Isolation, Identification, and Growth of Well‐Water Bacteria
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ABSTRACT Ground water from deep wells was sampled for the presence of heterotrophic bacteria. Over 500 bacteria were isolated from well‐water samples on a low‐nutrient medium (R2A). Gram‐negative, rod‐shaped, nonmotile bacteria predominated, and Acinetobacter spp. comprised 54% of the total number of isolates. Selected isolates were inoculated into unamended and carbon‐enriched well water, and growth was measured by acridine orange direct count (AODC). Carbon sources included glucose, acetate, pyruvate, and succinate in 100 μg carbon/liter and 1,000 μg carbon/liter concentrations. The isolates grew in unamended filtered well water within 24 hours, and growth of an Acinetobacter sp. was further stimulated (greater than two orders of magnitude within five days) in the carbon‐enriched well water.Keywords:
Isolation
Acridine orange
Carbon fibers
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Differential staining
Fluorescent staining
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Gram staining
Acridine
Fluorescent staining
Microbiological culture
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Acridine orange is a cell-permeable fluorescent dye that binds to nucleic acids, resulting in an altered spectral emission. Acridine orange staining has been shown to be highly selective for apoptotic cells in Drosophila ; however, the precise mechanism underlying this effect is not known. Advantages of acridine orange staining include the speed and ease of the staining. But there are disadvantages: It should be performed on unfixed tissue that therefore must be examined immediately, and multiple labeling cannot be performed. Slightly different protocols for the uptake of acridine orange are required for different developmental stages. Here, we present protocols for use of acridine orange to detect apoptosis in Drosophila embryos and in larval tissue. Slight modifications might be required for other Drosophila tissues.
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Total direct and direct viable counts of fresh and injured cultures of Escherichia coli were determined by image analysis in preparations stained with acridine orange, ethidium bromide and 4′,6‐diamidino‐2‐phenyl indole (DAPI). Cells stained with DAPI were not detected by image analysis. Fresh cultures stained with acridine orange or ethidium bromide gave comparable counts. Injured E. coli stained with ethidium bromide gave higher counts that with acridine orange. Injured cultures stained with acridine orange contain high proportions of green cells which are less easily detected than red cells in image analysis. In certain cases it may be better to use ethidium bromide, which stains all cells red, for direct viable counts by image analysis.
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Bacteriostatic activity of Acridine Orange against E. coli was inhibited by the addition of anionic surfactants. The inhibitory activity of surfactants decreased with a decrease in the carbon number of alkyl chain of the surfactants. The apparent concentration of Acridine Orange to inhibit the growth of E. coli was increased by the addition of surfactants. It was concluded that the complex formation between Acridine Orange and surfactants resulted in a decrease of free Acridine Orange in the culture media.
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