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Microbial Survey, Smear Preparation, and Simple Stain Essay

Essay Topic:

Instructional Objectives 1. Define Roccal = green, liquid disinfectant. Pathogen = an agent which causes disease. Wet Mount Slide = a microscope slide of a liquid specimen covered with a cover glass. Yeast = a single celled fungi. Budding = a true characteristic method of asexual reproduction among yeasts where budding of a new cell from a parent cell can be observed. Mold = multicellular masses of filamentous fungal growth. Hyphae = individual filaments of mold, generally comprised of more than one cell.Mycelium = the entire mass of the intermeshed hyphae. Colony = the sometimes circular body of fungal growth that is visible to the unaided eye. Can be comprised of thousands of hyphae and reproductive cells, yet is the result of an overgrowth of a single cell or reproductive spore. Reproductive Spore = a means of both reproduction and dissemination of molds, since they are readily carried about by air currents. Septa = hyphal cross walls which divide the filaments into separate cells. Petri Plate (dish) = a special covered dish in which mold is cultured.Medium = a solid nutrient used for culturing. Agar = a non-nutrient thickening agent which is thicker than gelatin but still quite soft. Smear = a thin film of microbial cells on a microscope slide. Fixing = passing the smear through the flame of the laboratory burner three times in rapid succession to heat fix the smear. Simple Staining = staining cells with a single dye so that they can be more readily observed. Basic Dye = (methylene blue) has positively charged chromophore group. A basic dye will be attracted to any negatively charged substance, such as bacterium.Acidic Dye = (eosin) have negatively charged chromophore groups, and thus are attracted to positively charged substances. Chromophore Group = chemical staining group. Bacilli = rod- shaped bacterium. Endospore = survival forms of the cells. Bacterial Spore = a released endospore. 2. Describe the steps in the preparation of a wet mount slide and name the three different specimens used. To prepare a wet mount slide you begin with the substance at hand. The specimens studied in the laboratory using this type of slide were a hay infusion, a yeast suspension, and the mold specimen.For the hay infusion you begin with placing two drops of the suspension in the center of a clean microscope slide using a transfer pipet. The specimen must be immediately covered with a cover glass completing the wet mount slide. The yeast suspension is transferred from the tube to the slide using a flame sterilized inoculating loop. Immediately cover the specimen with a cover glass. The stained yeast suspension is prepared the same way except that the suspension is mixed with a drop of lactophenol cotton blue placed on the slide prior to transferring the yeast.The mold must be cut from the petri plate and placed on top of the drop of lactophenol cotton blue already placed on the microscope slide. After it is covered it may be studied under the microscope. 3. State the scientific name of the yeast studied in the laboratory. Saccharomyces cerevisiae is the scientific name of the yeast studied in the laboratory. 4. Name the medium upon which the mold was cultured. Sabouraud agar is the medium upon which the mold was cultured. 5. Name the stain routinely employed on fungal specimens. Lactophenol cotton blue is a stain routinely used on fungal specimens. 6.List two methods by which the mold specimen was examined. The mold specimen was studied by observing the mold colony on the Petri dish directly under the microscope. It was also studied by placing a drop of lactophenol cotton blue directly on a microscope slide followed by a piece of the mold specimen from the Petri dish which is then covered and can be viewed under the microscope. 7. Name the specimen in which moving organisms were seen. Moving organisms were observed in the hay infusion. 8. List the steps in the proper transfer of microbes from a culture to a slide using an inoculating loop.To begin the proper transfer of microbes using the inoculating loop you must first light the laboratory burner. The flame will be used to sterilize the inoculating loop by heating until the loop is red hot. Wait for the loop to cool before using it to pick up a drop of the specimen. This specimen is placed on a clean microscope slide which is then covered with a cover glass. If it is stained place a drop of dye on the slide and then mix in the specimen. 9. Describe the procedures for the preparation of smears from both liquid and solid (agar) cultures.Liquid smears are prepared by first shaking the culture well and flame sterilizing the loop. After letting the loop cool a loopful of the liquid culture is transferred onto a clean slide. For a second time, flame sterilize the loop and transfer a loopful of liquid culture onto the same spot on the slide. Mix the loopfuls together and spread out to the size of a tack. Flame sterilize the loop and allow the smear to air dry without disturbance. The smear must then be heat fixed by passing the slide three times in rapid succession over the laboratory burner; it will be warm to touch.To begin the smear preparation for the solid agar culture two loopfuls of water needs to be placed on the slide. Flame sterilize the loop and let cool, open the Petri dish, and gently touch the flat side of the loop to the agar and slowly draw the loop toward you about a quarter of an inch. Mix the microbes on the loop into the water on the slide and spread it until it is dime sized. Again flame sterilize the loop and heat fix the smear after it has been air dried. 10. Describe the process of heat fixing a microbial smear. A smear is heat fixed by passing the slide over the laboratory burner three times in rapid succession. 1. Name the bacterium employed in the smear and simple staining exercises. The bacterium used in the smear and simple staining exercises is Bacillus megaterium. 12. List the materials and steps employed in simple staining. One microscope, two smears of the bacterium Bacillus megaterium, lens paper, sqeeze bottles of water, dropper bottle of methylene blue, staining pans with racks, paper towels, bottle of Roccal, immersion oil, and a bottle of 70% isopropyl alcohol are the materials necessary for the simple staining exercise.Begin by placing the smear slide (specimen up) on the rack in the staining pan, and then cover each smear with one or two drops of the methylene blue and let stand for one minute. Rinse the slide with water making sure all of the residual stain is removed from the slide, then blot dry. The slide is now able to be examined with the microscope. 13. Describe the difference in appearance of organisms or slides from broth and agar cultures. With the agar culture you are able to readily see mold growing on the surface without use of the microscope.Although the broth was cloudy (a sign of bacterial growth) it was not as easily examined with the unaided eye as was the agar culture. When viewed under the microscope, in the yeast broth you were able to clearly distinguish the nucleus within the cells and in some cases the process of budding was observed. Hyphae and a number of reproductive cells were able to be seen when examining the mold under the microscope. The nucleus of the cells was hard to distinguish, but septa were easily seen.

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