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importance of confirming morpholino findings with mutant exper-
iments [ 5 ].
Recently, several methods have been established to understand
the pathogen dynamics within an infected host. Multiple geneti-
cally labeled wild-type isogenic tagged strains (WITS) have been
successfully applied to modeling the dynamics of Salmonella
enterica infection. This approach has shown a great potential to
increase the understanding of the mechanisms that underpin infec-
tion processes [ 6 ]. Eight WITS simultaneously injected into a sin-
gle host permitted the enhanced resolution of bacterial
subpopulation tracing and dynamics in vivo. Alternatively, isogenic
strains labeled with different antibiotic resistance markers have
been used for population dynamic studies [ 7 , 8 ]. The use of genet-
ically tagged WITS requires laborious molecular biology tech-
niques such as qPCR, whereas our antibiotic resistant isogenic
strains can be quantified by simple plating on selective media.
Additionally, antibiotic resistance enables investigation into the
effects of antibiotic therapy on bacterial population dynamics [ 8 ].
In this chapter, we describe in detail the techniques used to
investigate pathogenesis, host–pathogen interaction and bacterial
within-host population dynamics using the zebrafish model of
infection.2 Materials
- E3 larval zebrafish medium (×10): 50 mM NaCl, 1.7 mM KCl,
3.3 mM CaCl 2 , 3.3 mM MgSO 4. Prepare the 10× stock and
subsequently dilute to 1× solution in distilled water. In order
to prevent fungal growth, supplement the E3 medium with
Methylene Blue to a final concentration of 0.00005% (w/v,
approximately four drops of 0.05% Methylene Blue per litre).
Autoclave and cool to approximately 28 °C before use. - Tricaine (zebrafish anesthetic). Prepare a stock solution of
0.4% (w/v) 3-amino benzoic acid ester (tricaine or MS322) in
20 mM Tris–HCl, adjust the pH to 7.0 and store at − 20 °C
(see Note 1). - Methylcellulose solution. Prepare the 3% (w/v) methylcellu-
lose solution in E3 (with Methylene Blue). Aliquot the clari-
fied solution into 20 mL syringes and freeze for long-term
storage. For use and short-term storage, keep in the zebrafish
incubator (see Note 2). - Low-melting point (LMP) agarose for mounting embryos
prior to microscopic imaging. Prepare 0.5% (w/v) LMP aga-
rose in E3 medium (without Methylene Blue), heat up the
Tomasz K. Prajsnar et al.