Materials 2 “Gram” Slides from Experiment 1

Crystal Violet

Deionized Water

Gram Iodine

Decolorizer

Safranin

*Sink or Disposable Plastic Container

*10% Bleach Solution

Labware 1/2 Bibulous Paper Sheet

Parafilm®

1 Pair of Disposable Gloves

*Lab Notebook (optional)

*Scissors

Note: You must provide the materials listed in *red.

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 deionized water for 30 seconds.

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

4. Rinse the slide with deionized 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 deionized 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 deionized 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 12 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 wa-

ter.

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

Figure 12: Gram-negative staining (left) and gram-positive staining (right) of two isolated bacteria cultures.

 

Data SheetExperiment 3 Data Sheet Table 3: Experiment 3 Staining Observations:

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