We use cookies on our website. Some are necessary for the operation of the website. You can also allow cookies for statistical purposes. You can adjust the data protection settings or agree to all cookies directly.
Data Protection Declaration
Imprint
Applications of flow cytometry in food microbiology
Project
Project code: MRI-MBT-08-1040 FlowFood2020
Contract period: 01.09.2019
- 31.08.2021
Purpose of research: Applied research
For the last 10 years, flow cytometry has also proven to be a powerful tool in food microbiology for the rapid analysis of bacterial populations at the cellular level. The aim of the project is to adapt this methodology to the following, different questions.
- WP1: Determination of absolute bacteria cell counts
The live-dead assay for the detection and enumeration of bacteria, as well as a determination of their physiological status, is well established. With the help of counting beads, it is possible to accurately and precisely determine bacteria concentration. With some adaptations, we have successfully enumerated bacteria in various food matrices including meat juice and milk.
- WP2: Bacteriophage as Biosensors
A fast and easy method to fluorescently tag bacteriophage has been established. With the tagged bacteriophage (PMBT14), a protocol to specifically fluorescently label the target bacteria was optimized. Initial problems of low specificity were overcome by making incremental improvements to the buffers and incubation protocol. It is now possible to accurately differentiate the host bacteria (Pseudomonas fluorescens DSM 50090) from other related and unrelated bacteria using flow cytometry and fluorescence microscopy. Using a confocal fluorescence laser microscope, it was even possible to observe the tagged bacteriophage as green fluorescent dots that bound to and infected its host bacteria. As the bacteriophage binding is very specific, it will be necessary to widen the repertoire of bacteria in order for this technique for routine bacteria identification. This can be done by using multiple bacteriophages as a bacteriophage cocktail.
Beside the use of tagged bacteriophages as biosensors to specifically identify bacteria, it is very likely that this technology will find application on other research topics. For example, the method to fluorescently tag bacteriophage is very easily transferable to other types of viruses and might help in virus-host interaction studies. Tagged bacteriophages binding to and infection of bacteria could be a rapid method to perform phage typing for use in biocontrol or phage therapy.
- WP3: Detection and characterization of viable but not culturable (VBNC) state in Listeria monocytogenes
In order to replicate previously published data, the VBNC state in L. monocytogenes was initially attempted by starvation of the cells in water. This is very time intensive and such a process could take months. After building up some experience with these attempts, it was clear that alternative methods for the induction and determination of the VBNC state in L. monocytogenes to be established. Using propidium iodide, which is a component of the live-dead assay, membrane integrity could be determined. Using CFDA-SE, an esterase assay has been established. A living cell will still possess esterase activity, and converts the CFDA-SE to a fluorescent form. This fluorescent is stable and could also be used to track cellular uptake of the bacteria. Glucose uptake assays using fluorescent glucose analog 2-NBDG is still to be tested. Using the available assays on hand, various disinfection agents will be tested at suboptimal dosis to induce the VBNC state in L. monocytogenes.
Initial attempts to use cell culture assays with human Caco-2 cells to detect infectiousness of L. monocytogenes were successful. Further experiments using VBNC bacteria will have to be performed in order to test for cellular uptake and possible reinvigoration of infectiousness.
Section overview
Subjects
- Food microbiology
Framework programme
Funding programme
Excutive institution
MRI - Department of Microbiology and Biotechnology (MRI-MBT)
