Direct Spotting

Direct spotting of the microorganism is the simplest method. It involves placing the organism from an isolated colony (working with a pure culture is critical) on a spot/well of the testing target, allowing it to dry, and then overlaying the microorganism with 1–2 µL of α-Cyano-4-hydroxycinnamic acid (CHCA). CHCA is a volatile solution that dries within three to five minutes and kills microorganisms. This process has been shown to produce reliable identifications for both gram positive and gram negative bacteria.³

While there is a slight learning curve, optimal microorganism spotting requires a smooth application from the target colony with an acceptable burden of microorganism. Also, when applying the microorganism to the target plate, it is important to distribute it evenly, as this will help minimize three-dimensional structures which can have a detrimental effect on the quality of spectra capture and result in unsuccessful identifications. Common techniques for inoculating organisms onto the target wells include the use of toothpicks, swabs, pipette tips, and plastic loops. All of these techniques can be used successfully but require some practice. VITEK has now produced a product called PICKME™ with disposable plastic tips called "nibs". At an American Society for Microbiology (ASM) conference in 2019, bioMerieux presented a poster that showed using this tool to prepare the spots resulted in 26–42% shorter time for sample spot preparation and reduced retesting by 33% due to better deposit uniformity.

Many laboratories apply two spots per sample to increase reliability in identifications.¹⁰