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The page below is a sample from the LabCE course Microbial Identification Using MALDI-TOF MS. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Specimen Preparation-Direct Spotting

A variety of methods can be used to prepare a microorganism for MALDI-TOF MS analysis. These include:
  1. Direct microorganism spotting on the target
  2. Direct formic acid overlay on the organism on the target
  3. Full chemical protein extraction
The process of extracting protein for analysis and microorganism identification varies by microorganism type and will be discussed accordingly.
Irrespective of the method used, once matrix has been applied to the template, our data shows storage as long as eight hours does not affect identifications.
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 a-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 (Tan et al. 2012). While there is a slight learning curve, optimal microorganism spotting requires smooth application from the target colony with an acceptable burden of microorganism. Also note, 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.
Many laboratories apply two spots per sample to increase reliability in identifications.