Endospore Stain Exercise 21 PDF

UJ Vl u Q: UJ >< UJ ENDOSPORE STAIN LEARNING OBJECTIVES----------- 1. Provide a definition of an endospore and describe how endospores contribute to survival of a species. 2. Describe the processes of sporulation an...

UJ Vl u Q: UJ >< UJ ENDOSPORE STAIN LEARNING OBJECTIVES----------- 1. Provide a definition of an endospore and describe how endospores contribute to survival of a species. 2. Describe the processes of sporulation and germination, including the conditions under which each process occurs and the genera of Bacteria that undergo these processes. 3. Explain the significance of endospores in the medical field and in the food industry. 4. Describe the conditions used to eliminate endospores from materials. 5. Identify the stains/reagents used in each step of the endospore stain and describe how each stain/reagent affects bacteria with and without endospores. 6. Differentiate between endospores and vegetative bacteria using bright light micros- copy. INTRODUCTION------------- Bacteria are dependent on nutrients present in a growth medium or environment for survival (see Exercise 3). Once a necessary nutrient is depleted, bacteria begin to die, and eventually, all the organisms in the culture will die. Two genera of bacteria, Bacillus and Clostridium, are different. Once conditions for growth become unfavorable, these bacteria produce endo- spores through the process of sporulation. Endospores are essentially dormant states which can withstand harsh conditions and allow for the survival of the species. It is important to note, however, that endospores are not produced as a means of reproduction, but simply help these bacteria survive inhospitable conditions. When environmental conditions become unfavorable, such as when a carbon, phosphate, or nitrogen source becomes depleted, when the pH decreases, or when peptide antibiot- ics are present, the vegetative cell begins to undergo sporulation. In this process, the organ- ism's chromosome and a small portion of cytoplasm become surrounded by two mem- branes, a thick layer of peptidoglycan, and three protein spore coats. Most of the water is removed from the endospore, while large quantities of calcium, DNA-binding proteins, and dipicolinic acid are deposited within the endospore. It is believed that the calcium and dip- icolinic acid are responsible for the heat resistance of endospores, but how they accomplish this is unknown. Depending on the bacterial species, the entire process of sporulation takes THE MICROBESAROUND US 141  EXERCISE 21 ENDOSPORE STAIN between 8-17 hours to occur. The location and size of an endospore varies with each species (Figure 21.1), and these characteristics are useful in helping to identify individual species. The shape of the endospore ranges from spherical to elliptical and the location of the endo- spore within the cell may be central, subterminal, or terminal. Endospores are extremely resistant to the effects of heat, drying, radiation, and chemicals. A fully mature endospore in the soil can exist for up to 100 years, and some endospores have been determined to be thousands of years old. a Spherical Cylindrical Spherical Spherical Cylindrical (central) (central) (terminal (subtermina/ (subtermina/) and swollen) and swollen) Figure 21.1. Examples of endospore shapes and locations within cells. Once environmental conditions improve, an endospore may undergo germination to yield a vegetative cell. It is thought that a small organic molecule or amino acid plus water provides the stimulus for germination. Germination to form a replicating vegetative cell occurs much more quickly than sporulation and takes about 90 minutes. The formation of endospores by members of Bacillus and Clostridium is important in both the medical field and the food industry. Endospores from the organism Bacillus anthracis, the causative agent of the disease anthrax, are considered to be one of the most dangerous agents in the field of germ warfare. Food scientists must employ conditions that destroy endospores during the canning of fruits and vegetables. Because of their resistance to heat (along with desiccation and disinfectant compounds), endospores can survive within improperly canned foods. Following the canning process, endospores may germinate inside the can and bacteria begin actively growing inside the can. Many endospore-producing bacteria also produce potent exotoxins during their growth and these exotoxins may accumulate within the canned item. Human consumption of these exotoxins can lead to life-threatening outcomes. Neither boiling nor common household disinfectants destroy endospores. There are some sterilizing agents, such as ethylene oxide, which can be used in medical facilities, but the most common method of ridding material of endospores is through the use of the auto- clave. Conditions inside an autoclave reach 121 °C at a pressure of 15 pounds per square inch. Autoclaves are widely used to sterilize materials in microbiology, human and veterinary medicine, dentistry, the food industry, and in tattoo parlors. 142 THE MICROBES AROUND US  EXERCISE 21 ENDOSPORE STAIN Bacterial endospores can be visualized microscopically as clear areas within bacteria when a simple stain is performed. However, the endospores are much easier to visualize when they are stained with the endospore stain. The primary stain of malachite green penetrates the tough walls of the endospore during the 10-minute incubation step. Washing the slide with water does not liberate the stain from the endospores. The safranin counterstain sim- ply stains the vegetative cells and does not affect the endospores. Accordingly, the bacterial cells themselves appear pink, while the endospores appear as green spots within the cells. The endospore stain will not be performed with the environmental isolates. The organisms growing on the agar plates were not deprived of nutrients and would not have begun the process of sporulation. TODAY'S PROCEDURE·----------- 1. Your graduate teaching assistant has set upa microscope focused on an endospore stained preparation of bacteria. It is each student's responsibility to view this slide. 2. Be sure to note the difference between the endospore and the bacterial cell itself. 3. Record all results. THE MICROBES AROUND US 143