Dear Blog and Blog readers,
Today, we completed a third gram stain of our environmental soil sample to determine if, in fact, it was gram positive, gram negative, or both.
Once we completed the heat fix as described on previous posts as well as the gram stain which is also described on previous posts, we determined that our environmental sample bacteria is still gram positive as it was before.
We sterilized our loop, and then prepared for the acid fast stain.
We began our acid fast stain by heat fixing a slide of unknown bacteria as depicted in previous posts. We then placed the slide over a beaker of boiling water.
Next, we placed a piece of bibulous paper and saturated it with Ziehl-Neelson carbolfuchsin. We allowed the slide to remain for 5 minutes . As the stain began to dry, we added more stain so that it would remain saturated for the duration of the 5 minutes.
After we were finished boiling, we removed the bibulous paper and placed it in a biohazard bag.
(John M. Hammot "Microbiology Lab Book")
We allowed the slide to cool and then we rinsed off the excess stain with deionized water.
Next, we added decolorizing "Acid Alcohol" drop by drop while holding the slide at a 45 degree angle until color stopped running off.
We immediately rinsed the slide when were done with the acid alcohol.
Then, we covered the slide with methylene blue for 2 minutes. And then we rinsed the slide with DI water after the 2 minutes were up.
Finally, we dabbed the slide with bibulous paper to remove the excess liquid.
Once we were through preparing the slide, we observed it under the microscope and determined that our unknown bacteria tested negative for the acid fast stain, which means we do not have a mycobacterium! We know this because our bacterium are spherical rather than rod shaped.
Citation
Lammert, John M. "Characteristic Features of Bacterial Growth in Culture."Techniques in Microbiology: A Student Handbook. San Francisco: Pearson Benjamin Cummings, 2007. 62.
No comments:
Post a Comment