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Assessment of damaged DNA and cell structure in Giardia lamblia trophozoites
Author(s) -
LENAGHAN SCOTT,
SUNDERMANN CHRISTINE
Publication year - 2005
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2005.05202003_1_47.x
Subject(s) - biology , dna , comet assay , lysis , dna damage , ethidium bromide , biophysics , agarose , microbiology and biotechnology , cytoplasm , fluorescence microscope , fluorescence , biochemistry , physics , quantum mechanics
Control of protozoal pathogens in water and food destined for human use employs chemical and physical agents for inactivation of trophozoites and cysts. γ radiation, when given in certain doses, damages DNA as well as other cell components. Previously, we characterized damage in irradiated Giardia lamblia trophozoites by using Nomarski interference and scanning electron microscopy. To further examine cellular damage, irradiated trophozoites were viewed with transmission electron microscopy. Degranulation of cytoplasm at higher irradiation doses was observed, however, axonemes, adhesive discs, and peripheral vesicles appeared morphologically similar in treated as well as control cells. In order to determine damage to the DNA, the comet assay was employed. The comet assay is a single cell gel electrophoresis assay that measures the number of single and double strand breaks in DNA. This was achieved by embedment of live cells in low melting point agarose, lysis of cells, and then addition of an alkali solution to allow the DNA to unwind. After ample time for unwinding, the cells were electrophoresed, thus pulling the smaller broken strands away from the supercoiled DNA. We used Sybr GreenR, a DNA specific fluorescent dye, and a signal‐enhancing camera to visualize the DNA. Lengths of comet tails (corresponding to number of strand breaks) were measured for quantification of damage.