Lab Exercise 8: The Gram Stain
OB JE C TIV E S
1. Understand the chemical and theoretical basis for differential
staining procedures, including the Gram Stain.
2. Perform a successful Gram Stain to differentiate between grampositive and gram-negative bacteria.
INTR O DU C TI O N
Christian Gram was a Danish physician of the 1800s who was trying to
develop a staining procedure that would differentiate bacterial cells from
eukaryotic ones in a stained tissue sample. Although he did not succeed
in this endeavor, he did discover a monumental staining technique that
distinguishes between different bacterial cell types based on the cell wall
structure. In the G ra m stai n, the two kinds of cells, gra m- posi tive and
gra m- ne ga tive, can be identified by their respective colors, purple and
red/pink, after performing the staining method. Gram positive bacteria
appear purple based on the fact that they retain a crystal violet-iodine
complex through decolorization with alcohol or acetone. In contrast,
alcohol or acetone removes the crystal violet-iodine complex from gramnegative bacteria. These bacteria must then be counterstained with a red
dye, safranin, after the decolorization step to have any visible color (i.e.
red/pink) during microscopy.
The basis of this difference in staining is the structure of the cell wall.
Both gram-positive and gram-negative cell walls contain a layer of
pe pti do glyca n; however, the gram-positive cell contains a very thick
layer, whereas the gram-negative cell contains a thin layer surrounded by
lipid-filled o ute r me mbra ne. This lipid-membrane of the gram-negative
cell is removed during the decolorization step, and the crystal violet-iodine
complex can then escape. Your instructor will talk more about cell wall
structure in the lecture portion of the class.
The Gram stain is one of the most important stains to master in
microbiology. It will be crucial in identifying your unknowns organisms
and is used frequently in this lab manual. In addition, it is still used on a
daily basis in medical and clinical microbiology labs for identification of
microbial pathogens. You will find that it takes some practice to get
reliable results on a Gram stain, so take the time now to become an
expert!
LAB EX E RC I SES
Ta ble s upplies
24-hour old culture of
Staphylococcus aureus
24-hour old culture of Bacillus
megaterium
24-hour old culture of Pseudomonas
aeruginosa
Indivi dual suppli es
Gram staining kit (crystal violet,
iodine, alcohol decolorizer, and
safranin)
Staining tray
Microscope slides
24-hour old culture of Branhamella
catarrhalis
Water bottle
Bibulous paper
Inoculating loop
1. Prepare lab bench by removing extraneous items and cleaning surface
with table disinfectant.
2. Draw a circle on the bottom of 3 slides to indicate area of smears. Label
the slides gram-positive, gram-negative, and mix.
3. Chose S. aureus or B. megaterium for your gram-positive organism.
Smear and heat fix onto slide (see Lab Exercise 6 for refresher on
smearing).
4. Chose P. aeruginosa or B. catarrhalis for your gram-negative organism.
Smear and heat-fix onto slide.
5. Chose one gram-negative and one gram-positive organism for your
“mix” sample. Using sterile technique, place the gram-positive sample on
the slide. Sterilize your loop, and then aseptically place gram-negative
sample in the same place on the same slide. Mix together, air-dry, and
heat-fix.
6. Place slides on staining tray and proceed with Gram stain:
1.
2.
3.
4.
5.
Flood smear with c rys tal viole t for 30 seconds.
Wash with water to remove excess dye.
Flood smear with Gra m’s io di ne for 1 minute.
Wash with water to remove excess dye.
Decolorize with a lco hol. IMP OR TANT STEP: Hold slide at
an angle. Get one full dropper of alcohol decolorizer and,
starting above the smear, slowly and steadily let alcohol
wash down over the smear for approximately 5 seconds.
IMMEDIATELY wash with water to stop decolorization
process.
6. Co unte rs tai n by flooding smear with saf ra ni n for 1
minute.
7. Wash with water to remove excess dye.
8. Blot with bibulous paper.
7. Examine all slides under 100x oil immersion lens.
DA TA A ND O B SE RVA TI O NS
1. Draw observations of Gram stains of Gram positive, Gram negative,
and mixed cultures.
Gram positive
Gram negative
Species _________________
Species _________________
Total magnification ___________
Total magnification ________
Gram positive/ Gram negative mix:
Species _________________
Total magnification ___________
DI SC USSI O N
1. Describe the purposes of each of the following reagents in a differential
staining procedure and name the specific type used in the Gram-stain:
a. Primary stain:
b. Counterstain:
c. Decolorizing agent:
d. Mordant:
2. How do gram-positive and gram-negative bacteria differ in cellular
structure? How does this contribute to their differential staining
properties?
3. What is the most critical step in the Gram-stain procedure? Why? If
this procedure is not done correctly, how might it affect the results?
4. How does culture age affect the results of a Gram stain?