Experiment 3 Gram Staining

Experiment Inventory

Materials Labware
2 “Gram” Slides from Experiment 1 1/2 Bibulous Paper Sheet

Crystal Violet Parafilm®

Gram Iodine Disposable Gloves

Decolorizer *Lab Notebook (optional)

Safranin *Scissors

*Distilled Water
Note: You must provide the materials listed in *red.
*Sink or Disposable Plastic Container

*Bleach

EXPERIMENT 3: GRAM STAINING

The Gram staining technique was invented by Dr. Hans Christian Gram (1853 – 1938), a scientist who worked in microbiology
labs with Robert Koch. Gram staining was a major breakthrough in the identification and classification of bacteria; it distinguishes
microbes broadly into Gram-positive and Gram-negative groups. These two groups can cause very different and important health
consequences. Gram-positive bacteria have thick cell walls made up of many (up to 40) layers of peptidoglycan. This thick cell wall
prevents microbes from losing the purple stain (upon destaining). Gram-negative bacteria have relatively thin cell walls with only a
few layers of peptidoglycan and an outer cell membrane. Gram-negative bacteria are visualized with a counterstain that creates a
reddish-pink color on the bacteria.

PROCEDURE

Note: If you are keeping a lab notebook, record the date, time, and experiment title on a fresh page before
you begin.

1. Over a sink or disposable plastic container, place several drops of crystal violet onto the smear on one of the “Gram” slides
so that the sample is completely covered. Incubate the sample in the dye for 1 minute.

2. Rinse the slide with distilled water for 30 seconds.

3. Saturate the smear with the Gram iodine, and wait 1 minute.

4. Rinse the slide with distilled water for 30 seconds.

5. Cover the smear with the decolorizer for 5 seconds. Do not let the decolorizer remain on the smear for more than 5 seconds
as this will completely destain even a Gram-positive organism.

6. Rinse the slide with distilled water, and wait 30 seconds.

7. Place several drops of the safranin stain onto the slide so that the smear is completely covered, and wait 1 minute.

8. Rinse the slide with distilled water for 30 seconds.

9. Use the bibulous paper to blot the excess water from the slide. Take caution not to disturb the sample.
 10. Repeat steps 1 – 9 with the remaining “Gram” slide.

11. If you have a microscope available, observe the stained slides under increasing magnification, and record what you see at
each magnification in Table 3, focusing on morphology and arrangement of the cells using terms learned in this lab. If no
microscope is available, refer to Figure 13 and take a photograph of your slide if required by your instructor. If keeping a lab
notebook, print out Table 3 and tape it into your lab notebook or re-create it by hand.

12. Place your slides in a disposable plastic container, and pour bleach over the surface until the sample is completely covered/
saturated. Allow the bleach to soak for approximately 20 minutes, and then rinse the bleach down the sink with running
water.

13. Wrap the slides in Parafilm® and dispose of them in the trash.

Figure 13: Gram-negative staining (left) and gram-positive staining (right) of two isolated bacteria cultures.
Data Sheet Experiment 3 Data Sheet

Table 3: Experiment 3 Staining Observations

Stain Used:

Observations: