Classification of Bacteria Using FT-IR

The classification of bacteria is generally based on the morphology and biochemical reactions of the bacteria. However, these measurements are time consuming, and require training and expertise. Infrared (IR) spectroscopic measurements of bacteria followed by a formal chemometrics analysis could offer advantages of speed and consistency. Such an analysis was first tried in the late 1950s, with some success, but with limitations imposed both by the instrumentation and by the post-processing tools available (1). The IR analysis of bacteria underwent resurgence in the 1980s with the advent of Fourier transform infrared (FT-IR) instruments. The higher signal to noise and generally better performance, coupled with significant advances in computing power, made the application to bacterial analysis feasible. Hopkinson et al., and Naumann et al. were pioneers (1988) in using FT-IR for identification and differentiation of microorganisms (2,3). Recent work has shown the method is effective with pathogenic and nonpathogenic bacteria. The food industry, especially groups like cheese producers, has a high interest in bacterial screening. The basic concern is that the correct strain of bacteria, and no other, is present in a food product. Europe is leading a push for tighter regulations that would require food producers to increase the sampling frequency. The potential speed and consistency advantages of FT-IR analyses are thus of great interest. The standard infrared methodology is based on transmission of light through a bacteria sample dried onto a ZnSe window. Regardless of sample preparation methods, only about 50 samples per day can be processed. However, Thermo Scientific offers a powerful set of high-throughput screening (HTS) hardware and software tools which could greatly accelerate this rate, helping meet the needs of the increased sample load. The throughput improvement is largely realized through automation of the data collection and analysis. The hardware utilizes a Micro Well Plate Reader stage inserted into a NicoletTM 6700 FT-IR spectrometer, as seen in Figure 1. The sample plates and reader stage are based on the industry standard 96-well plate footprint, which allows the user to mount many samples on a single IR-transmission plate. With proper loading, multiple strains can be tested simultaneously, and replicate measurements can be made automatically to mitigate reproducibility concerns. The Array AutomationTM software allows a high level of control over the experiment. Users can choose the standard 96-well format or define customized well-plate formats for data collection. Data collection parameters can be entered for the whole plate, or can be tailored for each well. Array Automation interfaces directly with Thermo Scientific’s OMNICTM Spectroscopy software for collection and processing, and to the TQ AnalystTM chemometrics software for analysis. A plate can be loaded, and the data collection and analysis will then proceed without further user intervention.