What is the main reason E. coli cysts were barely visible on a permanent stained smear?

The main reason E. coli cysts were barely visible on a permanent stained smear can be attributed to the fixation process. If the cysts were poorly fixed, their structural components might not adhere well to the slide or retain their morphological features during the staining process. Proper fixation is crucial in microscopy because it preserves the cellular structure and enhances contrast, allowing for better visualization of the organisms under a microscope. If fixation is inadequate, the cysts may appear distorted or may not take up the stain effectively, leading to a lack of visibility.

The other considerations, while they may be relevant in other contexts, do not address the specific issue of visibility tied to the staining process as effectively.