How to Gram Stain Bacterial Cells

Alyson Frederick

Nicole Lambert
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Introduction

The gram stain is vital to bacteriology and the identification of bacterial

cell species. This stain is used to differentiate bacteria and divide cells into two
groups: gram positive and gram negative. These two groups vary in their cell
wall structure, which affects how well the cells retain the dye used for staining.
Determining which group a bacterial species belongs to is crucial in diagnosing
a disease and prescribing effective antibiotics.

Warning: Throughout this procedure, basic laboratory safety measures should be

followed:

 Tie back long hair

 Do not wear loose-fitted clothing
 Sanitize lab benches before and after completion
 Wash hands before leaving the lab
 Avoid getting any dye on clothing; Dye will permanently stain fabrics

Materials

The staining procedure requires many materials which should be

prepared before continuing to the actual procedure. All materials should be
accessible throughout the staining process and while making observations.

 Distilled water- in a small squirt bottle.

 Bacterial culture- examples include yogurt samples, pure cultures, or swabs.
 Inoculating loop - used to transfer bacterial cells.
 Bunsen burner- attached to a gas line
 Striker- used to light the Bunsen burner
 Glass slides
 Staining box- slides are placed on the staining
box during application of dyes
 Pipettes- used to flood slides with dyes or rinse
with distilled water
 Crystal violet- purple dye used first; stains all
cells

Figure 1: Inoculating loop

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 Iodine- enhances the crystal violet dye

 Alcohol- decolorizes gram negative cells
 Safranin- stains gram negative cells red
 Bibulous paper- used to dry off glass slides
 Paper towels- used for disinfecting lab benches
 Microscope- used to view cells after staining
 Kim-wipes- used to clean the microscope lens
 Immersion Oil- use only for the highest magnification lens of the microscope
 Disinfectant- bleach or 70% ethyl alcohol

Preparing for the Procedure

1. Assemble all of the materials needed for the procedure (see Materials
section above).

2. Sanitize the table/lab bench using disinfectant and a paper towel. Allow
the surface to air dry.

Staining Procedure
1. Turn on the gas to the Bunsen burner and use the striker to ignite the
flame. This will be used later, so set it aside (still burning) for now.

2. Put a drop of distilled water onto a glass slide using the squirt bottle.

Note: Do not add more than a drop because it must be able to air dry later.

3. Return to the flaming Bunsen burner. Take

the inoculating loop and briefly hold it in
the flame as shown in Figure 2 so that the
entire wire has, at some point, glowed red.
This should take approximately 3-10
seconds. Your loop is now sterilized and
ready to transfer bacterial cells.

Figure 2: How to
properly sterilize a loop
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4. Allow the loop to cool. Dip the loop into the

culture and transfer the cells to the drop of
water on the glass slide. Using the loop, mix
the cells into the drop of water and spread the
mixture across the middle third of the slide
(see Figure 3).

Note: Spreading this mixture will produce a thin

layer of cells that can more easily be stained
and observed. It will also facilitate drying of
the slide.

Figure 3: Transferring
cells to the glass slide.

5. Allow the slide to air dry. This may take up to several minutes. After the
slide is completely dry, quickly pass the slide over the top of the flame
three times. This makes the cells adhere to the slide.

6. Place the slide on top of the wire screen covering the staining box. Using
a pipette, flood the slide with the crystal violet stain so that the entire
cell layer is saturated with dye. Allow the flooded slide to sit for 30
seconds.

7. Rinse the slide with distilled water until all of the dye has been removed
and the water running off the slide appears colorless. Gently blot the
slide dry with Bibulous paper.

Note: Make sure to gently blot it so that the cell layer is not removed from the
slide.

8. Place the slide on the staining box again and saturate the slide with
iodine. Allow the slide to sit for 30 seconds.

9. Once again, rinse the slide off with distilled water and gently blot it dry.
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10. Next, hold the slide at a 45⁰ angle above the staining box and slowly drip
a steady stream of alcohol over the slide. Watch the color of the alcohol
running off of the slide. When it becomes colorless, stop rinsing the slide
with alcohol.

Note: Do not rinse the slide for more than 15-20 seconds. Decolorizing for
longer amounts of time may yield poor results.

11. Immediately rinse the slide with distilled water to remove the alcohol
and gently blot it dry.

12. Place the slide on the staining box again and saturate the slide with
safranin. Allow the slide to sit for 60 seconds.

13. Rinse the slide with distilled water and gently blot it dry.

14. Allow the slide to completely dry.

Observing the Cells

Study Figure 4 and familiarize yourself with the different parts of the
microscope before continuing.

Figure 4: The different parts of the microscope.

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1. Place the slide on the stage of the microscope.

2. Rotate the nosepiece so that the lowest magnification objective lens

(labeled 10X) is above the slide. Use the coarse adjustment knob to raise
the slide as close to the lens as possible. While looking into eyepiece, use
the knob to slowly lower the stage until the cells on the slide come into
focus.

Note: At this magnification, you will not be able to see individual cells. Instead,
it will appear as a colored, indistinct mass.

3. Next, rotate the nosepiece so that the next highest magnification objective
(labeled 45X) is placed over the slide. While looking into the eyepiece,
use the fine adjustment knob to focus the cells. You may also need to
use the coarse adjustment knob to bring the specimen into focus.

Caution: Slowly adjust the coarse adjustment knob when focusing to avoid
hitting the slide with the objective lens.

4. Once it is in focus, partially rotate the nosepiece to position the slide

between the 45X objective and the highest magnification objective (labeled
100X).

5. Apply a drop of immersion oil to the middle of the slide and rotate the
nosepiece to position the 100X objective over the slide.

Note: The objective lens should come in contact with the oil.

6. Use the fine adjustment knob to focus the cells on the slide.

7. Observe the results.

Interpreting the Results

At the end of the staining and observation procedures, you will be able to see
the stained cells. This allows you to determine whether the bacteria are gram
positive or gram negative. Figure 5 shows what the cells would look like at each
step of the staining process based on whether they were gram positive or gram
negative. Figure 6 shows examples of gram positive and gram negative bacteria.
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Using the highest magnification lens allows you to easily view the cells and
observe their color, shape, and arrangement. However, only the color of the
cells is important for determining the result of the stain. Gram positive cells
will have a purple color while gram negative cells have a red or pink color.

Note: Insufficient decolorization may result in false positives: gram negative

cells that are purple (appear gram positive). Likewise, excessive decolorizing
may result in false negatives: gram positive cells that are red (appear gram
negative).

Figure 5: Gram positive and Gram negative bacterial

cells at each stage of the staining process.
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Figure 6: Gram positive (left) and Gram negative (right) bacterial cells.

You have successfully completed a gram stain! For further information or for
any questions regarding this process, refer to a microbiology lab manual or
textbook.

Cleaning Up
1. Clean the oil off of the microscope lens using a Kim-Wipe.

2. Disinfect the table/lab bench with the disinfectant and a paper towel.

3. Dispose the glass slide into a sharps container (found in every

microbiology lab).

4. Place any bacterial cultures and contaminated materials in the

biohazardous waste container.

Enjoy your microbiological experience!

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Works Cited

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