Abstract
Isothermal microcalorimetry (IMC) is regarded as a promising diagnostic tool for fast detection of bacterial contaminations in various matrices. Based on a reference detection time determined by visual inspection of bacterial growth on solid medium, we investigated the strict aerobically growing Pseudomonas putida mt-2 KT2440 in a static 4-mL ampoule system on solid and liquid media by IMC to evaluate the three main options to reduce the detection time of bacterial contamination. Firstly, the sample preparation (e.g. membrane filtration) leads to an elevated number of bacteria in the measuring ampoule and thus to a reduced detection time. Secondly, the amount of substrate and oxygen has been investigated by varying the filling volume of medium in the calorimetric ampoule. Here, we were able to show how biophysical characteristics like the substrate and oxygen diffusion determined the shape of heat flow signals and thus the detection time. Finally, the technical framework determines the sensitivity of the IMC instrument. We examined the impact of four different detection threshold values (2, 10, 50 and 100 µW) on the detection time as a function of the initial number of bacteria presented in the ampoule and the filling volume.
Highlights
A calorimeter is capable of detecting the reaction heat released or taken up by any type of physical, chemical or biological process in real-time [1]
As our study dealt with the detection time of aerobic bacterial growth using microcalorimetry, a reference value for the detection time of P. putida mt-2 KT2440 was determined by CFU counting by five independent observers
It is important to mention that our study aims at the earliest detection of bacterial growth; our considerations are restricted to exponential growth at the beginning of the metabolically determining heat flow
Summary
A calorimeter is capable of detecting the reaction heat released or taken up by any type of physical, chemical or biological process in real-time [1] This has led to numerous applications in a wide range of fields, including life sciences [2,3,4,5,6], materials science [7], biotechnology [8, 9] and medical diagnostics [10, 11]. Detecting bacterial contaminations is possible by monitoring the growth of the bacteria in real time by cultivation in liquid (LC) [15, 19,20,21,22] or solid (SC) [23,24,25] medium This benefits from the exponential growth forming substantial biomass from a single bacterial cell within a short time [26]. We identify the factors influencing the detection time in 4-mL static ampoules in microcalorimetric real-time monitoring using the example of Pseudomonas putida mt-2 KT2440
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