Sept 27

Today we prepared a Gram stain of both our environmental sample and of our unknown sample.

The materials we used to prepare a Gram stain included:

1. Two slides: one with a fixed smear of our unknown sample, and one with a fixed smear of our environmental sample.
2. Safranin stain
3. Crystal Violet stain
4. 95% Ethanol solution.
5. Gram's Iodine
6. Distilled water


(NOTE: Look to our previous postings for demonstrations as to how to fix bacteria to a slide.)

1.After fixing the bacteria to the slide, we covered the smear with crystal violet and let it sit for about 20 seconds.

2. We rinsed the slide with water to remove excess crystal violet, and covered the smear with Gram's iodine for 1 minute. We then rinsed the slide with distilled water to remove any excess iodine solution.

3. Then, while holding the slide at a 45 degree angle, we de-colorized the slide with an 95% ethanol solution, applying the solution drop by drop until colored solution stopped running down the slide. After decolorizing the slide, we immediately rinsed the slide with distilled water to remove the decolorizing agent.

4. We covered the smear with Safranin for about 1 minute, and rinsed the slide off with distilled water to remove any excess Safranin, and blotted the water off the slide using bibulous paper.

5. Lastly, we examined the stained smear under the microscope using the oil immersion lens.

We concluded that our unknown sample was a Gram negative because the stain stayed Red.

Pic: GRAM STAIN

 Safranin agent on slide