Creating an Endospore and an Acid-fast Stain

Welcome Back! This week, though it started off slow, really heated up when we started our stains! To understand even more about our unknown bacteria, we created Endospore and Acid-fast stains.

The Endospore Stain: 

To start the stain we followed the same beginning procedure as the simple stain (smearing our bacteria, letting it air dry, and heat-fixing). This procedure is found in the previous blog pertaining to simple stains. We then placed the slide over a beaker full of boiling water (suspended with a slide drying rack), covering the smear with Bibulous paper. We saturated the paper with drops of Malachite Green dye.

We continuously added dye to the paper to keep it saturated for 5-6 minutes. It was important not to let all of the stain evaporate, or the slide would not properly set. After the time was over, we removed the slide with the forceps, removing the paper from the slide as well.

We let the slide cool, and then rinsed the slide (for about 30 seconds) with deoxidized water to remove excess dye from the slide. It is important not to rinse too thoroughly or all of the dye will be removed from your slide.

After the slide was rinsed, we suspended the slide on a drying rack over the sink and added Safranin dye directly onto the slide, letting it sit for 60-90 seconds. This dye is a very deep red color. When the allotted time was through, we rinsed the slide carefully and blotted with Bibulous paper.

The results: 

Notice the colonies of our small cocci bacteria. Also, note the color of our bacteria in this sample. The Green Malachite dye remains visible when endospores are present. Since all of the green dye was rinsed away and the bacteria was only visibly stained with the Safranin, there are no endospores present in our bacteria.

This picture was taken from http://archive.microbelibrary.org. This is a website that shows many more examples of stains, if interested. Notice how the endospores in this picture are stained green. This obviously contrasts with our sample, not having any visible green endospores.

The Acid-fast Stain:


To start this procedure, we once again began with the steps for making a simple stain. After, we placed the slide over a beaker of boiling water suspended by a slide drying rack. Then we covered the slide with Bibulous paper and saturated the paper with Ziehl-Neelsen Carbolfuchsin.

Like the last procedure, we had to keep the slide saturated with the dye while it steamed, but this time for 3-5 minutes. After the allotted time we removed the slide carefully from the rack and removed the Bibulous paper, throwing the used paper away in the designated receptacle.

After cooling, we rinsed the slide with deoxidized water to remove the excess stain. Also to remove more stain, we used Acid-alcohol. To do this we held the slide at an angle over the sink, and added Acid-alcohol to it until the magenta color stopped running.

Immediately we rinsed the slide free of decolorizing agent so that the rest of our process was not inhibited. We then covered the slide directly with Methylene Blue for 2 minutes. After this time frame, we rinsed the slide clean of excess dye and blotted the slide dry.

The results: 

Once again the small cocci colonies are present within our slide, but this time with a different stain color and meaning. The blue appearance reveals that our bacteria is Non-acid-fast. If the magenta stain remained after using the Acid-alcohol, then our bacteria would be considered Acid-fast. In an Acid-fast bacteria the first dye (red) is trapped by waxes in the cell membrane.

Join us next week for another exciting installment of Microbiology Lab! See you then!