A Comparison of Heat versus Methanol Fixation for Gram Staining Bacteria

Gram staining bacteria is a fundamental technique introduced in general biology and microbiology laboratory courses. Two common problems students encounter when Gram staining bacteria are (1) having a difficult time locating bacterial cells on the microscope slide and (2) over-decolorizing bacterial cells during the staining procedure such that gram-positive bacteria, which should appear purple in color, are pink instead. In this study, we examined whether the method of fixation (heat versus methanol) that is used to adhere bacteria to the slide prior to staining might influence the staining results. We found that significantly greater numbers of "Staphylococcus aureus" (gram-positive) and "Escherichia coli" (gram-negative) cells adhered to slides following methanol fixation compared to slides that were heat-fixed. Additionally, methanol-fixed cells of "Staphylococcus aureus" were consistently stained the correct color (a dark purple) while the staining of heat-fixed cells was more variable with cells ranging in color from purple to pink. Overall, our results indicate that students are more likely to successfully visualize and Gram stain bacteria if the cells are fixed with methanol rather than heat. (Contains 3 figures.)