MALDI imaging of microbial colonies on agar media is a growing application for mass spectrometry imaging; however, matrix deposition on agar is often complicated. Typically, matrix is applied to wet agar using a fine sieve to dust matrix powder over the top of the slide, which often yields low to medium signal intensity and poor reproducibility. Using the HTX TM-Sprayer to deposit matrix on microbial colonies prior to mass spectrometry imaging allows for easier and more reproducible matrix application.