Amanda Ratliff
Lab Report #1
3-5-12
Gram Staining Lab Report
Introduction:
To better understand and recognize the difference between gram positive
and gram negative cells we will be using a gram staining technique to stain slides of
B. Cereus, S.epidermidis, E.coli, and a mixture of E. coli and S. epidermidis. Due to
previous gram staining experiments done on these organisms by a number of
scientist with similar results, My hypotheses is that B. cereus, and S. epidermidis will
stain a crystal violet color proving it has a Gram positive cell wall structure and that
E. Coli will be stained with the safranin pink color due to it’s gram negative cell wall
structure.
Materials and Methods:
Materials that will need to be obtained for this experiment include:
Gloves and lab coat
Test tube rack contain the 3 organisms
Organisms: E. coli, B. cereus, and S. epidermidis
4 clean slides labeled appropriately
Crystal violet stain, Iodine, Alcohol, safranin, and water
Loop
Bunsen burner and striker
Slide holder
Bibulous paper
Microscope and immersion oil
Using sterile technique make general smears of a slide containing E. coli, one
slide containing B. cereus, one slide containing S. epidermidis and one slides with a
mixture or E. coli and S. epidermidis. Let all four slides completely dry and then heat
fix by holding the slide with a slide holder and running it over the Bunsen burner
flame approximately 3 times and let cool.
Step 1: Once the slide has cooled, apply crystal violet dye for approximately
30 seconds then rinse with H20.
Step 2: Apply the iodine mordant for one minute and rinse with H20 again.
Step 3: decolorize with alcohol wash for no more than 15 seconds (or until
light purple pools at the edge of the slide.
Sept 4: Counterstain with safranin for 45 seconds to 1.5 minutes then rinse
with H20.
Step 5: Blot dry with bibulous paper
Sept 6: Examine with microscope including oil immersion.
Step 7: Repeat for all slides
Results:
After examining each slide under the microscope it was the apparent that the
staining was successful, E. coli and B. cereus were both a deep purple color from the
crystal violet stain, and S. epidermidis was a deep pink color from the counter stain
safranin. And the slide with both E. coli and S. epidermidis had both pink and purple
cells.
Discussion:
After preforming gram-staining technique on the provided organisms, it was
found that this experiment matched the results of previous gram staining results.
When observing E. coli under the microscope it was apparent that the deep pink tint
it obtained meant it was a gram negative cell, therefore having a thin layer of
peptidoglycan were the crystal violet was easily decolorized out of the cell by the
alcohol and then stained by the counterstain, safranin. Resulting in a pink stained
cell. When observing B. cereus it as apparent that the deep purple stain meant it was
a gram negative cell, containing a thick peptidoglycan layer which the increased
affinity to the stain by the mordant restricted the affect of the alcohol decolorizer.
Explaining the purple stained cell. And when observing the mixture slide of E. Coli
and S. epidermidis it was apparent that mixtures of gram positive and gramnegative cells were present because the slide contained some purple cells and some
pink cells. Since it was already determined that E. coli was the gram negative cell we
can then come to the conclusion that S. epidermidis is the gram positive cell. This
was confirmed when observing the slide with only S. epidermidis on it, the slide
contained only purple stained cells meaning it was also a gram positive cell
structure having a thick peptidoglycan layer.
Extensions:
To further continue the experiment, it would be interesting to determine
major sources of theses specific organisms. To know what illnesses or disease
contain a gram positive or gram negative cells.
References:
Gram-staining procedure (Figure 3.10a)
Copyright The Benjamin/Cummings Publishing Company, Inc., from Tortora, Funke,
Case, Microbiology: An introduction