FISH performance is carried out easily within few steps. First the water sample
has to be fixed. According to Amann [5], for most gram-negative bacteria paraformal-
dehyde is good to be used whereas ethanol is better for gram-positive bacteria. The fixation step is very important, otherwise degradation of ribosomes is risked. This
could lead to underestimation of the fluorescence signal. For some water samples, it is
necessary to perform enzymatic digestion after fixation. Especially this is required for
samples with very dense biofilm formations that impede direct diffusion of the probe
into the target cell. Enzymes like lysozyme or trypsin break down most polysaccha-
rides and proteins, respectively, and therefore can enhance the transport of the probe
through the biofilm into the cell. The hybridization step is then directly carried out on
the sample itself. Therefore the sample is spotted on teflon coated microscope slides
and air-dried. In a next step, the sample is dewatered through stepwise incubation in
ethanol from low to high percentage, i.e. from 50% to 98%, respectively. After de-
watering, the hybridization step is carried out through adding a mixture of salts,
formamide and detergents, and including the probe directly into the sample, which
will be then incubated in the dark for at least two hours. Subsequently, the sample has
to be washed of residual probes and can be analyzed under the fluorescence micro-
scope.
FISH performance is carried out easily within few steps. First the water sample has to be fixed. According to Amann [5], for most gram-negative bacteria paraformal-dehyde is good to be used whereas ethanol is better for gram-positive bacteria. The fixation step is very important, otherwise degradation of ribosomes is risked. This could lead to underestimation of the fluorescence signal. For some water samples, it is necessary to perform enzymatic digestion after fixation. Especially this is required for samples with very dense biofilm formations that impede direct diffusion of the probe into the target cell. Enzymes like lysozyme or trypsin break down most polysaccha-rides and proteins, respectively, and therefore can enhance the transport of the probe through the biofilm into the cell. The hybridization step is then directly carried out on the sample itself. Therefore the sample is spotted on teflon coated microscope slides and air-dried. In a next step, the sample is dewatered through stepwise incubation in ethanol from low to high percentage, i.e. from 50% to 98%, respectively. After de-watering, the hybridization step is carried out through adding a mixture of salts, formamide and detergents, and including the probe directly into the sample, which will be then incubated in the dark for at least two hours. Subsequently, the sample has to be washed of residual probes and can be analyzed under the fluorescence micro-ขอบเขตการ
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