Day 4

Today we prepared 3 different types of stain: Acid-Fast, Endospore, and Capsule stain.  We began with the capsule stain.  In order to prepare this, we made a Negative stain where we placed nigrosin at the end of a clean slide.  Then we added a small amount of bacteria into the drop, mixed it, and spread it across the slide.

Using this negative stain, we prepared a capsule stain by covering the spread smear with safranin.  Then we rinsed off the excess stain and blotted off the excess water. 

Next we prepared an acid-fast stain.  First, we fixed a bacteria smear on the slide.  We put bibulous paper on the slide and saturated it with Ziehl-Neelsen carbolfuchsin while holding it over boiling water for 3 minutes.

  After the slide cooled, we removed the paper and rinsed the slide with water.  We then decolorized the slide with acid-alcohol and immediately rinsed it with water.  Then the covered the smear with methylene blue for 2 minutes and removed the excess dye with water.

The final stain was the endospore stain.  We took a slide fixed with a bacteria smear, covered it with bibulous paper, and for 5 minutes added malachite green stain.  We did this while holding the smear over boiling water. 

Then we rinsed the slide for 30 seconds and covered the smear with safranin for 60 seconds.  To finish, we rinsed the excess safranin off the slide and blotted the water to dry it.

These are the 3 completed slides:

Lesson of the day: don't spill the dye!!!