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Gram Stain Lab
Purpose:
By following the Gram stain procedure I will determine which type(s)
of bacteria are in the cultures grown in the science lab.
Background Information:
A. Differential stains are very useful in microbiology because they can
be used to distinguish between groups of bacteria. The Gram stain is
one of the most important steps in the characterization and
identification of bacteria.
The Gram stain separates bacteria into one of two groups:
1)
Gram-positive bacteria
These retain the color of the first stain used (Crystal Violet).
2)
Gram-negative bacteria
These assume the color of a second stain (Safranin).
Bacteria stain differently because of differences in the structure and
chemical composition of their cell walls.
B. The size and shape of bacteria are also used to identify them.
C. Three possible species I will find are:
1)
Escherichia coli (digest food in your intestines)
- straight rods, arranged singly or in pairs
- cells stain Gram-negative (pink)
2)
Staphylococcus epidermis (found normally on skin)
- spherical shape
- cells stain Gram-positive (blue-violet)
3)
Bacillus megaterium (found in soil)
- straight rods, arranged singly or in pairs
- cells stain Gram-positive (blue-violet)
Procedure:
Part I -- Culture Transfer:
A. Wash, rinse and dry a slide.
B. Pass the end of the inoculating loop through the flame of a Bunsen
burner until it is red hot.
C. Let it cool.
D. Put a drop of water on the slide.
E. Use the inoculating loop to pick up a small amount of the bacteria
culture from the petri dish.
F. Add the bacteria on the loop into the water drop on the slide,
mixing it thoroughly, and spreading it out into a large, thin layer.
G. Air dry the slide on the warming tray until the water is gone.
H. Holding the slide in a clothespin, heat fix the bacteria by passing
the bottom of the slide through the flame of the Bunsen burner for
5 seconds.
I. Let it cool. Leave the clothespin on it for the staining portion.
Part II – Staining:
Step 1
To be done over the sink!!
Crystal Violet
60 seconds
Cover the smear with an even
layer of stain. Keep it horizontal.
This is the primary stain that enters the
bacterial cells making them visible.
Step 2
Water Rinse
brief
Rinse slide with a gentle stream
of water. NO RUBBING!
Step 3
Gram’s Iodine
60 seconds
Cover the smear with an even
layer of stain. Keep it horizontal.
This stain forms a chemical complex with
the crystal violet that helps the dye attach
to the bacteria’s cell wall, membrane, and
cytoplasm.
Step 4
Water Rinse
thorough
Rinse slide for several seconds
with a gentle stream of water.
NO RUBBING!
Step 5
95% Ethanol
10 seconds
Cover the smear with an even
layer of stain. Keep it horizontal.
This draws out the dye from the bacteria
cells. Since the Gram negative bacteria
have a much thinner wall, the dye is
removed more rapidly from them.
Step 6
Water Rinse
thorough
Immediately rinse slide for
several seconds with a gentle
stream of water. NO RUBBING!
If the Ethanol remains for more than
10 seconds, it will remove all of the dye
from each type of bacteria. If done
correctly, the Gram positive cells will
still be dark blue, while the Gram negative
cells will be clear and difficult to see.
Step 7
Safranin
60 seconds
Cover the smear with an even
layer of stain. Keep it horizontal.
This stain acts as a counterstain to color
the clear, Gram negative bacteria red.
Step 8
Water Rinse
thorough
Immediately rinse slide for
several seconds with a gentle
stream of water. NO RUBBING!
Step 9
Dry The Slide
carefully
Carefully blot the slide dry with
some paper towel. NO RUBBING!
Step 10
Remove the clothespin for the slide and place a cover slip over
the area that you plan to view through the microscope.
Step 11
When finished with the lab, wash the slide and cover slip
carefully in the tub of soapy water provided.
Dry, and put them away.
Observations:
1.
I took bacteria from dish __________.
Get the smear in focus using the 4x objective lens. Then switch to the
10x objective lens and refocus. Now, draw and label what you observe.
Bacteria @ 100x
2.
Switch to the 40X objective lens. Draw and label what you observe.
The color and shape of the bacteria should be exact in your
drawings!
Conclusion:
Bacteria @ 400X
Explain how it went, what you observed, what you learned. Be sure to
include what type of bacteria you had, and how you identified them.