Lab Exercise 9: Differential stains for visualization of cell structures

OBJECTIVES

1. Understand the chemical basis for the spore and capsule stains. 2. Perform a successful spore stain to differentiate between bacterial spores and

vegetative cells. 3. Perform a successful capsule stain to distinguish capsular material from the

bacterial cell. INTRODUCTION The spore stain Certain bacterial species, most commonly gram-positive bacilli such as those of the genera Bacillus and Clostridium, undergo a complex developmental cycle that produces a resting endospore when faced with environmental adversity. The process of sporulation allows the bacteria to survive in harsh environmental conditions such as low nutrients, high temperatures, UV radiation, acids and toxic chemicals. If conditions improve, the spore may germinate to form a new vegetative cell and growth will resume. Endospores are very dehydrated structures that are not metabolically active. They possess a protein coat, called an exosporium, that forms a barrier around the spore. Since endospores are not easily destroyed by heat or chemicals, they define the conditions necessary to extablish sterility. For example, to destroy endospores by heating, they must be exposed for 15-20 minutes to steam under pressure, which generates temperatures of 121° C. Such conditions are produced in an autoclave, discussed in Lab Exercise 3. These resistant properties also mean they are not easily penetrated by stains. Endospores will not readily stain with any color during the Gram stain procedure, thus appearing as clear ovals in a stained cell. However, if heat is applied while staining with malachite green, the stain penetrates the endospore and becomes trapped. The malachite green is not removed by subsequent washing with decolorizing agents or water. In this instance, heat is acting as a mordant to facilitate the uptake of the stain.

The capsule 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. Staining of a bacterial capsule is not achieved using basic staining protocols. If smears are heat fixed prior to staining, the capsule shrinks or is destroyed and can’t be seen. However, you can combine the simple stain with a negative stain using gentle heat fixation to achieve visualization of the capsule. The final result is that capsules will appear as a clear halo-like structure surrounding purple cells against a dark background. LAB EXERCISES I. Spore stain

Table supplies Individual supplies 3-5 day old cultures of multiple Bacillus spp.

Spore staining kit (malachite green and safranin)

Microscope slides Staining tray Water bottle Bibulous paper Inoculating loop 1. Prepare lab bench by removing extraneous items and cleaning surface with table

disinfectant (this will be omitted from future protocols but should always be done).

2. Prepare smears of a Bacillus species. Air-dry and heat-fix. 3. Proceed with Schaeffer- Fulton spore stain:

1. Place your slide on a can of steaming hot water. 2. Immediately flood warm smear with malachite green and let steam

thoroughly for 5 minutes. You may place a small piece of paper towel over the smear to keep the stain in place. Make sure the stain does not evaporate completely, keep flooded with stain.

3. After the 5 minutes of steaming with stain, you need to wash slide with water to decolorize and remove excess dye.

4. Counterstain by flooding smear with safranin for 30 seconds. 5. Wash with water to remove excess dye. 6. Blot dry with bibulous paper.

4. Examine all slides under 100x oil immersion lens. II. Capsule stain

Table supplies Individual supplies Culture of Klebsiella pneumoniae

Nigrosin dye or India Ink

Crystal violet Microscope slides Staining tray Water bottle Bibulous paper Inoculating loop

1. Add one drop of nigrosin dye to a clean slide. 2. Mix in a loopful of K. pneumoniae and spread the mixture evenly on the slide to at least the size of a quarter. 3. Let slide air dry. 4. Gently heat fix the smear by passing through the flame 1-2 times. 5. Flood smear with crystal violet for one minute. 6. Gently rinse off excess dye with water. 7. Gently blot dry with bibulous paper. 8. Examine slide under 100x oil immersion lens.

DATA AND OBSERVATIONS 1. Draw observations from spore stain of 2 Bacillus spp. Differentiate spores from vegetative cells. Bacillus spp. 1 Bacillus spp. 2 Species _________________ Species _________________ Total magnification ___________ Total magnification ___________ 2. Draw observations from capsule stain of K. pneumoniae. Klebiella pnuemoniae Total magnification ___________

DISCUSSION 1. How does the culture age affect the results of a spore stain? 2. What prevents the vegetative cell from appearing green in the finished endospore stain? 3. Bacillus anthracis, the causative agent of anthrax, is an endospores former. Why does this trait enhance its capabilities as a biological weapon? 4. How might a capsule contribute to pathogenicity?