Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

The fish experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Chongqing and the Institutional Animal Care and Use Committee of Chongqing Medical University, China, as well as the standards for experimental animals issued by the State Bureau of Quality and Technical Supervision (Approval ID: GB14922-2001 to GBT14927-2001). Zebrafish (Danio rerio, wild type, AB strain) were sourced from the Institute of Hydrobiology, Chinese Academy of Sciences, and maintained in the laboratory following previously reported procedures³⁶.

1. Adult zebrafish maintenance and embryo collection

NOTE: Drawing upon modifications from previous studies and reports^16,37,38,39, the maintenance of adult zebrafish, larvae rearing, and practices for germ-free (GF) models in our laboratory were carried out following the steps outlined below. The ultra-pure flow system for fish culture was commercially obtained (see Table of Materials). The water, maintained with a balanced pH, salt, and alkali, undergoes filtration during cycling to ensure clean conditions.

1. Maintain adult fish with the following standard procedure in the auto cycle system (see Table of Materials) with a photoperiod of the dark: light = 10 h: 14 h at 28 °C ± 0.5 °C and fed with fresh incubated Artemia sp. twice daily.
2. Put the sexually mature adult zebrafish (male: female = 2:1) into tanks with fresh and clean water the night before.
3. Let the fish spawn the next morning (approximately 8:30 a.m.) under regular light stimulation in the laboratory. After 1-2 h, transfer the fish to another tank.
4. Collect embryos into water from the auto-cycling system as the rearing medium in a culture dish using a disposable Pasteur pipette.

2. Generation of GF zebrafish from embryos to larvae

NOTE: The procedure for generating germ-free (GF) fish is outlined in Figure 1, while the basic developmental indexes of conventional (CR) and GF models are presented in Supplementary Figure 1. Please follow the steps outlined below in a benchtop biological safety cabinet or laminar flow hood using a sterile aseptic technique in a clean room.

1. Wash the embryos using the fish culture water and remove the feces impurities and dead or unfertilized eggs.
2. Transfer the embryos at 2 hpf to sterilized plates (up to 480 embryos per sterile dish with a 10 cm diameter) filled with antibiotic germ-free zebrafish medium (AB-GZM, see Table of Materials) solution and culture at 28.5 °C for 6-8 h.
   NOTE: AB-GZM is used to treat the embryos for several hours to eliminate the surface microorganisms, and GZM without antibiotics is used to culture GF fish in subsequence. Usually, the perfected culture time is 6 h).
3. Exclude eggs with white spots or a whitish appearance, and select those that are normally developed, displaying transparent and intact envelopes for further processing.
4. Rinse the embryos using AB-GZM three times within 10 min.
5. Soak the embryos in 0.2 g/L povidone-iodine solution (PVP-I) for 1 min (see Table of Materials).
   NOTE: Higher concentration and longer than 2 min will influence the death and hatching rate of embryos.
6. Wash the embryos using germ-free zebrafish medium (GZM) three times within 10 min.
7. Add embryos into sodium hypochlorite (NaClO) working solution, tightly cover the 50 mL microcentrifuge tubes, and bleach for 15 min. During this period, suspend the embryos in bleach solution by gently shaking them.
8. Wash the embryos using GZM three times within 10 min.
9. Transfer the embryos to sterile 6-well plates with 5 embryos and 10 mL of GZM per well. Culture them in an ultra-clean platform or incubator with a photoperiod of dark: light = 10 h: 14 h at 28 °C ± 0.5 °C.The culture density will influence the sterile rate.
10. Renew the culture medium daily by removing the spent GZM, collecting it as samples for sterile status detection, and replenishing each well with the same volume of sterile GZM.
11. Record the basic developmental indexes of GF embryos/larvae, including the survival rate, hatching rate, heartbeats, body length and weight, etc¹⁹.
12. Transfer the GF fish to suitable containers (dishes, cell culture flasks, or glass bottles with covers) with expanded volume, providing sterile food throughout the growth process.

3. Maintenance of GF zebrafish from larvae to juvenile

1. Renew the GZM without feeding before 7 dpf and daily detect the sterile states (see step 5).
2. Feed zebrafish larvae from 8 dpf to juvenile stages with sterile egg yolk (10 µL prepared stock solution per well) once daily before changing GZM.
3. Feed GF juveniles from 14 dpf with sterilized egg yolk mixed with GF Artemia sp. once daily (1-3 Artemia/larvae, see step 4), gradually increasing food mass with the growth and development of GF fish.
4. Provide egg yolk until all fish can consume Artemia nauplii.
   NOTE: GF Artemia should undergo sterility testing before use. Assess the number of Artemia nauplii left, observe pursuit and capturing progress, and examine the fish body with consumed food to indicate feeding success.
5. From 28 dpf to early adult stages, feed GF Artemia once daily (3-5 Artemia/larvae) and clean untapped or dead Artemia, and dead fish into the waste GZM as daily samples.
6. During GF fish growth, change the 6-well plates after 7 dpf to sterile plates or cell culture flasks from 8 to 21 dpf, and then to sterile bottles after 21 dpf. Increase culture medium and food mass according to the number and weight of GF fish in each container.
7. Renew GZM daily and check samples to ensure the success of GF models.
   NOTE: Maintaining germ-free (GF) fish models until early adulthood and sexual maturity is challenging, with a low average survival rate at 30 dpf, typically less than 10%. This is primarily attributed to weak immunity, delayed development, and impaired intestinal function.

4. Preparation of GF Artemia nauplii as the sterile food for chronic GF fish models

NOTE: The cultivation and preparation method of germ-free (GF) Artemia is outlined in Figure 2. Note that all the following steps are conducted in a benchtop biological safety cabinet, using a sterile aseptic technique.

1. Rinse 0.25 g of Artemia cysts using 2.4 g/L sodium hypochlorite (NaClO, see Table of Materials) for 5 min.
2. Filter the rinsed Artemia cysts with a sterilized ultra-dense filter (about 170 meshes in aperture) and wash with sterilized ultrapure water three times, each time for 1-2 min.
3. Transfer the washed Artemia cysts into 100 mL of 2.5% sterilized saline water, mix, and package for aseptic hatching according to the steps mentioned below.
4. Pack the 50 mL treated Artemia cysts mixture solution into a sterile bottle with a volume of 100 mL, seal it with film, and incubate in a shaking incubator at 150 rpm, 30 °C for 18 h to 24 h with light.
5. Extract approximately 30 mL of hatching Artemia nauplii from the middle and lower layers using a disposable Pasteur pipette.
   NOTE: Prevent floating eggs and empty shells from reaching the upper layer. The indexes of germ-free (GF) Artemia cysts and nauplii incubated for 24 h are presented in Figure 3.
6. Filter the Artemia with a sterilizing filter and wash them in a sterilizing beaker with sterilizing ultrapure water three times, about 30 s to 1 min for each time. Finally, add 4 mL of sterilized ultrapure water to prepare the Artemia mixed solution.
7. Using a disposable Pasteur pipette, retrieve actively swimming Artemia from the upper layer, wash, and prepare the nauplii solution for feeding and aseptic identification.

5. Identification of GF fish models at the whole life stages

NOTE: Throughout the entire life stages of germ-free (GF) fish, key indexes and daily sample detections are crucial to ensure the success of the models. This step summarizes the common classification of samples and the usual methods for identification (Figure 4).

1. Observe and count the survival status and rate of GF zebrafish larvae daily. In experiments calculating related rates, GF fish are cultured in 24- or 48-well plates with one fish per well.
   1. Survival rate = (number of live fish at the time of observation / total number of eggs) × 100%.
   2. Sterility rate = (number of sterile fish/number of surviving fish) × 100%.
      NOTE: This protocol focuses on the sterile rate of live fish. The number of sterile fish is determined by counting successful GF fish after multiple checks among the live fish. Any dead fish or live fish GF models that have failed due to bacterial presence are eliminated during subsequent GF procedures.
2. Collect the following samples of GF zebrafish larvae.
   1. Culture medium of GF zebrafish from each well or bottle container.
   2. Fish food, such as sterile egg yolk and GF Artemia.
   3. Waste medium, including fragments of egg membrane after hatching and food residue or feces during the feeding periods from larvae to juvenile fish.
   4. Dead fish samples to identify the presence of bacteria.
   5. Fish medium and agents used in embryo treatment as the sterile control.
   6. Samples from materials, operating platform, and incubator, etc.
3. Detect samples collected daily using multiple methods.
   1. First, use the spread plate method and culture plates in an incubator at 30 °C.
      1. Coat with 20-50 µL of waste medium sample on the Trypticase Soy Agar (TSA) plate (see Table of Materials) under aerobic conditions.
      2. Coat with 20-50 µL of waste medium sample on the TSA plate under anaerobic conditions.
      3. Coat with 20-50 µL of waste medium sample on the blood TSA plates (see Table of Materials) under aerobic conditions.
      4. Coat with 20-50 µL of waste medium sample on the blood TSA plates under anaerobic conditions.
   2. Second, use the liquid culture method.
      1. Take 200 µL of sample into aerobic tubes with Brain Heart Infusion Broth (BHI) medium (see Table of Materials) and culture in the shaking incubator at 30 °C, 150-180 rpm.
      2. Add 200 µL of sample into anaerobic tubes with BHI medium and culture in the incubator at 30 °C.
      3. Add 200 µL of sample into aerobic tubes with Trypticase Soy Broth (TSB) medium, then culture in the shaking incubator at 30 °C, 150-180 rpm.
      4. Add 200 µL of sample into anaerobic tubes with TSB medium and culture in the incubator at 30 °C.
   3. Third, use molecular techniques-16s polymerase chain reaction (PCR) assay.
      1. Analyze the wastewater sample with two pairs of primers 27F and 1492R, as well as 63F and 1387R (Table 1).
         NOTE: The PCR master mix was prepared according to Table 2 using a commercially available DNA polymerase kit (see Table of Materials), and the PCR machine was set up with the program specified in Table 3.
      2. After the PCR program is completed, examine the products by 1% agarose gel electrophoresis⁴⁰ at target bands of 1465 bp (using primers 27F and 1492R) or 1324 bp (using primers 63F and 1387R) to infer the sterility of samples.
4. Record and observe sterility tests, including plate and medium culture from 24 h to 3 days and 7 days, during which no colony on plates and no turbidity in liquid indicate the successful construction of GF models. Observe and record the plate and medium at 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h.

6. Identification of GF Artemia samples before feeding on GF fish

NOTE: To detect the GF Artemia samples, which are indispensable as live food before feeding on GF fish (Figure 4), follow the steps below.

1. Collect samples of GF Artemia models.
   1. Untreated Artemia cysts.
   2. Treated GF Artemia cysts.
   3. Hatched GF Artemia nauplii.
   4. Sterilized ultrapure water as a control.
2. Detect GF Artemia with the following methods.
   1. Use the spread plate method to detect the samples by coating them on TSA plates and blood TSA plates under aerobic and anaerobic conditions. Incubate them at 30 °C.
   2. Use a liquid medium to detect the samples in aerobic and anaerobic TSB and BHI tubes. Culture in a shaker at 150-180 rpm, 30 °C.
   3. Use PCR assay to detect the presence of bacteria in the samples collected using 16s ribosomal RNA target genes primers 27F and 1492R, 63F and 1387R (Table 1). The volume and program are shown in Table 2 and Table 3.
3. Record the results: Detect the PCR product by 1% agarose gel electrophoresis⁴⁰ for target bands measuring 1465 bp (using primers 27F and 1492R) or 1324 bp (using primers 63F and 1387R). The results of plates and liquid medium can ensure the sterile status of GF Artemia before being used as GF fish food.

7. Isolation and identification of intestinal bacterial strains from zebrafish

NOTE: To explore intestinal functions in vitro and in vivo, it is necessary to isolate bacterial strains from zebrafish, which also provide the species source for potential probiotics screened by infection using GF animals (Figure 5). In this protocol, we describe the traditional dissection method to obtain intestinal contents, while the samples can also be collected directly from live anesthetized fish (data not shown).

1. Choose healthy adult zebrafish and wash them with sterile water. Perform the following steps in the clean bench.
2. Anesthetize the fish with 100 mg/L MS-222 for 5-10 min.
3. Use 75% alcohol to treat the fish's body surface.
4. Dissect the fish intestines and extrude the intestinal contents with TSB or BHI medium.
5. Incubate the intestinal supernatant by applying TSA, blood plate, TSB, and BHI medium in aerobic and anaerobic conditions.
6. Translate the bacteria to gain the signal strain and record the characteristics of different colonies.
7. Store the bacteria within 30% glycerol at -80 °C.
8. Then, identify the intestinal bacteria by genomic DNA extraction and PCR sequencing.
   NOTE: The main steps of bacterial genomic DNA extraction include liquid centrifugation, RNase A and Protease K dissociation, ethyl alcohol extraction, rinsing, and dissolution using commercially available kits following the manufacturer's instructions (see Table of Materials).
9. Analyze the 16S sequences using the National Center for Biotechnology Information (NCBI) and the Basic Local Alignment Search Tool (BLAST) with the Bacteria and Archaea database^40,41(see Table of Materials).
10. Pick the best-matched bacteria and characters to identify the isolated intestinal strains at the genus level.
11. Detect the metabolic functions of bacterial strains using commercially available kits (see Table of Materials).

8. Flu-label, infection, and colonization of bacteria in GF zebrafish intestines

NOTE: Before infecting GF zebrafish, the isolated bacteria were modified with dyes and counted, making microbial colonization and migration visible continuously and in vivo (Figure 6).

1. Choose potential beneficial bacteria or dominant strains with high abundance in the host to infect the GF fish models.
   NOTE: The bacteria used in this study were Aeromonas sp. and Vibrio sp. (see Table of Materials), selected from the 8 genera that were isolated and identified from adult wild-type zebrafish in the laboratory⁴².
2. Label the fresh bacteria with CM-Dil dyes (see Table of Materials) and expose them to GF zebrafish at 5 dpf with a concentration of 10⁶-10⁸ Colony-Forming Units (CFUs)/mL.
3. Observe the fluorescence under the fluorescent microscope with 3× magnification to indicate the colonization and distribution of bacteria in the gut of GF zebrafish larvae from 6 dpf and 14 dpf.
4. Manually count the number of bacteria in GF fish by plate culture at each time point.
5. Detect the colonies and sequences to ensure the infection was successful with the target bacterial strains.
6. Collect samples of GF fish with bacterial infection for subsequent assays, including neuro-behavior, developmental indexes, histopathological analysis, and hormone measurements, etc^43,44,45.
   NOTE: The bacteria used in the infection experiment should be newly recovered and freshly cultured by the liquid medium.