I would like to do some CBD staining for microscopy.
My protocol says that I should add formaldehyde methanol to my water samples and fix for 1h before filtering through 0.5um polycarbonate filters. Is it possible to reverse this and first filter and then fix the filters somehow? I am worried about the large amount of toxic waste I produce by incubating 500 ml water samples in formaldehyde-methanol.
I am not familiar with CBD staining or what it stands for.
I think you should be able to filter the water sample first through the filter.
Next, filter about 25-50 ml of formaldehyde-methano through the filter.
This will fix the the organisms on to the filter.
According to Davis and Sieburth (1982), many eukaryotic picoplankton such as flagellates that lack a rigid cell wall cannot survive filtration or freezing without fixation.
I always refer "Handbook of Methods in Aquatic Microbial Ecology".
All depend on the type of organisms you want to collect.
In a previous work, to isolate bacteria from marine water samples, I kept the samples (500 mL bottles) cooled on ice (4°C) and used 100 ml on small filter (0.22-0.4 um for bacteria maybe better) using air pump system, with 4 or 5 replicates. I used autoclaved graduated to measure the 100 ml volume and this tool was rinced with the sample water before use, to avoid contamination. After filtration, the wet filter was placed into an Eppendorf using sterilized pincette, then I added 1 ml of glutarldehyde / or formaldehyde to fix the cells. Resuspend cells in Eppend. Then bacteria are counted. They also can be stocked at -80°C in glycerol solution.
For protist, flagellates or other picoplankton, as Kazama san pointed out, you need to fix them before gentle filtration (use low pressure) because the cell is easily disrupted. You can collect cells by centrifugation then remove supernatant and fix the cells.
Many thank's for your replies! I am interested in cyanobacterial cells and fungal sporangia. I suppose these organisms are similarly sensitive as what is described by Davis and Sieburth (1982) as Takehiro mentioned (thank you!). Maybe, I can filter with very very low underpressure and then fix. The problem is that I cannot do a trial run, stain and see if it worked. I will take a look in "Handbook of Methods in Aquatic Microbial Ecology".
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