Why was the smear not subjected to heat fixation for negative stain preparation?

Heat fixation is not used for negative stain preparation because the heat can cause the cells to shrink or distort, making it difficult to accurately observe their morphology. Negative staining involves using a negatively charged dye that is repelled by the negatively charged bacterial cell walls, creating a contrast between the cells and the background. Heat fixation is not necessary for negative staining as the cells are not being fixed to the slide, but rather the stain is being used to highlight the cells without altering their structure.

Negative stain preparation does not require heat fixation of the smear because the negative stain solution precipitates around the biological material, creating contrast without the need for heat-induced protein denaturation. Heat fixation, which is typically used in positive staining techniques, aims to preserve the sample's morphology by coagulating proteins and enhancing the adhesion of the specimen to the slide. In contrast, negative staining relies on the exclusion of the stain from the hydrated regions of the sample, resulting in a dark background surrounding the unstained components. Heat fixation would disrupt this delicate balance and potentially damage the specimen's structure.