Table 1.

Summary of the in vitro tissue culture technique developed for the explant and cell suspension of tissues of the zebra mussel (Dreissena polymorpha)

Dissection & decontamination

Scrape mussel clean under running water. Place in sterile water

Place in antibiotic sol. (2×) for 2 hr in laminar flow partially on ice

Rinse mussel in ethanol 70°, allow to dry

In laminar flow, rinse mussel with 10 mL sterile H2O, dissect tissues

Place tissues (digestive gland, gill, gonad) in sterile buffer solution

Trim tissue to ensure as pure a sample as possible

Rinse in petri dish of sterile buffer solution. Cut tissue into 1–2 mm2 pieces.

Antibiotic sol. (separately)

×4 (10 mL of anti-b sol.) 30 min

×2 (5 mL of anti-b sol. + 5 mL buffer sol.) 20 min

×1 (2.5 mL anti-b sol.  + 7.5 mL buffer sol.) 10 min

Rinse in sterile buffer sol.

Dissociation with pronase

Add tissue to 0.025% pronase in buffer sol. (12.5 mg in 50 mL) with antibiotic (×1)

Keep separate organs from different animals in separate tubes

Store at slight angle at 4 °C for specified time

Filter sample liquid through autoclaved gauze 60 μm (slowly) into centrifuge tube

Rinse tube and gauze with buffer solution

Centrifuge filtered liquid for 3 min at 1,200 rpm

Remove liquid, add buffer sol. and re-centrifuge, 3 min at 1,200 rpm. ×2

Remove supernatant

Add media, mix cells using pipette. Calculate cell density using a haemacytometer, adjust media volume if needed

Place cell suspensions in culture, 1 mL in petri (8.8 cm2) and 0.3 mL in 24 well multiwell plate. Add 0.5 and 0.2 mL media (respectively) after 24 h 15 °C incubation

Explant

Take tissue material from gauze and place in petri (8.8 cm2) with buffer solution

Cut up tissue into 1 mm2 using scalpel

Place 10–12 explants in petri dish. If drying place drop of media on explant

Leave for 10 min

Add 1 mL of media and slowly immerse explant. Add 0.5 mL after 24 h incubation

Incubate at 15 °C

Sol solution