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Observing Bacteria under the Microscope
1. Spread a thin film from the culture on one half of a microscope slide, using the inoculating
loop or needle.
2. Let the film dry.
3. Flame-fix the film by passing the smeared end of the slide through a Bunsen flame several times.
4. Flood the slide with 1% Methylene Blue stain (several drops will do).
5. Let stand for several minutes, then dip the slide into a container of water to remove the
excess stain.
6. Blot dry. Do not rub, as this may remove the prepared culture from the slide. Add a drop
of immersion oil and examine under high power (100X).
This procedure gives a quick look at bacteria and will easily demonstrate bacteria shape.
Gram Staining Procedure for Bacteria
1. Flame-fix a bacterial smear as described above.
2. Add several drops of Gram's Crystal Violet stain.
3. Let stand for one minute, then rinse by dipping in water.
4. Add several drops of Gram's Iodine Solution to "set" the stain.
5. Let stand one minute, then rinse by dipping in water.
6. Tilt the slide, then add 95% alcohol as a destain. Add drop by drop, letting the alcohol flow over
the stained smear. Do this until there is no more color in the runoff. This step is to remove the color
from gram-negative bacteria. The gram-positive organisms will retain the purple color.
7. Counterstain with Gram's Safranin stain. Allow to stand for about 30 seconds, then rinse as before.
Blot away excess moisture and allow to dry.
8. Add a drop of immersion oil and examine under high power (100x).
The cell walls of gram-negative bacteria are chemically different. They have a higher lipid content,
which allows the alcohol destain to wash the stain out of the cell. The most accurate determinations
using the Gram technique are done with newer cultures, preferably less than 24 hours old, although
to view spores in Bacillus sp. or Clortridium sp. older cultures are necessary.
Bacteria
