Soil Microbial Ecology - Soil Molecular Ecology Laboratory

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METHOD
1. Place a small drop of clear water on a clean microscope slide using the
inoculating loop.
2. Flame the inoculating loop, let it cool, and transfer a small portion of a bacterial
colony to the drop of water.
3. Using a circular motion, mix and spread the resulting cell suspension to cover an
area about the size of a dime. Flame the loop again immediately.
4. Allow this smear to air dry and then heat fixes it by passing it briefly through the
flame of the burner several times. Add a few drops of methylene blue to cover the
smear. Use sparingly to avoid making a mess with spillage.
5. After the dye has been on the smear for 4 min, gently rinse it in a stream of water
in the sink.
6. Blot the slide dry using a pad of bibulous paper. Examine the stained smear at the
different magnifications on the microscope. Be sure to use the oil immersion
objective to view the preparation.
PREPARATION OF WET MOUNTS:
It is not always desirable to view only stained preparations of bacteria since no
information is gained as to whether or not the organisms are motile i.e. if they have
flagella (the organelle of locomotion in bacteria: singular = flagellum). To determine
whether an organism is motile, it is usually observed in a wet mount.
METHOD
Place a small drop of water on a microscope slide using a flamed inoculating loop.
Flame the loop, let it cool and add some bacterial from a culture to the drop of water.
Mix but do not spread the drop out. Alternatively, if you have a broth culture, simply
place several loop-full onto the center of the slide to form a drop.
After you have placed the cell suspension on the slide, simply place a cover slip over the
drop to obtain as few air bubbles as possible. This is most easily done by bringing one
edge of the cover slip into contact with the edge of the drop and then laying the cover slip
down on an angle so the fluid flows evenly across the slide.
GRAM STAIN FOR BACTERIA
There are basically two types of bacteria, gram + (purple color) and gram - (no purple
color) when stained with Crystal Violet and Iodine. This is mainly due to the chemical