it on microscope.
4. The observation was observed with low power (10X) to locate a good field. A drop of
oil was added and the oil immersion lens was swung into the oil. The fine focus was
only used to bring the image into clear focus.

d. Gram Stain
1. The smears with Crystal Violet was stained 1 minute.
2. The smears with Crystal Violet was rinsed with water.
3. The smears with Gram’s Iodine was stained 1 minute.
4. The slide was rinsed carefully with acetone.
5. The slide was rinsed with water.
6. The slide was countered-stained with safranin 10 seconds.
7. The slide was rinsed with water and air dry. The slide was completely dried before
put it on microscope.
8. The power lens (10X) was used for find a good field. A drop of oil was added. Then,
the 100X oil immersion lens was submerged into the oil by rotating the nosepiece.
The fine adjustment was only used to bring the image into clear focus.
e. Endospore stain
1. A smear of culture given was heated, a clean glass slide was took and prepared.
2. The heat-fixed slide was placed on top of a small can of boiling water, which will
serve as a steam source, and the smear was flooded with Malachite green.
3. Malachite green was added as necessary. The slide was kept from drying out and was
steamed for 5 minutes
4. The excess stain was rinsed from slide with a gentle stream of water from faucet. A
clothespin was used to hold the slide it will be hot.
5. The decolorized slide was placed on the staining rack in the small sink and flood with
safranin.
6. The excess water was shaked off and the slide was blotted dried with bibulous paper.
7. The slide was examined under oil immersion and the results was recorded. The spore
should appear green within and around red vegetative cells.