Antibiotic Resistance Protocols (Methods in Molecular Biology)

153

For bacterial population dynamics experiments, zebrafish embryos

should be infected as normal, but care should be taken to place

infected fish with exact amount of E3 (e.g., 100 μL, see Note 19).

1. Transfer infected embryos individually into homogenizer tubes
   together with 100 μL of E3 from a well at different time inter-
   vals (if time-course experiment is required), or while the
   embryo is heavily infected/dead (for within-host population
   dynamics).


2. Freeze collected samples at − 20 °C.


3. After collecting all samples, defrost them for approximately
   30 min prior to use.


4. Homogenize defrosted embryos using a homogenizer, e.g.,
   PreCellys 24-Dual. Typical homogenization time is 30 s.


5. Serially dilute up to 3 orders of magnitude (“neat” plus 3 deci-
   mal dilutions) using 96-well plates and 8-channel pipette (see
   Note 20).


6. Plate out 5 μL starting with a row of most diluted samples
   onto BHI agar supplemented with appropriate antibiotics.


7. Incubate the agar plates overnight at 37 °C.


8. Count colonies of convenient dilution and determine number
   of CFU per embryo depending on dilution factor used for
   counting colonies.


9. Prepare 1% (w/v) low-melting point agarose solution in E3
   medium (without Methylene blue) (see Note 21).


10. Anesthetize embryos using tricaine as instructed previously (see
    Subheading 3.4).


11. Place embryos into a glass bottom dish and immerse embryos
    in low-melting point agarose solution (see Note 22).


12. Wait for approximately 5 min (until the agarose solidifies) and
    cover embedded embryos with E3 solution supplemented with
    tricaine.


13. Use 2× objectives when imaging the entire embryo body (see
    Fig. 2a), 10× for large section of embryos (see Fig. 2c), and
    60× for imaging bacterial and host cellular interactions (see
    Fig. 2b).

GraphPad Prism graphing and statistics software is commonly used

to present and analyze data.

1. For presenting survival curves and comparing virulence of dif-
   ferent bacterial strains, use a Kaplan–Meier survival plot, and
   perform a pair-wise statistical analysis using Log-rank test. A
   Bonferroni correction is recommended when performing mul-

3.6 Determination
of In Vivo Total
and Relative Bacterial
Loads

3.7 Imaging of S.
aureus-Infected
Zebrafish In Vivo
Using Fluorescence
Microscopy

3.8 Data
Presentation
and Statistical
Analysis

Infection Model in Zebrafish