Rapid and accurate identification of bacterial and yeast isolates is needed to advance public health. Touch spray is a novel ambient ionization method developed. It is performed by using any metal probe capable of transferring material from the object of interest to the surface of the probe for analysis. In this particular application, the bacterial colonies are directly sampled from culture dishes. Touch spray ionization can also be used to detect suspended micro-organisms in various solvents, such as methanol, water, dimethyl formamide and acetonitrile. Detected lipid profiles are characteristic for various bacteria genera. Touch spray has been utilized efficiently to distinguish among gram-negative and gram-positive bacteria in both of positive mode and negative modes. As an example of species differentiation, Staphylococcus aureus, Staphylococcus hominis, and Staphylococcus epidermidis are readily distinguished through their lipid patterns utilizing multivariant statistical analyses. In a parallel study, individual bacterial colonies are transferred to a filter paper cut into a sharp point and analyzed using paper spray-MS. Comparison of the mass-spectral data obtained from paper spray and touch spray ionization techniques show high degree of similarity and reproducibility.