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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?