Acid-fast & Endospore Stains

This week started off with an endospore stain. We first begin a simple stain (smearing, air-drying & heat fixing-mentioned in previous posts!) and than placed the slide in a slide drying rack suspended over a beaker filled with boiling water. After covering the smear with Bibulous paper we wet the paper with Malachite Green dye. We continued to add dye to the paper, keeping it damp for around 5 minutes. We did not let all of the stain evaporate, therefore the slide could properly set but not kill the bacteria! After 5 minutes, we removed the slide with forceps (twisers) and set the slide on paper towels to cool down. The slide was than rinsed with distilled water and remove some excess Malachite Green from the slide. Then we added Safranin dye for 60 to 90 seconds over a sink on a drying rack. Once correct time had passed, we rinsed with distilled water and let dry. Once viewed under a microscope it was apparent that the Green Malachite dye had all rinsed away and only the Safranin stained bacteria remained. This meant that bacteria 'L' did not hace endospores as the Green Malachite dye reminds when there are endospores.

Next we did an acid-fast stain. We started with a simple stain and than suspended it over boiling water on a drying rack. Once covered with Bibulous paper, we saturated it with Ziehl-Neelsen Carbolfuchsin. We continued to keep the slide wet with dye for 3 to 5 minutes. Once time was up, we used forceps to remove the slide to cool down and to throw away the Bibulous paper into the proper waste. The slide was then rinsed with distilled water to remove excess dye and to remove even more stain we took acid-alcohol and dropped it over the slide, which we placed at a 45 degree angle over the sink, until the pink color of the Ziehl-Carbolfuchsin was done running off. After once again using distilled water to rinse, we made sure to cover the smear with Methylene Blue for 2 minutes, rinsed it clean with more distilled water and than blotted dry with Bibulous paper.

Since bacteria 'L' remained dark pink after using acid-alcohol it is determined that our bacteria is acid-fast! This means the dark pink dye was trapped by the waxes in the cell membrane of bacteria 'L'!

Methylene Blue rinsing off!           

Annie B