Gram Staining Procedure

Gram staining is the most essential and all around used staining technique in bacteriology lab. Gram staining was firstly presented by Christian Gram in 1883. This method is utilized to distinguish between gram positive and gram negative bacteria which have consistent differences in their cell wall. Gram positive bacteria stain blue-purple and gram negative bacteria stain pink-red. There are a few objectives as to why we ought to do this experiment; to gain knowledge of the differences between gram positive and gram negative bacteria, develop the lab skill, and be more acquainted with the gram staining procedure. There are also several different materials that we use in this experiment such as absolute methanol, Gram Crystal Violet, Gram Decolorizer,

Be more acquainted with gram staining procedure

4. To understand how the gram stain reaction influences gram positive and gram negative bacteria based on the biochemical and structural differences of their cell walls

Material:

1. Gram stain reagents

2. Gram Crystal Violet

3. Gram Iodine

4. Gram Safranin

5. Microscope slides

6. Gram Decolorizer

7. Absolute methanol

Procedure:

1. Cultures were taken from the mouth around your tooth or inside your nose of a person in each group

2. We then rolled the swab across the slide, thin and evenly

3. The smear was air dried

4. The smear was then flooded with absolute methanol and then drying before we stained it

5. The smear was placed with crystal violet

6. The crystal violet from the slide with tap water and drain off excess water were washed after staining fro 1 minute

7. Each smear was flooded with iodine

8. Rinse the iodine from the slide with tap water

9. Rinse the smear with gram decolorizer until the solution rinses colorlessly from the slide

10. Immediately rinse with cold tap water

11. Flood the smear with gram safranin and then rinse it with cold tap water

12. Blot off excess water with paper towel and allow it to air dry

13. Examine

Of course, before we began the experiment, it is crucial for us to perform aseptic technique in order to minimize contamination. In the wake of setting up the smears of the microorganisms, the smears were air dried. Then we stain both smears using crystal violet dyes. Crystal violet dyes are basic dyes that have positively charges particles that helps them to bind to negatively charged particles. The following step is to add iodine to the smear. Next, we washed the iodine with tap water and dried off the excess water. After that, absolute methanol was being added to act as a decolorizing agent. For gram positive bacteria, the addition of alcohol dehydrated the layer of peptidoglycan which thus would trap the CVI complex. This causes the gram positive bacteria to appear to be purple as the CVI complex are being held. The addition of alcohol is not to be over 15 seconds as this would break the cell wall of the bacteria, thus resulting in no stain to be observed. The slides are washed with water and dried off. In the next step, safranin was utilized to counterstain the smear. This is to enable the gram negative bacteria to be visualized easily as it can be stained pink. The gram positive bacteria does not being stained pink when safranin was being introduced because the peptidoglycan layer as of now have CVI complex. At that point, the slides were washed utilizing tap water and dried off.