Lab 2: Gram Stain

Background:
Bacterial cells are translucent so staining is required to make them more visible
under the microscope. There are many techniques which will visualize bacterial cells
under a microscope. However, a Gram stain is an example of a differential stain which
can differentiate between two types of bacteria as well as visualize the bacteria. The
Gram stain is able to differentiate cells into two classes (Gram positive or Gram negative)
based on the structure of their cell walls. Two dyes are used in a Gram stain, a primary
dye (Crystal Violet) and a counter stain (Safranin). The primary dye is basic; therefore
it is positively charge and interacts with the negatively charged cell wall. After the
primary stain is applied a mordant (Gram’s Iodine) is added. A mordant facilitates
further interactions between the primary dye and the cell wall. The mordant used in a
Gram stain crystallizes the dye that has penetrated the microbe.
Gram positive and Gram negative bacteria are able to be differentiated due to
differences in their peptidoglycan layer. Gram positive cells have a larger and more
highly cross-linked peptidoglycan which contains smaller pores. The smaller pores
enable Gram positive cells to retain the primary dye (Crystal Violet, a purple dye) when
the samples are washed with alcohol. Gram negative cells do not possess the thick
peptidoglycan layer and therefore have larger pores. Hence, they cannot retain the
primary dye and become colorless after being washed with alcohol. Gram negative
bacteria are re-colorized using a counter stain (Safranin, a red dye). Due to the cell wall
properties of each type of cell Gram positive bacteria will be purple and Gram negative
bacteria will be red/pink after performing the Gram stain. Gram staining is usually the
first and most important step in identifying bacteria; therefore, you will be expected to
memorize the procedure.

Figure 2.1

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Guidelines:
• The dyes you will be working with are very potent and will stain your
clothes as well as your skin. Be careful with the dye and wear latex gloves
if you don’t want dye getting on your skin
• Do not overheat the slide when heat-fixing. Excessive heat can destroy the
cells and make it impossible to identify them.
• Do not over decolorize with the alcohol. If you leave the ethanol on for too
long the Gram positive cells may decolorize causing misidentification of
the bacteria.
• The time lengths given for dyeing and decolorizing are approximate and
may need to be varied depending on your results.
• After each use of the oil immersion lens use the lens paper to wipe the oil
off of the lens.

Materials:

• Microscope Slide
• Samples of the bacteria to be Gram stained:
o Bacillus subtilis
o Staphylococcus epidermidis
o Escherichia coli
• Crystal Violet
• Gram’s Iodine
• Ethanol
• Safranin
• Wash Bottle filled with water
• Mineral Oil (for oil immersion lens)
• Bibulous Paper
• Lens Paper

Procedure:

• Prepare a Gram stain

1) Using an inoculating loop prepare smears of the bacteria by spreading a thin
suspension of each bacteria on a glass slide
2) Heat-fix the smears by passing them through the Bunsen burner flame a few
times.
3) Place the slides on the staining rack.
4) Cover the smears with the primary dye, crystal violet, and let it stand for 30
seconds.
5) Rinse the slides with water for 5 seconds.
6) Apply the mordant, Gram’s Iodine, by covering the smears and let it stand
for 1 minute.

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 7) Rinse the slides with water for 5 seconds.
8) Decolorize with ethanol for 15-30 seconds. Add the ethanol slowly until
the crystal violet no longer floats up from the slide. Be careful not to
decolorize for too long.
9) Rinse the slides with water for 5 seconds.
10) Cover the smears with the counter stain, safranin, and let stand for 1
minute.
11) Rinse the slides with water for 5 seconds.
12) Blot dry using the bibulous paper.
13) Place slide on the stage of the microscope.
14) View your specimen under the microscope under each of the objective
lenses (4x, 10x, 40x, and 100x). NOTE: Before using the oil immersion
lens oil must be applied to the slide:
 Move the 40x objective lens out of position so that no objective
lenses are over the specimen.
 Place a drop of oil directly on the slide where you were viewing the
specimen.
 Move the 100x oil immersion objective lens into position.
 Use the fine adjustment knob to bring your specimen into focus.
15) Record what you see under the oil immersion lens for each species of
bacteria in your notebook. Be sure to clean the oil off of the lens after every
use with lens paper.

Critical Thinking:

1) Suppose you Gram stained a sample from a pure culture and observed a field of red
and purple cocci. Adjacent cells were not always the same color. What do you
conclude?

2) Suppose you are viewing a Gram stained field of red rods and purple cocci through
the microscope. What do you conclude?

3) Since you can't identify bacteria from a Gram stain, why might a physician perform a
Gram stain on a sample before prescribing an antibiotic?

4) If you Gram stained human cells, what would happen?