155- Volumes of 0.5–1 nL are typically used and morpholino con-
centrations vary between 200 μM and 1 mM to effectively
knock down gene activity without causing off-target effects.
0.5% (v/v) solution of Phenol Red can be added to the mor-
pholino solution (at 1:10 ratio) to “colorize” the injecting
material and therefore facilitate the injections. Same dye can be
added to bacterial suspensions. - Mortality rates of MO-injected embryos may reach 50%, there-
fore inject double the number required. - There is no need to use antibiotics if genomic markers are
used. Bacteria with antibiotic markers located on a plasmid still
require supplementing appropriate antibiotics in liquid media. - OD of 1 at 600 nm corresponds to bacterial concentration of
approximately 2 × 108 CFU/mL. - Carefully pinch one side of the fish chorion with one hand and
‘peel’ it open with the other. If fish is damaged in any way,
remove immediately into bleach solution (should be prepared
in a bucket near your setup). - Very carefully decant the embryos out onto a line of methylcel-
lulose. Do not break the surface tension between pipette and
methylcellulose. Slowly drag the pipette back across the line of
methylcellulose. This should separate the fish out from the ini-
tial drop you made. Use this method all the way along the line
and you should have a well dispersed line of fish. - Prior to testing the dose control, ensure needle is unblocked
and is producing visible suspension by ejecting into methylcel-
lulose. Number of pulses injected into the PBS solution should
depend on expected bacterial counts. - The needle should approach the embryo from the dorsolateral
side (see Fig. 2a). Between injections into embryos, keep ensur-
ing that needle is unblocked and is producing visible suspen-
sion by ejecting into methylcellulose. - To remove zebrafish embryos from methylcellulose use a
Pasteur pipette to place a few drops of E3 onto the line of fish.
Carefully aspirate the fish into the pipette. Try not to bring any
air bubbles with the injected zebrafish. Carefully place the fish
back into a petri dish with fresh E3 solution, pumping up and
down slowly to aid their removal from the pipette. - Check whether embryos are not still stuck in viscous methyl-
cellulose material. If they still are, use a pipette to gently stream
some E3 over them to remove them. - In experiments regarding determination of bacterial numbers
within infected embryos, it is important to collect an embryo
with a known amount of E3 medium (for subsequent serial
dilution factors and colony counts). It is convenient, therefore,
Infection Model in Zebrafish