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Biology 230W Euglena Protocol

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Biology 230W Euglena Protocol JSM090711 ARE CHLOROPLASTS DERIVED FROM PROKARYOTES? THE EFFECT OF STREPTOMYOCIN ON BLEACHING THE CHLOROPLAST SYSTEM OF Euglena gracilis Jacqueline S. McLaughlin and Missy Coyle Department of Biology, Penn State Lehigh Valley Biology 230W, Fall 2011 September 7, 2011 Purpose: Euglena are unicellular organisms that belong to the Kingdom Protista. Euglena are autotrophic under some conditions and heterotrophic under other conditions. For example, when grown in light and viewed under a microscope Euglena is green as a result of chlorophyll in its chloroplasts, which is used for sugar production during photosynthesis. In the absence of light, Euglena loses its chlorophyll and vigorously consumes food such as bacteria from its habitat. There is substantial evidence from research to support endosymbiosis – the theory that mitochondria and plastids, including chloroplasts originated as prokaryotic cells engulfed by an ancestral eukaryotic cell. The engulfed cell and its host cell then evolved into a single organism. The discovery of specific chloroplast DNA (Brawermann & Eisenstadt, 1964; Edelman, Schiff & Epstein, 1965) and the presence in chloroplasts of 70S ribosomes which otherwise are found only in mitochondria and in prokaryotic microorganisms (Boardman, Francki & Wildman, 1965; Kuntzel & Noll, 1967) exemplify some of this evidence. Additionally, these 70S ribosomes have been found to be different from the 80S ribosomes which are found in the cytoplasm of Euglena and all other plants and animals not only in their sedimentation coefficient, but also in their composition of rRNA and protein (Taylor, Glasow & Storck, 1966) and submicroscopic architecture (Ris & Plaut, 1962). Classic experiments on Euglena to further support endosymbiosis as the explanation of the chloroplast is the finding that antibiotics “bleach” the chloroplast system of Euglena gracilis without significantly inhibiting cell division or viability of the cell. The work herein sets out to replicate the work of Ebinger et al. (1969) that found that streptomycin (SM) at concentrations of 10 ug/ml, 100 ug/ml, and 500 ug/ml successfully bleached colonies of E. gracilis after a five to six day period of cultivation. Materials: Euglena gracilis cultures (Carolina Biological, or similar); Euglena media (for stock culture); Pen-strep 10,000 units (mg)/mL (Hyclone SV30010); Syracuse dishes; Parafilm (with holes); microscope slides; coverslips; compound microscope Procedure: 1. E. gracilis stock culture: Prepare Euglena media by mixing and boiling 40 wheat grains, 35 rice grains and 5 mL of dry evaporated non-fat milk in 1L of distilled water. Boil for 5 minutes. Let the mixture stand overnight and then add Euglena. Keep in a well-lit area but out of direct sunlight. Overheating will kill Euglena. 2. Experimental and control cultures: Prepare two control cultures (10 mL each): Euglena with light and no SM; Euglena without light and no SM. Prepare three experimental cultures (10 mL each): Euglena with light and 10 ug/mL SM; Euglena with light and 100 ug/mL; and, Euglena with light and 500 ug/mL. 3. Light microscopy: After five to six days, make a wet mount of each of the five cultures and compare the control and experimental groups. Draw and label results in lab notebook. Take photomicrographs. Controlled variables: Indirect sunlight for all SM treated dishes, ambient temperature (~22°C), volume (10mL stock culture per Syracuse dish) Note: Euglena will be evenly distributed in 50mL culture dishes by gentle mixing, then aliquot into the 10mL Syracuse dishes. Data Interpretation: Observations will be recorded as microscopic labeled drawings and photomicrographs. Analysis of these materials will allow for the determination of the following: (1) which environment(s) altered the chloroplasts of E. gracilis after five to six days in culture; and, (2) which environment did not alter the chloroplasts of E. gracilis after five to six days in culture. This data will be compared to the original findings of Ebinger et al. (1969) and reasons for the results obtained will be suggested and articulated. References: Boardman, N.K., Franki, R.I.B. &Wildman, S.G. (1964) Protein synthesis by cell-free extracts from tobacco leaves. 2. Association of activity with chloroplast ribosomes. Biochemistry, 5: 872-878. Brawerman, G & Eisenstadt, J.M. (1964). Deoxyribonucleic acid from the chloroplasts of Euglena gracilis. Biochimica et biophysica acta, 91: 477-485. Endelman, M., Schiff, J.A. & Epstein, H.T. (1965) Studies of chloroplast development in Euglena. XII. Two types of satellite DNA. Journal of Molecular Biology, 11: 769-774. Epringer, L. (1972). Are plastids from prokaryotic micro-organisms? Action of antibiotics on chloroplasts of Euglena gracilis? Journal of General Microbiology, 71: 35-52. Kuntzel, H. & Noll, H. (1967) mitochondrial and cytoplasmic polysomes from Neurospora crassa. Nature, 215: 1340-1345..
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