151- Transfer 40 mL of culture into 50 mL centrifuge tube, and
spin down at 5000 × g for 10 min at 4 °C. - Based on OD 600 readings, resuspend samples in appropriate
amount of PBS (see Note 12) to achieve bacterial concentra-
tions of 1.5 × 109 CFU/mL. - Mix together an appropriate amount of each strain on ice to
create a suspension of bacteria at the ratio required by your
experiment (usually 1:1:1). Vortex well, both before and after
mixing. - When zebrafish embryos reach approximately 28 hpf, using a
pair of forceps, dechorionate them manually under the stereo-
microscope (see Note 13). Remove empty chorions from the
petri dish afterward. - 5–10 min prior to bacterial injections at 30 hpf, anesthetize
zebrafish embryos by addition of tricaine to a final concentra-
tion of 0.02% (w/v). Due to photosensitivity of tricaine, cover
the dish immediately with a dark object to prevent exposure to
light. - Apply an approx. 1 cm wide line of methylcellulose onto a
microscope slide from one end to the other. - Collect the anesthetized zebrafish embryos into a 3 mL Pasteur
pipette and place approximately 30 embryos along the line of
methylcellulose (see Note 14). Remove any excess liquid water
with paper tissue. - Load a glass injection needle with approximately 5 μL of previ-
ously prepared bacterial suspension using 20 μL microloader
pipette tip. - Mount the needle on the injector nozzle installed on the
micromanipulator. - Break the needle tip to ideally to form oblique angled tip (see
Note 8). - Calibrate the droplet size using a calibration slide.
- Place the slide with mounted fish on methylcellulose onto the
injecting stage. - Inject a number of pulses into 1 mL of PBS for dose control
(see Note 15) prior to zebrafish injections. - Inject bacteria into zebrafish yolk sac circulation valley (see
Fig. 2a and Note 16). - After injecting a set of embryos repeat step 5 for dose control
at the end of the set. - Remove the injected set of embryos from the slide, remove
embryos from methylcellulose (see Note 17) and transfer
them into E3 solution in a petri dish.
3.4 Preinfection
Preparation
of Zebrafish Embryos
3.5 Bacterial
Injections
into Zebrafish
Embryos
Infection Model in Zebrafish