1. Access to the Zebrafish Center is allowed only to authorized persons: it is therefore forbidden to introduce unauthorized persons without first contacting the managers of the Center (Francesco Argenton, Natascia Tiso, Luisa Dalla Valle, Massimo Santoro, Martina Milanetto, Paolo Bonaldo, and Enrico Moro).

Concerning this point, we wish to underline the visit of underage students, which must be made known to the afore-mentioned managers, and appropriately organized.

2. Access to the Facility is allowed only for groups with a financed project in which adequate resources are provided for this service. The projects must be approved by the OPBA (Animal Welfare Body) and by the Italian Ministry of Health. Only the personnel indicated in OPBA request is admitted to the Zebrafish Center.

3. It is mandatory, except for Bachelor's students, to attend a specific course for zebrafish handling, periodically held at the Department of Biology.

4. Tutors must periodically inform the Facility Manager about additional students, who will be directed to the training course, held by Dr. Martina Milanetto.

5. Bachelor's students can access the Facility only if accompanied by their tutor and cannot handle fish (over 6 dpf); Master's students can manipulate fish only in the presence of their tutor or co-tutor included in the OPBA project.

6. Before entering the main room, dedicated clogs (to be left in the lockers after use) or shoe covers (for occasional visitors) and lab coats must be worn. Shoes must be left in the appropriate locker, located before the line on the floor. Clean clogs (for exclusive use in the facility), located in the cabinet, must be worn after the line. Disposable or personal lab coats and shoe covers are available in the same closet. The line of tape present in the area in front of the fish room (changing room) cannot be crossed with shoes and clothing not adequately protected by overshoes and lab coats.

7. Direct contact between operators and animals is absolutely forbidden. The use of gloves is recommended for all operations.

8. Before and after handling fish or water in the system, the hands should be washed with soap and water.

9. All users are required to monitor the health status of the fish. All fish presenting symptoms of disease must be promptly eliminated, in agreement with the personnel responsible for the specific area and for the entire facility (see the file "monitoring of fish morbidity and mortality"). The eliminated fish must be indicated on the original tank, specifying the date of elimination and, during the monthly census, it must be indicated in the register.

10. Non-carriers, sick fish, or any other individual to be eliminated, must be placed, using a fish net, in a "fish to be euthanized" tank and promptly disposed, according to the Facility regulations.

11. Fish with serious diseases must be collected and sent to the Zoo-prophylactic Institute for an accurate analysis of the pathogens.

12. For fish in good health, fish nets from the left bucket should be used, while for sick or dead fish, fish nets from the right bucket (paper label) should be used. Fish nets should be placed back in the correct bucket. Fish nets in the buckets are immersed in a solution containing sodium hypochlorite and should be rinsed before use. For each aquarium cabinet, a different net should be used, to be then placed in sodium hypochlorite.

13. Fish should be exposed only to water from the aquarium system, as tap water contains chlorine, which is harmful to fish.

14. Do not leave fish tanks completely unattended for a long time, especially if placed on the table.

15. In the small tanks, containing fish under genotyping, the name of the responsible person and the date on which the biopsy was performed must be indicated.

16. Guidelines for fish expansion in room 1:

• for each tank the label must indicate, completely and clearly: name of the line, date of birth and initials of the owner;

• larvae up to 5 dpf must be kept in plates in the incubator;

• larvae up to 15 dpf can be grown in a tank in the incubator;

• fish from 15 dpf to 2-3 months can be transferred to the nursery and then to definitive tanks, upon agreement with Dr. Martina Milanetto to define the destination tank;

• fish kept without flow under the table or in shelves require tank cleaning performed by the owner at least once a week.

17. Guidelines for fish expansion in room 2:

(see the file "fish expansion and feeding guidelines in room 2")

• for each tank the label must indicate, in a complete and clear way: the name of the line, date of birth and initials of the owner.

• only fish subjected to bleaching can be introduced (see the file "Embryo Surface Sterilization") or derived from crosses exclusively involving fish from room 2.

• cabinets 1 and 2 can accommodate embryos from the 24 hpf stage, which will subsequently be moved to cabinets 3 and 4 once they have reached the appropriate size or number.

• cabinets 3 and 4 are intended for growing fish, which must be moved to the hand-fed cabinets once they reach adulthood (red label).

18. Guidelines for fish feeding in room 1:

• 1-6 dpf: food not necessary;

• 1-2 wpf: dry food for larvae;

• from 3 wpf: dry food for larvae and brine shrimps;

• 6-8 wpf (depending on the size of the fish): dry food for adults and brine shrimps.

• In the nursery, the brine shrimps should be administered to all tanks; concerning the dry food, tanks with blue stamp require dry food for larvae;

• Fish in small tanks without flow should be fed with brine shrimps and not with dry food.

19. Guidelines for feeding fish in room 2:

• Cabinets 1-4 are fed by a robot with different dry food, depending on the stage of development, based on the label affixed to the tank by the owner, according to the indications of the files "expansion guidelines and feeding fish in room 2" and "Triton date matrix positioning".

• Cabinets 5-15 will only receive manually distributed dry food for adults;

• Tanks with blue or yellow label (small fish) must be cleaned by the owner using a specific vacuum pump (see the file "cleaning trays").

20. The tanks to be cleaned must be washed quickly with a brush and handed over to the Plaisant technical staff for washing in the dishwasher.

21. Each group can micro-inject twice a week, after having booked the micro-injection session in the Google Calendar. If the day before the injection there are no reservations, users can make additional injection sessions. In a given week, users can book an injector with Google Calendar only for the following week.

22. Each group can use a maximum of 10 wild type fish for each injection session, taking them from the tanks destined for the day of mating, whether using them for wt crosses (= 5 pairs max) or using them for outcross (= 10 pairs max). Before setting up the crosses, especially if these are numerous, the availability of fish, tanks and space for the mating must be evaluated, so as not to preclude other people from the possibility of using fish. In room 1 the mating tanks must be placed in Cabinet 9 or, in the absence of space, on the table. In room 2 tanks must be placed in the "transparent shelf" or, in the absence of space, on the table. Crosses must be set up from 15.30 onwards using, in case of shared wild fish, only those with the label corresponding to the day of the week in which the couples are prepared (the fish with the "inj" label have rested a week and are therefore recommended for microinjection, while those without "inj" can be used for other purposes).

23. After mating, the fish must be reintroduced into the original tanks, taking care not to mistake the tank. If an error occurs or if abnormalities in the phenotype occur, notify the staff and isolate the fish for further checks.

24. The tanks placed in the breeding shelf will not receive food, so it is not recommended to leave them in this shelf for more than a day.

25. After use, the tanks should be cleaned without using detergents, dried and stored neatly on the correct shelf.

26. At the end of the work, the floor should be dried and the work surface should be left tidy and dry. The activities must be written in the register, specifying name, group, line used, number of fish (not pairs!) and approximate number of eggs obtained; all crosses should be reported (for injections, but also for treatments, in situ hybridization, transgenic propagation...). Crosses for the expansion of shared lines should be reported in the sheets labeled "facility". Finally, the door of the Center must be closed.

27. The basket for sharp objects should only be used for needles and sharp objects and not for pipettes and tips.

28. The plates containing the micro-injection needles are in a cabinet in the micro-injection room 2. Each plate that users take should be replaced with an empty plate, which will then be filled by Dr. Martina Milanetto.

29. Shared wild type fish may not be used during rest periods; all other fish can be used instead. In addition, each group can raise up to two tanks of their own wild type fish (maximum density = 50 fish / tank), which can be used at any time, both during the rest periods and during the weeks of "regular fishing".

30. During the own fish feeding turn, the sheet with the task list must be consulted and filled out (in the green / blue notebook) (see files "weekend duties reminder, room 1" and " weekend duties reminder, room 2").

EVERYBODY MUST SEEK TO MAKE THIS REGULATION BE RESPECTED AND ANYONE WHO IS PRESENT DURING ANY INFRINGEMENT WILL BE CONSIDERED EQUALLY RESPONSIBLE.

Authorized users can book microscopes and microinjection systems via specific booking sheets and on-line links (Google Calendar: zebrafishpadova@gmail.com).