Week of 2/21


Introduction

This week in lab, we performed a gram stain on bacteria D.Ficus. Being that it was my first week

in the lab, I needed to learn proper labeling and basic techniques used in this lab. The purpose

of gram staining is to verify the inoculated colonies have no contaminants. The gram stain

allows for differentiation between gram-positive and gram-negative cells. The first two dyes used

in a gram stain, violet and iodine, form the CV-I, or crystal violet complex. The crystal violet

complex cannot be easily rinsed from gram-positive bacteria because the thick peptidoglycan

wall traps it. Gram-negative bacteria, however, have a thin layer of peptidoglycan in their cell

wall, which cannot hold the dye. The final dye, safranin, is simply meant to stain the

gram-negative cells so they will still show up under the microscope despite having been rinsed

of the CV-I complex. Since D.Ficus is a gram-positive bacteria, the expected result is that all of

the cells on the slide will be violet, and none pink. Since most common bacterial contaminants in

cell cultures are gram-negative, a slide consisting of violet cells only would imply that there was

likely no contamination during the inoculation of the bacteria.

Procedure

The gram staining procedure is as follows:

1. On the opposite side from where you will be placing your sample, label your slides with

your initials and draw a circle where you want to place the sample.

2. Swipe your sample onto the slide using an inoculation loop and aseptic technique.

3. Heat fix the cells to the slide by waving the slide over a lit bunsen burner 3 times.

4. Stain the slide first with crystal violet, allowing the dye to sit for 60 seconds.

5. Rinse by running deionized water down the slide from above where the sample is for

about 10 seconds.

6. Stain the slide with iodine for 60 seconds to create the CV-I complex.

7. This time, rinse as before but with ethanol instead of deionized water.

8. Stain one last time with safranin for 60 seconds.

9. Rinse once more with deionized water.

Results

The gram stain of Deinococcus ficus produced a slide of only violet-dyed bacteria, all assumed

to be the D. ficus species. We concluded that, since there were no gram-negative bacteria,

there was no contamination and the inoculation of this bacteria was successful.

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