Chloe Evetts (8990-4674)
May 21, 2013
Gram Stain, Bacterial Shapes and Arrangement
The purpose of this experiment is to identify Gram negative and Gram positive
specimens on a slide. Because the two types of bacteria have different characteristics
to their cell walls, the stains react differently with the specimens. Gram positive bacteria
has a membrane surrounded by a thick cell wall of peptidoglycan. Gram negative
bacteria has an inner membrane covered by a thin layer of peptidoglycan and then
another outer membrane. The difference in thickness of the peptidoglycan layers
contributes the different results of the two types of bacteria after staining. Gram positive
bacteria hold the original purple stain because of its thick cell wall. Gram negative
bacteria, however, will shed this stain once the alcohol is applied and then will easily
take up the new pink stain. When viewing our resulting slide, gram positive will appear
purple, and gram negative bacteria will appear pink. This procedure is very important to
the medical community and to our society because bacteria are sensitive to certain
types of antibiotics, based on if they are gram positive or negative. It is helpful to know
what type of bacteria is present before prescribing antibiotics.
Gram staining is an effective method for identifying bacteria because the different types
of bacteria (gram negative, and gram positive) respond differently to staining. By the
end of this experiment, we should be able to successfully identify the gram negative and
gram positive bacteria within the sample.
1. Light Bunsen burner to create an aseptic environment, and flame inoculating loop
to sterilize it. Cool loop.
2. Gram stain can be performed from liquid culture, from cells grown on an agar
plate, or from an environmental sample like your cheek swab. Select sample to choose
from. Follow below procedures to successfully obtain a sample.
o Cells from liquid culture: Vortex culture. Use your sterilized loop or a pipette and
add a drop of culture (10-20 μl) to your slide. Spread around and make a thin layer of
bacteria cells on a clean glass slide from a fresh liquid culture
o Cells from agar plate/colony: add a drop of water (10-20 μl) with a pipette or
sterilized loop to the glass slide. Use sterilized loop and touch lightly the surface of a
single isolated colony on an agar plate and mix thoroughly with the drop of water.
o Cheek swab: to examine the cells and bacteria inside of your cheek or plaque from
your teeth use a pipette tip and rub it on the inside of your cheek or along the gum line of your teeth. Mix the content with a drop of water on your slide
3. Allow the bacterial suspension to air dry completely.
4. Hold slide with cloth pin and use heat fixation to heat-fix the bacteria to the slide.
Do so by gliding the slide through the Bunsen burner. Place slide on staining rack
following the heat fixation.
5. Gently flood the smears with crystal violet (primary stain) and let stand for 1
minute. Tilt slide and let crystal violet run off.
6. Gently flood the smears with Gram’s iodine (mordant) and let stand for 1 minute.
7. Hold the slide with the clothespin, tilted 90 and add decolorizer drop by drop to the
slides until the decolorizer runs clear and no color comes of the slide (about 10-30 sec).
8. Rinse the smears in a gentle and indirect stream of tap water for 2 seconds.
9. Gently flood the smears with safranin (counterstain) and let stand for 30 to 60
10. Rinse the smears in a gentle and indirect stream of tap water for 2 seconds.
11. Air dry and blot (DO NOT RUB) the smear dry with bibulous paper.
12. Examine dry slide under 100x oil immersion without a cover slip.
13. Gram-positive bacteria appear blue or violet and gram-negative bacteria appear
The results of this gram stain were clusters of cocci gram positive bacteria.
By effectively completing the gram stain procedure, I was able to identify whether the
bacteria present in my unknown was either gram positive or gram negative. The
bacteria present showed up purple (due to the crystal violet stain). This means that it is
gram positive. The morphology is cocci, and