Tiny Cells, Big Challenges: Mastering Microscopy for Low Biomass Samples

First, let's talk about the tiny world of cells. When scientists work with low-density, low-biomass material, they face a big challenge. The cells they study are often as scarce as the background contamination in their lab. This makes it super hard to get accurate cell counts. Imagine trying to spot a few needles in a haystack. That's what researchers deal with when they're near the detection limit. Background contamination becomes a major issue. It's like trying to find a few friends in a crowded room full of strangers. So, what did they do? They decided to tackle this problem head-on. They focused on the cell mounting process for epifluorescence microscopy. This process involves microscope slides, coverslips, and various solutions. First, they checked out the microscope slides and coverslips. They tested them before and after cleaning them with autoclaving, detergent, and ethanol. They also tested the solutions used in sample mounting. These included 4', 6-diamidino-2-phenylindole, phosphate buffered saline, and immersion oil. They tested these solutions before and after autoclaving, as well as after filtering them once and thrice with a 0. 2 µm membrane filter. What did they find? By combining detergent and ethanol rinses of glassware and triple filtering of all solutions, they were able to cut down the background contamination. They managed to reduce it from 1×104(±4. 3×103) cells to 302(±312) cells per filter paper. That's a huge improvement! But they didn't stop there. They wanted to make sure their method worked. So, they tested it with low biomass glacial sediment samples from Renardbreen, Svalbard. The cell concentrations were 1. 8×105(±2. 9×104) cells g-1. This was close to the detection limit of epifluorescence microscopy. This whole process shows how important it is to reduce background contamination. It's crucial for getting accurate cell counts. By doing this, scientists can make sure their findings are reliable.