High-throughput determination of dry mass of single bacterial cells by ultrathin membrane resonators

How bacteria are able to maintain their size remains an open question. Techniques that can measure the biomass (dry mass) of single cells with high precision and high-throughput are demanded to elucidate this question. Here, we present a technological approach that combines the transport, guiding and focusing of individual bacteria from solution to the surface of an ultrathin silicon nitride membrane resonator in vacuum. The resonance frequencies of the membrane undergo abrupt variations at the instants where single cells land on the membrane surface. The resonator design displays a quasi-symmetric rectangular shape with an extraordinary capture area of 0.14 mm2, while maintaining a high mass resolution of 0.7 fg (1 fg = 10−15 g) to precisely resolve the dry mass of single cells. The small rectangularity of the membrane provides unprecedented frequency density of vibration modes that enables to retrieve the mass of individual cells with high accuracy by specially developed inverse problem theory. We apply this approach for profiling the dry mass distribution in Staphylococcus epidermidis and Escherichia coli cells. The technique allows the determination of the dry mass of single bacterial cells with an accuracy of about 1% at an unparalleled throughput of 20 cells/min. Finally, we revisit Koch & Schaechter model developed during 60 s to assess the intrinsic sources of stochasticity that originate cell size heterogeneity in steady-state populations. The results reveal the importance of mass resolution to correctly describe these mechanisms.
This work was supported by the European Union’s Horizon 2020 Research and Innovation Program under Grant Agreement No. 731868—VIRUSCAN and by the ERC CoG Grant 681275 “LIQUIDMASS”. We acknowledge the service from the Micro and Nanofabrication Laboratory an X-SEM laboratory at IMN-CNM funded by the Comunidad de Madrid (Project S2018/NMT-4291 TEC2SPACE) and by MINECO (Project CSIC13-4E-1794 with support from FEDER, FSE). E.G.S. acknowledges financial support by the Spanish Science and Innovation Ministry through Ramón y Cajal grant RYC-2019-026626-I.