Kristina SimonovicBIO305W Sec 16Heather Gillette02-27-18MATERIALS AND METHODSFive stains were performed on the unknown environmental isolate including, simple, Gram’s reaction, capsule, endospore, and acid-fast. Each slide was prepared by dry-mounting and heat-fixing the organism onto the slide, excluding the capsule stain, which was dry-mounted after emulsifying the culture in Congo red stain. In addition, Trypticase Soy Agar (TSA) was the media used to cultivate each of the cultures for the five stains excluding the endospore stain, in which B. megaterium was grown on a stress TSA plate. Each of the stains were viewed through a100x objective lens under oil immersion.The simple stain was performed in order to determine the cell’s morphology. The unknown environmental isolate was stained with methylene blue and no control organisms were used for preparing this slide. While there is no negative result to a simple stain, a positive result specifies the morphology of the cell observable by the blue stain. The purpose of the Gram’s reaction stain was to determine the thickness of the peptidoglycan layer in the cell wall. The two positive control organisms used were Bacillus megaterium and Staphylococcus epidermidis and the negative control organism used was Escherichia coli. The slide was stained with crystal violet as the primary stain and Gram’s iodineas the mordant, decolorized with 70% ethyl alcohol, and counterstained with safranin. A positive result, indicated by purple cocci cells, determines a thick peptidoglycan layer, while a negative result, specified by pink rod-shaped cells, determines a thin peptidoglycan layer.The capsule stain was performed in order to determine the organisms’ ability to produce capsules. There were no control organisms used in the preparation of this slide. The culture was emulsified in Congo red as the primary stain, and air-dried with Maneval’s stain as the counterstain. A positive result, indicated by halos surrounding red cells on a blue background, suggests that the cell is capable of producing a capsule, while a negative result, indicated by red cells lacking halos on a blue background, suggests that the cell is incapable of producing a capsule.The purpose of the endospore stain was to determine the organisms’ ability to release endospores, store genetic information, and survive environmental stress. The positive control organism used for this slide was B. megaterium grown on a stress TSA plate, and no negative control was used. The slide was covered with filter paper and steamed with malachite green as the primary stain, decolorized with deionized water, and counterstained with safranin. A positive result, indicated by green spores located on pink cells, implies that the cell is capable of producing spores, storing genetic information, and surviving environmental stress, while a negative result, indicated by solely pink cells, implies that the cell is incapable of producing spores, storing genetic information, and surviving environmental stress.The acid-fast stain was performed in order to determine the presence of mycolic acid in the cell wall. The positive control organism used for this slide was Mycobacterium smegmatis and the negative control organism used was B. megaterium. The slide was covered in filter paper,steamed with carbol fuschia as the primary stain, decolorized with hydrochloric acid, and counterstained with methylene blue. A positive result, indicated by pink cells, specifies some amount of mycolic acid present in the cell wall, while a negative result, indicated by blue cells,determines that there is no mycolic acid present in the cell wall. All methods are from Shand andFitchett (2017).RESULTSFive staining techniques were performed on the unknown environmental isolate in order to define specific characteristics about the organism, including cell morphology, thickness of peptidoglycan layer and presence of mycolic acid in the cell wall, and the organisms’ ability to produce spores as well as capsules. The result of the simple stain was rod morphology characterized by blue rod-like cells (Table 1). The Gram’s reaction stain was positive indicated by purple stained rod-shaped cells (Table 1). The capsule stain had a negative result represented by red cells that lacked halos around them (Table 1). The result of the endospore stain was positive observed by the green spores located on the pink cells (Table 1). The acid-fast stain was a negative result indicated by the presence of blue cells (Table 1). Table 1. The results and observations of five staining techniques that characterize the unknown environmental isolate collected in Flagstaff, Arizona. Characteristics Observations ResultsCell morphology Blue Cells RodGram reaction Purple Rods PositiveCapsule Blue Background, Red Cells w/o Halos NegativeEndospore Pink Cells with Green Spores PositiveAcid-fast Blue Cells NegativeREFERENCESHawksworth DL. 1991. The Biodiversity of microorganisms and invertebrates: its role in sustainable agriculture. WEFSA. 1(29):133-133.Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST. 2000. Bergey’s manual of determinative bacteriology. 9th ed. Philadelphia: Lippincott Williams & Wilkins.Shand R, Fitchett L. 2017. BIO 205L: fundamental techniques and experiments in microbiology. 3rd ed. Minneapolis: bluedoor, LLC.Van Der Heijden M, Bardgett R, Van Straalen N. 2007. The unseen majority: soil microbes as drivers of plant diversity and productivity in Terrestrial ecosystems. 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