Precise analysis of the aquatic cells and their responses to the toxic chemicals, i.e., water disinfective agents, is of crucial importance due to their role in the ecosystem. We demonstrate the application of the droplets based millifluidic tool for isolating and longtime monitoring of single Paramecium tetraurelia cells using a large number of water-in-oil emulsion droplets. Due to the automated monitoring of the fluorescence signal, the droplets containing cells are distinguished from the empty reservoirs. A viability indicator is used to follow the metabolic dynamic of the cells in every single droplet. Finally, we perform ecotoxicity tests in droplets, exposing the encapsulated paramecia cells to silver nitrate for determination of EC50 levels, and compare the output with the conventional microtiter plate assay.
References
1.
J.
Johnke
, Y.
Cohen
, M.
de Leeuw
, A.
Kushmaro
, E.
Jurkevitch
, and A.
Chatzinotas
, “Multiple micro-predators controlling bacterial communities in the environment
,” Curr. Opin. Biotechnol.
27
, 185
–190
(2014
).2.
C. R.
Curds
, “The ecology and role of protozoa in aerobic sewage treatment processes
,” Annu. Rev. Microbiol.
36
(1
), 27
–28
(1982
).3.
L. M.
Campbell
, R. E.
Hecky
, J.
Nyaundi
, R.
Muggide
, and D. G.
Dixon
, “Distribution and food-web transfer of mercury in Napoleon and Winam gulfs, Lake Victoria, East Africa
,” J. Great Lakes Res.
29
, 267
–283
(2003
).4.
N. S.
Rodriguez
, K. R.
Bright
, D. C.
Slack
, D. R.
Uhlmann
, and C. P.
Gerba
, Appl. Environ. Microbiol.
74
, 1639
–1641
(2008
).5.
O.
Bondarenko
, K.
Juganson
, A.
Ivask
, K.
Kasemets
, M.
Mortimer
, and A.
Kahru
, “Toxicity of Ag, CuO and ZnO nanoparticles to selected environmentally relevant test organisms and mammalian cells in vitro: A critical review
,” Arch. Toxicol.
87
, 1181
–1200
(2013
).6.
N.
Musee
, M.
Thwala
, and N.
Nota
, “Nanowastes and the environment: Potential new waste management paradigm
,” J. Environ. Monit.
13
(5
), 1164
–1183
(2011
).7.
C.
Burkart
, W.
von Tümpling
, T.
Berendonk
, and D.
Jungmann
, “Nanoparticles in wastewater treatment plants: A novel acute toxicity test for ciliates and its implementation in risk assessment
,” Environ. Sci. Pollut. Res.
22
(10
), 7485
–7494
(2015
).8.
N. Rao
Amanchi
, “A low cost microbiotest for screening behavioural and ecotoxicological responses of Paramecium caudatum and Oxytricha fallax to azadirachtin
,” Adv. Appl. Sci. Res.
1
(2
), 124
–131
(2010
).9.
Z. L.
Gong
, Y.
Chen
, Y.
Yan
, S. Y.
Pei
, D.
Wu
, M.
Zhang
, and Q.
Wang
, “Toxicity of three heavy metal pollutants of the pharmaceutical wastewater to Paramecium caudatum
,” Adv. Mater. Res.
937
, 571
–577
(2014
).10.
C.
Bueno
, M. L.
Villegas
, S. G.
Bertolotti
, C. M.
Previtali
, M. G.
Neumann
, and M. V.
Encinas
, “The excited-state interaction of resazurin and resorufin with amines in aqueous solutions. Photophysics and photochemical reactions
,” Photochem. Photobiol.
76
(4
), 385
–390
(2002
).11.
G. M.
Whitesides
, “Overview the origins and the future of microfluidics
,” Nature
442
, 368
–373
(2006
).12.
P.
Garstecki
, M. J.
Fuerstman
, H. A.
Stonec
, and G. M.
Whitesides
, “Formation of droplets and bubbles in a microfluidic T-junction—Scaling and mechanism of break-up
,” Lab Chip
6
, 437
–446
(2006
).13.
D. R.
Link
, E.
Grasland-Mongrain
, A.
Duri
, F.
Sarrazin
, Z.
Cheng
, G.
Cristobal
, M.
Marquez
, and D. A.
Weitz
, “Electric control of droplets in microfluidic devices
,” Angew. Chem., Int. Ed.
45
, 2556
–2560
(2006
).14.
S.
Jakiela
, T. S.
Kaminski
, O.
Cybulski
, D. B.
Weibel
, and P.
Garstecki
, “Bacterial growth and adaptation in microdroplet chemostats
,” Angew. Chem.
125
, 9076
–9079
(2013
).15.
L.
Boitard
, D.
Cottinet
, C.
Kleonschmitt
, N.
Bremond
, J.
Baudry
, G.
Yvert
, and J.
Bibette
, “Monitoring single-cell bioenergetics via the coarsening of emulsion droplets
,” Proc. Natl. Acad. Sci. U.S.A.
109
, 7181
–7186
(2012
).16.
S.
Köster
, F. E.
Angilè
, H.
Duan
, J. J.
Agresti
, A.
Wintner
, C.
Schmitz
, A. C.
Rowat
, C. A.
Merten
, D.
Pisignano
, A. D.
Griffiths
, and D. A.
Weitz
, “Drop-based microfluidic devices for encapsulation of single cells
,” Lab Chip
8
, 1110
–1115
(2008
).17.
G. S.
Du
, J. Z.
Pan
, S. P.
Zhao
, Y.
Zhu
, J. M. J.
den Toonder
, and Q.
Fang
, “Cell-based drug combination screening with a microfluidic droplet array system
,” Anal. Chem.
85
, 6740
–6747
(2013
).18.
M. S.
Art
, D. K.
Kang
, J.
Hong
, H.
Park
, R. J.
Leatherbarrow
, J. B.
Edel
, S. I.
Chang
, and A. J.
deMello
, “Analysis of protein-protein interactions by using droplet-based microfluidics
,” ChemBioChem
10
, 1605
–1611
(2009
).19.
J. J.
Agresti
, E.
Antipov
, A. R.
Abate
, K.
Ahn
, A. C.
Rowat
, J. C.
Baret
, M.
Marquez
, A. M.
Klibanov
, A. D.
Griffiths
, and D. A.
Weitz
, “Ultrahigh-throughput screening in drop-based microfluidics for directed evolution
,” PNAS
107
, 4004
–4009
(2010
).20.
S. P.
Damodaran
, S.
Eberhard
, L.
Boitard
, J. G.
Rodriguez
, Y.
Wang
, N.
Bremond
, J.
Baudry
, J.
Bibette
, and F.-A.
Wollman
, “A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii
,” PLoS One
10
(3
), 1
–28
(2015
).21.
L.
Baraban
, F.
Bertholle
, M. L. M.
Salverda
, N.
Bremond
, P.
Panizza
, J.
Baudry
, J. A. G. M.
de Visser
, and J.
Bibette
, “Millifluidic droplet analyser for microbiology
,” Lab Chip
11
, 4057
–4062
(2011
).22.
C. D.
Kelly
and O.
Rahn
, “The growth rate of individual bacterial cells
,” J. Bacteriol.
23
(2
), 147
–153
(1932
).23.
M. E.
Lidstrom
and Mi. C.
Konopka
, “The role of physiological heterogeneity in microbial population behavior
,” Nat. Chem. Biol.
6
, 705
–712
(2010
).24.
F.
Buettner
, K. N.
Natarajan
, F. P.
Casale
, V.
Proserpio
, A.
Scialdone
, F. J.
Theis
, S. A.
Teichmann
, J. C.
Marioni
, and O.
Stegle
, “Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells
,” Nat. Biotechnol.
33
, 155
–160
(2015
).25.
C. O.
Hendren
, X.
Mesnard
, J.
Dröge
, and M. R.
Wiesner
, “Estimating production data for five engineered nanomaterials as a basis for exposure assessment
,” Environ. Sci. Technol.
45
(7
), 2562
–2569
(2011
).26.
C. O.
Hendren
, A. R.
Badireddya
, E.
Casmanb
, and M. R.
Wiesner
, “Modeling nanomaterial fate in wastewater treatment: Monte Carlo simulation of nano-Ag concentration as a function of surface chemistry
,” Sci. Total Environ.
449
, 418
–425
(2013
).27.
F.
Gottschalk
, T.
Sonderer
, R. W.
Scholz
, and B.
Nowack
, “Modeled environmental concentrations of engineered nanomaterials (TiO2, ZnO, Ag, CNT, fullerenes) for different regions
,” Environ. Sci. Technol.
43
(24
), 9216
–9222
(2009
).28.
N. C.
Mueller
and B.
Nowack
, “Exposure modeling of engineered nanoparticles in the environment
,” Environ. Sci. Technol.
42
(12
), 4447
–4453
(2008
).29.
E. S.
Kaneshiro
, L. S.
Beischel
, S. J.
Merkel
, and D. E.
Rhoads
, “The fatty acid composition of Paramecium aurelia cells and cilia: Changes with culture age
,” J. Protozool.
26
(1
), 147
–158
(1979
).30.
A. T.
Soldo
and W. J.
Van Wagtendonk
, “A method for the mass collection of axenically cultivated Paramecium aurelia
,” J. Protozool.
16
(3
), 500
–506
(1969
).31.
Biological Test Method, Environment Canada, EPS 1/RM/33 Second Edition,
2013
.32.
Y.
Huang
, C. C.
Reyes-Aldasoro
, G.
Persoone
, and D.
Wlodkowic
, “Integrated microfluidic technology for sub-lethal and behavioral marine ecotoxicity biotests
,” Proc. SPIE
9518
, 95180F
(2015
).33.
S. K.
Mahto
, V.
Charwat
, P.
Ertl
, B.
Rothen-Rutishauser
, S. W.
Rhee
, and J.
Sznitman
, “Microfluidic platforms for advanced risk assessments of nanomaterials
,” Nanotoxicology
9
(3
), 381
–395
(2015
).34.
F.
Zhu
, A.
Wigh
, T.
Friedrich
, A.
Devaux
, S.
Bony
, D.
Nugegoda
, J.
Kaslin
, and D.
Wlodkowic
, “Automated lab-on-a-chip technology for fish embryo toxicity tests performed under continuous microperfusion (μFET)
,” Environ. Sci. Technol.
49
(24
), 14570
–14578
(2015
).35.
Y.
Skhiri
, P.
Gruner
, B.
Semin
, Q.
Brosseau
, D.
Pekin
, L.
Mazutis
, V.
Goust
, F.
Kleinschmidt
, A.
El Harrak
, J. B.
Hutchison
, E.
Mayot
, J. F.
Bartolo
, A. D.
Griffiths
, V.
Taly
, and J. C.
Baret
, “Dynamics of molecular transport by surfactants in emulsions
,” Soft Matter
8
, 10618
–10627
(2012
).36.
See supplementary material at http://dx.doi.org/10.1063/1.4944869 for movies and additional figures. Supplementary materials contain several subchapters: 1. Detection of the optical signal. Enclosed Fig. 1S describes the principle for collection of the emission light, using virtual filtering. 2. Adjustment of cell density. 3. Determination of the droplets' filling factor. Enclosed Fig. 2S describes the fill rate of the droplet compared with theoretical values of the Poisson distribution for the cell densities. 4. Ecotoxicity assays. Enclosed Fig. 3S shows the fluorescence signal of droplets with different concentrations of AgNO3. Supplementary video S1 demonstrates the single paramecia encapsulated inside a droplet. Supplementary video S2 corresponds to the case, when two paramecia cells filling the droplet. Finally, supplementary video S3 shows the division events of single paramecia cells, encapsulated inside the droplet.
© 2016 AIP Publishing LLC.
2016
AIP Publishing LLC
You do not currently have access to this content.