Antibiotic Resistance Protocols (Methods in Molecular Biology)

150

1. Collect freshly laid embryos from the fish tank into a petri dish
   (see Note 7).


2. Decant aquarium water from the dish and refill plate with fresh
   E3 solution.


3. Transfer embryos into fresh petri dishes (approximately 60
   embryos per dish), use a stereomicroscope to ensure embryos
   are at the similar developmental stage.


4. Top up with E3 and place petri dishes into incubator, kept at a
   constant 28 °C.

Microinjections of morpholinos are performed using a pneumatic

micropump, a micromanipulator and a dissecting microscope (sim-

ilarly to S. aureus intravenous injections).

1. Collect zebrafish embryos from aquarium facility promptly
   after eggs being laid to obtain eggs at no later than 1–4 cell
   stage.


2. Load a glass injection needle with approximately 3 μL of mor-
   pholino solution using 20 μL microloader pipette tip.


3. Mount the needle on the injector nozzle installed on the
   micromanipulator.


4. Break the needle to ideally form oblique angled tip (see Note
   8 ).


5. Calibrate the droplet size using a calibration slide (see Note 9).


6. Using an edge of microscopic slide placed inside a petri dish
   lid, line up approximately 50 embryos against the side of the
   slide and remove excess liquid.


7. Inject 0.5–1 nL of morpholino solution into the center of
   embryo yolk (see Note 9).


8. After injections, remove the lined-up embryos from the micro-
   scopic slide edge by gently applying E3 using a Pasteur pipette.


9. Transfer injected eggs into a fresh petri dish.


10. Repeat steps 5–8, depending on number of knockdown
    embryos required (see Note 10).

For within-host bacterial population studies use wild-type isogenic

strains labeled with either different antibiotic markers (construc-

tion described in McVicker et al. submitted protocol) or with dif-

ferent fluorescence markers [ 6 ].

1. Grow an overnight starter culture by inoculating a few bacte-
   rial colonies into in 5–10 mL of BHI and placing into 37 °C
   with 250 rpm orbital shaking (see Note 11).


2. Inoculate 50 mL of fresh BHI with the 0.5 mL of starter cul-
   ture (1:100) and grow for 2 h until the optical density at
   600 nm (OD 600 ) reaches approximately 1 (see Note 12).

3.1 Maintenance
of Embryonic
Zebrafish

3.2 Morpholino
Injections

3.3 Preparation
of Bacterial Inoculum

Tomasz K. Prajsnar et al.