Identifying bacteria using a flow cytometer

A new, easy-to-adapt and inexpensive analytical procedure has been developed by researchers from the Institute of Physical Chemistry of the Polish Academy of Sciences in Warsaw. The main role is played by innovative bioconjugates -- luminescent, magnetic microparticles coated with appropriately selected bacteriophages.

The detection device in the new technique for identifying bacteria is a flow cytometer. Despite the seemingly formidable name it is quite a simple and relatively inexpensive piece of equipment, available in many hospitals, where it is used commonly in blood tests. In the cytometer the sample is passed through a nozzle from which the stream that flows is so narrow that all the larger particles in the solution, particularly cells, have to flow one by one. The stream is lit by lasers and surrounded by detectors that record the light reflected from individual particles, scattered to the sides and emitted by them.

The main problem was to develop a method for labelling the bacteria to be easy to intercept them in the test sample and identify them with great certainty with the aid of the cytometer. To do this, researchers decided to construct special bioconjugates, i.e. complexes formed by the combination of microparticles with biomolecules. The biological element was a bacteriophage, which is a virus infecting a particular species of bacteria (the IPC PAS experimenters used the T4 bacteriophage, attacking Escherichia coli bacteria). The bacteriophages were coupled with microparticles capable of emitting light that could be easily registered on the cytometer and exhibiting magnetic properties. The latter was essential, because it made it possible to separate the bioconjugates from other particles in the sample with a simple magnet.

With the method, the sample -- it can be a physiological fluid obtained from the patient, or a food product, e.g. carrot juice -- is diluted and then a small amount of previously prepared bioconjugates is added to the solution. After this, it takes a short while for the bioconjugates to attach to the bacteria. Next, a magnet is applied to the test-tube with the liquid and all bioconjugates are attracted, including those with the attached bacteria. After pouring out the remainder of the sample and re-dilution of the separated precipitate, the solution is passed through the cytometer.

For further details see:

Marta Janczuk, Łukasz Richter, Grażyna Hoser, Jerzy Kawiak, Marcin Łoś, Joanna Niedziółka-Jönsson, Jan Paczesny, Robert Hołyst. Bacteriophage-Based Bioconjugates as a Flow Cytometry Probe for Fast Bacteria Detection. Bioconjugate Chemistry, 2016; DOI:10.1021/acs.bioconjchem.6b00596

Posted by Dr. Tim Sandle