TITLE: Protocol (A): Zebrafish infections into the otic vesicle (2 dpf)
AUTHORS: Désirée A. Schmitz, Tobias Wechsler, Hongwei Bran Li, Bjoern H. Menze, Rolf Kümmerli

[DESCRIPTION]
This protocol details the zebrafish infections into the otic vesicle.

[GUIDELINES]

 

Table SP1. Media preparation of E3 zebrafish water (60x stock and 1x), pronase for dechorionation, PTU to avoid pigmentation, tricaine for anesthetizing.

 

 

 


[STEPS]
SECTION: Part 0: Material preparation
1. Media: prepare all required media according to Table SP1 (E3 zebrafish water, PTU to avoid pigmentation, pronase for dechorionation, tricaine for anesthetizing).
SECTION: Part 0: Material preparation
2. Hair loop manipulators (Fig. SP1A): tape (e.g. using 19 mm wide tape) a piece of hair as a 4-7 mm loop as an extension onto e.g. the wooden end of a cotton swab (ROTILAB, 150mm long) (see image of hairloop tool).

 

SECTION: Part 0: Material preparation
3. Needles (Fig. SP1B): Using the micropipette puller device, pull filamented borosilicate glass microcapillary injection needles (Science Products, 0.58mm diameter, GB100F-10) with the following settings: air pressure=500, heat=609, pull=200, velocity=100, time=50.

 

SECTION: Part 0: Material preparation
4. Agarose molds (Fig. SP1C right): prepare as many agarose molds as there are treatments using a negative rubber mold (Fig. SP1C left):  

 

SECTION: Part 0: Material preparation
4.1. Heat 1.5% agarose in 100 mL ddH2O in a beaker in a microwave in 30 s bursts until the liquid is completely clear (no turbidity visible)
SECTION: Part 0: Material preparation
4.2. Carefully pour the heated agarose onto the rubber mold with channels from Ellett & Irimia (2017, Zebrafish) avoiding the formation of any bubbles.
SECTION: Part 0: Material preparation
4.3. Once it has solidified, peel the agarose mold off the rubber mold.
SECTION: Part 0: Material preparation
4.4. Store at 4 °C in a petri dish wrapped in parafilm until use.

 

SECTION: Part 1: Zebrafish pre-experiment preparation (1 day post fertilization (dpf))
5. In the morning, move the zebrafish to a petri dish (LGG Labware, 90x16 mm, sterile) filled with about 20 mL E3 zebrafish water plus N-Phenylthiourea (PTU) (final PTU concentration: 0.003%) to avoid their pigmentation (see Table 1 on how to prepare E3 water and PTU). 
SECTION: Part 1: Zebrafish pre-experiment preparation (1 day post fertilization (dpf))
6. In the evening, add 20 µL pronase (30 mg/mL) to the zebrafish (final pronase concentration: 0.03 mg/mL) and mix well to ensure that they will dechorionate (hatch from the chorion, which is the protective membrane around the zebrafish embryo) until the next morning. 
SECTION: Part 2: Preparation of bacterial suspensions (2 dpf)
7. Take fluorescently tagged bacterial species from 25% glycerol stocks that were stored at -80 °C and either streak them on an agar plate to pick a single colony (after overnight growth at 37 °C) for the following inoculation or directly inoculate them into 10 mL fresh LB medium. 
SECTION: Part 2: Preparation of bacterial suspensions (2 dpf)
8. Grow   at 37 °C and 170 rpm with aeration until they reach the stationary phase. 
Depending on the biological question being asked, exponentially growing cells could also be used.

SECTION: Part 2: Preparation of bacterial suspensions (2 dpf)
9. The next day, wash cells by centrifuging them at 7500 rcf, 3 min . Discard the supernatant and replace it with 0.8% NaCl to resuspend the cell pellet.  
SECTION: Part 2: Preparation of bacterial suspensions (2 dpf)
10. Repeat step 9. 
SECTION: Part 2: Preparation of bacterial suspensions (2 dpf)
11. Measure the optical density at a wavelength of 600 nm and adjust it to reach the desired cell numbers per bacterial species (to be determined in a pre-experiment). 
SECTION: Part 2: Preparation of bacterial suspensions (2 dpf)
12. Mix 9 µL per bacterial culture or 0.8% NaCl solution (as control) with 1 µL of 0.5% phenol red (final concentration: 0.05%) to visualize whether an infection is successful and remains local in the inner ear structure, the otic vesicle, of the zebrafish. 

 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
13. Add approximately 9 mL of E3 zebrafish water with PTU (final PTU concentration: 0.003%) and 10 drops of the thawed anesthetic tricaine (ethyl 3-aminobenzoate methanesulfonate salt analytical standard, 4000 mg/L) using a Pasteur pipette (LLG Labware, 3 mL, unsterile) into a small petri dish (Greiner, 60x15mm, sterile).
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
14. Add 10-20 2 dpf zebrafish into this dish to anesthetize them (Fig. SP2A). 

 


 


SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
15. For the first treatment, fill a glass needle (pulled borosilicate glass microcapillary injection needle (Science Products, 0.58mm diameter, GB100F-10)) with 10 µL of the 0.05% phenol red and 0.8% NaCl (control) or bacterial solution using a microloader tip (Eppendorf). 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
16. Put the filled glass needle into the micromanipulator, screwing it tight but not too tight to avoid breaking. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
17. Position the needle in the field of view of the stereomicroscope using the micromanipulator. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
18. Going from the lowest to the highest magnification, focus on the tip of the glass needle. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
19. Once it is possible to distinguish the two walls of the needle at a final magnification of 56X, break off the tip of the glass needle using high-precision tweezers (e.g., RubisTech Switzerland, model 5-SA)(Fig. SP2C). 
 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
20. Connect the injection pump with the micromanipulator (and thus the glass needle) via the tubing of the pump. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
21. Put a drop of oil (VWR Chemicals, 10S) on a microscopy slide with a cavity (76x26 mm) that sits on an inverted lid of a petri dish (LGG Labware, 90x16 mm, sterile) and place it under the stereomicroscope. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
22. Move the needle into the drop of oil using the micromanipulator and press ‘Clean’ on the injection pump to move the liquid into the tip of the needle. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
23. Determine the desired drop size by injecting drops into the oil and adjusting either the injection time (to between 1 s-5 s) or injection pressure (to between 200-400 hPa). In case the solution in the needle gets aspirated back in or leaks, adjust the injection back pressure (to between 20-30 hPa). At a magnification of 56X, a drop that has the diameter of five stripes on the ruler corresponds to a volume of 1 nL. This can be determined because the liquid to inject will be a perfect sphere within the oil.  

 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
24. Whenever the needle is loaded and not in use, leave it in the oil to prevent evaporation of the liquid and the clogging of the needle. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
25. Put a few drops of the E3-PTU-tricaine solution from the petri dish onto the agarose mold.  
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
26. Make sure to wet the channels that will be used for the injection (lateral for the otic vesicle) using a hair loop manipulator. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
28. Load the zebrafish into the channels using a hair loop manipulator, turning all zebrafish onto the same side so that the same otic vesicle can be injected and imaged later on (left or right otic vesicle)(Fig. SP2B). 
 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
29. Repeat step 14 to anesthetize the next batch of zebrafish for the same treatment. If it is a different treatment, this step is not necessary yet. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
30. To determine the number of bacterial cells injected at the beginning of the injections, add a drop from the needle into 1 mL of 0.8% NaCl within a 1.5 mL Eppendorf tube.  
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
31. Repeat step 30 to have at least duplicates for plating.  

 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
32. Place the mold under the objective of the stereomicroscope.  
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
33. Using a magnification of 56X, position the needle into the otic vesicle and inject (Fig. SP2D).

 


 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
34. Using a Pasteur pipette, aspirate the injected zebrafish and place them either individually into wells of a 48-well plate or five zebrafish individuals per well in a 24-well plate that is filled with E3 zebrafish water. 

 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
35. Repeat steps 27-34 if more zebrafish receiving the same treatment need to be injected. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
36. To determine the number of bacterial cells injected at the end of the injections, add a drop from the needle into 1 mL of 0.8% NaCl within a 1.5 mL Eppendorf tube.  

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
37. Repeat step 36 to have at least duplicates for plating. 

 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
38. Repeat steps 14-37 for each treatment. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
39. Once all zebrafish from all treatments have been injected, check whether any zebrafish in the well-plate(s) have been damaged and remove them. 
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
40. Incubate the well plate(s) at 28 °C either in the dark or with a 840 min light and 600 min dark cycle.

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
41. If necessary, further dilute and then plate 100 µL of the bacterial solution added to the 1 mL of 0.8% NaCl for all treatments in duplicates on 1.5% LB-agar plates to enumerate and confirm the infectious dose for each experiment. 

 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
42. Incubate plates at 37 °C (or the temperature required for your bacteria’s growth) 5 s. 

SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
43. Count CFU from agar plates.
SECTION: Part 3: Zebrafish infections into the otic vesicle (2 dpf)
27. Move the 2 dpf zebrafish from the small petri dish onto the large circle of the agarose mold for loading using a Pasteur pipette.