Acid Fast Stain

In order to find out if our bacteria is acid fast or non-acid fast we had to do an acid fast stain.

We first smeared some of our bacteria on a slide and heat fixed it to the slide (just as we have been doing for all our stains) so the bacteria will not be washed off by all of the liquids it will be encountering. Once the slide was heat fixed we put a square of bibulous paper on the slide and then put the slide on a drying rack that was resting over a beaker filled with boiling water.

Transporting our slide was really tricky because our forceps didn't hold onto our slide completely, so we were very careful in transporting our slide and very anxious that we might drop it and have to start over again!

Then we proceeded to drop Kinyoun's carbolfuchsin solution over the slide for 5 minutes, all the while making sure the slide doesn't ever get too dry. 

We were sure to constantly drop the solution on the paper (so it would look like this) to make sure that it never dried out.

Next, with the slide still over the beaker of boiling water, we added Ziehl-Neelsen carbolfuchisn. We used the same method for this stain. For three minutes we watched and added more drops to our slide in order to make sure the solution wouldn't evaporate and dry out the slide. 

Using forceps we very carefully removed our slide from over the beaker of boiling water. (Which proved to be quite difficult for us, but in the end we were able to do it) We then placed the slide on a drying rack and removed the bibulous paper, disposing of it in a bio hazard bag. After letting the slide cool for a few minutes, we rinsed the slide off with water to remove any access stain. 

Then we proceeded to use a decolorizing agent, holding the slide at a 45 degree angle we dropped the agent on the slide until the color stopped running. After we washed off the slide with de-ionized water to remove the decolorizing agent. 

Next we covered the slide with methylene blue for 2 minutes, flooding the slide with it and then letting it sit. 

After 2 minutes we rinsed off the excess stain with water. To remove the water we then put our slide between two pieced of bibulous paper and gently blotted it to remove any water, but not too excessively so we would not ruin the stain we had made.

Now it is time to observe what we obtained by looking at our stained bacteria under the microscope, using the oil immersion lens!

Upon observing our bacteria, we could clearly see that all the bacteria had been stained blue, and retained none of the red staining. We concluded from this that our bacteria is in fact no-acid fast.