Exercise 15 Lab Activity Guide

Title and Date: Exercise #15- Gram Stain Technique 02/24/2022

Purpose:
The purpose of this exercise is to differentiate between gram-positive and gram-negative
bacteria and to determine cell size, shape, and arrangement
Methods:
Follow Protocol on pages 92-95
1. Prepare air-dried, heat fixed smears if selected bacterial organisms on the clean slides,
as outlined in Exercise 11
2. Flood the Smears with crystal violet (the primary stain) and allow the stain to remain for
1 minute as indicated in 15.1A
a. Rinse with water
b. Do not blot the slide dry; simply gently shake off excess water
3. Flood the smears with Gram’s iodine solution for 1 minute (15.1C). Rinse with wter, but
do not blot
4. Decolorize the smears with 95% ethyl alcohol as follows:
a. Pour a few drops of alcohol on the smears, and rock the slide back and orth for
15 seconds
b. Allow the alcohol to drip off, then repeat this discoloration procedure for another
15-second period. Continue until runoff is no longer purple, but this may be
difficult doing it the first time
5. Rinse the slide with a gentle stream of water but do not blot. Excessive decolorization
may cause gram-positive bacteria to lose their color (appear gram negative) and
insufficient decolorization may allow gram-negative bacteria to retain a purple color
(appear gram positive). At this point, gram positive bacteria retain the crystal violet
iodine complex while gram negative bacteria lose the complex and would appear
transparent if observed with the microscope
6. Flood the smears with safranin (the counterstain), and allow the stain to remain for 30
seconds
a. Rinse gently with water, andblot the slide using the bibulous paper
b. Safranin, a red dye, will stain bacteria that lost the crystal violet-iodine complex
during decolorization. It will have no effect on the color of the gram-positive
bacteria
7. Without using a coverslip, examine the smears under the low-power (10X) objective lens
on your microscope to orient yourself, then move to the high power (40x) lens, and
finally the oil immersion (100x) lens.
8. Scan the slide thoroughly to locate areas where the bacteria are widely separated
a. Gram positive bacteria will appear blue to purple, while gram negative bacteria
will be orange to red. If both are present, determine most prominent color
b. Be sure to confine your work to areas containing thin smears of bacteria, since
thicker areas may resist decolorization due to the heavy concentration of bacteria
c. Prepare representations of the organism in the Results section. Include the
complete binomial name of the organism, The Gram reaction, and the
magnification
 d. If the slide will be retained, remove the oil by gently blotting the slide. Otherwise,
clean the slide with soap and water and dry with paper towels
e. Label the slide with your name, organism’s name, the date, and the designation
Gram stain
Results:

All 3 Slides (Ascending-Descending with the

corresponding species to the right) pictured
after the gram staining process

E. coli - Oil Immersion Lens

- E. coli is gram-negative and that is
very clear by the overall red color of
the stain under the oil immersion lens
- The E. coli appears to me to be
bacillus in shape because the cells
are rod shaped
- The cell arrangement appears to be in
one big cluster in the middle
- Edges of the stain show some diplo
(pairs) and strepto (chains)
arrangement as well
 B. megaterium - Oil Immersion Lens
- B. megaterium is gram-positive due to
the purple color of the stain
- Cells are arranged in one big cluster
with some smaller chains
- Shape appears to me to be Spirillum,
but it is difficult to tell

M. luteus - Oil Immersion Lens

- M. luteus is gram-positive by the
purple color of the stain
- Cells are definitely in one big cluster in
this image
- Shapes appear to be bacillus, but
again it is difficult to tell from this
image

Conclusions: In this experiment, I successfully performed three gram stains of three different
bacterias from bacterial smear to microscopic viewing under oil immersion. E. coli was the only
bacteria from exercise 15 that appeared gram-negative, indicated by its pink/red color. The
other two species proved gram-negative when seen by their purple colors. Size is still difficult for
me to determine through a microscope, but I was able to successfully indicate perceived
arrangement and shape of all three bacteria that were viewed.

Next Step(s): Next steps are to examine further into recognizing what it means when stains
appear gram-negative versus gram-positive, and to continue this process further to determine
more features of different experimental bacteria in the lab.