Download Lab Exercise 7: The Negative Stain and Capsule Stain

Lab Exercise 7: The Negative Stain and Capsule Stain
O BJECTIVES
1.
2.
3.
Perform a negative staining procedure
Understand the benefits of using negative stains.
Perform a successful capsule stain to distinguish capsular material from the bacterial cell.
INTRODUCTION
As mentioned in lab exercise 6, not all dyes stain the bacterial cell. However, acidic dyes can be useful for
observing the size and shape of bacterial cells and for cells such as spirochaetes that do not readily stain
with ordinary dyes. In these negative stains, the background area around the cell appears dark or opaque.
Cells appear as transparent against this dark background, and this allows you to observe such extracellular
features as capsules. Negative stains are also not heat fixed, to avoid shrinkage of the cells and destruction
of the capsule.
The capsul e stain
Some bacterial cells are surrounded by an extracellular slime layer called a capsule or glycocalyx. This
structure can play a protective role for certain pathogenic bacteria. The capsule prevents phagocytosis by
white blood cells, as well as provide a means for attachment to solid surfaces in the environment. For
example, Streptococcus mutans can attach to the surface of a tooth by its capsular material, resulting in the
formation of dental plaque, which contributes to the decay of teeth in humans. Most capsules are composed
of polysaccharides and in some cases polypeptides with unique amino acids. Evidence suggests that all
bacterial cells have some amount of slime layer, but in most cases the amount is not enough to be readily
discernible.
.
Negative Stain:
LAB EXERCISES
Table supplies
Cultures of Bacillus megaterium
Cultures of Staphylococcus
aureus
Individual supplies
Microscope slides
Nigrosin dye
Staining tray
Sterile toothpicks
Protocol:
1. Place one drop of nigrosin onto the end of a clean slide.
2. Using sterile technique, transfer one loopful of either, oral
scraping of your teeth, Klebsiella pneumoniae or B.
megaterium into the drop of nigrosin and mix.
3. Spread the mixture evenly into the drop of nigrosin.
4. Repeat steps 2 and 3 using another species if desired for
contrast.
5. Let the preparations air-dry. DO NOT heat fix.
6. Examine all stained slides under 100x oil immersion lens.
II. Capsule stai n
Table supplies
Culture of Klebsiella pneumoniae
Individual supplies
Copper sulfate 20%
Crystal violet
Microscope slides
Staining tray
Water bottle
Bibulous paper
Inoculating loop
1. Add one drop of crystal violet dye to a clean slide just like the negative stain (toward one end).
2. With a sterile toothpick scrap between your teeth for plaque
OR use a loopful of K. pneumoniae. Mix and spread the
mixture evenly over the slide like in the negative stain.
3. Let slide air dry for 5 to 7 minutes.
4. Gently rinse off excess dye with Copper sulfate, and allow
the copper sulfate to pool on the smear for about 1 minute.
7. Gently blot dry with bibulous paper.
8. Examine slide under 100x oil immersion lens.
DATA AND OBSERVATIONS
1. Draw observations of cells from your plaque sample and sample organisms.
Plaque sample
Total magnification ___________
B. megaterium
Total magnification ___________
2. Draw observations from capsule stain of K. pneumoniae or plaque bacteria.
Klebiella pnuemoniae
Total magnification ___________
DISCUSSION
1. Why are negative stains useful?
2. Do you heat fix a negative stain? Why?
3. What color is the capsule itself? Why?