classified as a Biosafety Level 2 (BSL2) microorganism. It is
cultivated at 32
C; under these conditions, the doubling time
of M. marinum is approximately 10 h.
3. LoFlo is a low fluorescence medium and is less autofluorescent
than HL5c and, therefore, induces less phototoxicity. The use
of this medium should be restricted to time-lapse recordings
lasting up to a few hours. The low nutrient content might cause
cell starvation; so for longer recordings, it is more appropriate
to add 50% HL5c.
4. The design of every part of the InfectChip device has been
described in detail in the following chapter [25]. The coverslips
(25 mm, Menzel-Gl€ aser) are first cleaned with oxygen plasma
treatment and then coated with a resin (SU8 1060 Gersteltech). The resin is then baked following these steps: soft-bake,
photoresist exposure, post-exposure bake, photoresist development, and finally hard bake. For the microfluidic fabrication, a
PDMS elastomer is mixed with a curing agent to a 10:1 ratio.
The mix is poured onto the mold and placed under vacuum.
The microfluidic device is heated up to 80
C for 50 min. Then,
the two connecting holes are made using a biopsy puncher and
the two-silicon tubings are connected.
5. Visualization of rapid events is performed with a spinning disc
confocal microscope (Intelligent Imaging Innovations Marianas
SDC mounted on an inverted microscope (Leica DMIRE2)).
Green and red fluorophores can be visualized thanks to the two
laser lines at 488 and 561 nm, respectively. Both fluorescence
channels can be excited sequentially to avoid interferences in
their detection. The microscope is equipped with the SlideBook
version 6.0 software (Intelligent Imaging Innovations). It allows
a precise control of neutral density and intensity of the lasers as
well as intensification and gain of each individual signal.
6. The doubling time of D. discoideum is about 8–12 h [26]. New
cultures should be started from frozen cell aliquots about every
4–6 weeks.
7. The correlation between bacteria biomass and the fluorescence/luminescence signals was confirmed by the determination of coefficients of correlation between CFU counting,
fluorescence measurements (RFU), bioluminescence (RLU),
and optical density (OD) in M. marinum cultures, and results
can be found in supplementary Fig. S4 of the following article
[18]. M. marinum cultures expressing either the bacterial Lux
or GFP were diluted to an OD600 of 0.2. Samples were taken
at regular intervals during growth. Comparison of CFU/RFU,
CFU/RLU, RFU/OD, RLU/OD, and CFU/OD all demonstrated a linear relation.
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Manon Mottet et al.
cultivated at 32
C; under these conditions, the doubling time
of M. marinum is approximately 10 h.
3. LoFlo is a low fluorescence medium and is less autofluorescent
than HL5c and, therefore, induces less phototoxicity. The use
of this medium should be restricted to time-lapse recordings
lasting up to a few hours. The low nutrient content might cause
cell starvation; so for longer recordings, it is more appropriate
to add 50% HL5c.
4. The design of every part of the InfectChip device has been
described in detail in the following chapter [25]. The coverslips
(25 mm, Menzel-Gl€ aser) are first cleaned with oxygen plasma
treatment and then coated with a resin (SU8 1060 Gersteltech). The resin is then baked following these steps: soft-bake,
photoresist exposure, post-exposure bake, photoresist development, and finally hard bake. For the microfluidic fabrication, a
PDMS elastomer is mixed with a curing agent to a 10:1 ratio.
The mix is poured onto the mold and placed under vacuum.
The microfluidic device is heated up to 80
C for 50 min. Then,
the two connecting holes are made using a biopsy puncher and
the two-silicon tubings are connected.
5. Visualization of rapid events is performed with a spinning disc
confocal microscope (Intelligent Imaging Innovations Marianas
SDC mounted on an inverted microscope (Leica DMIRE2)).
Green and red fluorophores can be visualized thanks to the two
laser lines at 488 and 561 nm, respectively. Both fluorescence
channels can be excited sequentially to avoid interferences in
their detection. The microscope is equipped with the SlideBook
version 6.0 software (Intelligent Imaging Innovations). It allows
a precise control of neutral density and intensity of the lasers as
well as intensification and gain of each individual signal.
6. The doubling time of D. discoideum is about 8–12 h [26]. New
cultures should be started from frozen cell aliquots about every
4–6 weeks.
7. The correlation between bacteria biomass and the fluorescence/luminescence signals was confirmed by the determination of coefficients of correlation between CFU counting,
fluorescence measurements (RFU), bioluminescence (RLU),
and optical density (OD) in M. marinum cultures, and results
can be found in supplementary Fig. S4 of the following article
[18]. M. marinum cultures expressing either the bacterial Lux
or GFP were diluted to an OD600 of 0.2. Samples were taken
at regular intervals during growth. Comparison of CFU/RFU,
CFU/RLU, RFU/OD, RLU/OD, and CFU/OD all demonstrated a linear relation.
198
Manon Mottet et al.