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.