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tiple comparisons (e.g., a range of mutants tested versus one
control parental strain).- For bacterial numbers recovered from infected embryos Mann–
Whitney test is recommended as the numbers do not follow the
Gaussian distribution (two categories of low bacterial numbers
within surviving embryos and high bacterial numbers which
succumb to infection).
4 Notes
- Working stock of tricaine can be kept at room temperature, but
since tricaine is light-sensitive, it needs to be kept in the dark
(the container should be wrapped in tin foil). In addition, a
petri dish with anesthetized zebrafish embryos should be also
covered to protect it from direct light. - To facilitate methylcellulose solubilization, use several rounds
of partial freezing and thawing to dissolve the white clumps of
methylcellulose. Freeze for approximately 30 min (until layer
of ice is formed on the surface) and mix thoroughly while
thawing. Repeat this action for around five times. - We do not recommend the use of forceps with very sharp tips
(e.g., “biology” type in Fine Science Tools catalogue) as they
tend to damage zebrafish embryos while dechorionating (see
Subheading 3.4). - Use pipettes with an opening of approx. 2 mm, as smaller size
could be damaging to zebrafish embryos while transferring. - In order to achieve optimal needle thickness and shape for suc-
cessful injections, establishing the correct parameters on the
needle puller is essential. Use thin and long needles for intrave-
nous injections. Our settings on a Sutter Instrument Model
P-97 puller are: Heat 430, Pull 225, Velocity 150, Time 225. - The glass bottom should be very thin (ideally glass thickness
No. 0) to enable high magnification imaging using 60× oil
objective. - For morpholino injections, zebrafish embryos have to be col-
lected very early (within half an hour of laying), to make sure
injections are performed before embryos reach the 4-cell stage.
If MO injections are not required, embryos can be collected
up to a few hours later. - Using the dissecting microscope set on the highest magnifica-
tion (e.g., 50×), gently scrape the tip of the glass needle with
forceps. Ensure that only small amount of the needle tip is
broken off.
Tomasz K. Prajsnar et al.