Embryo culture condition

• Embryos are kept in Falcon 35-3001 (or Corning 430165 and equivalents) tissue culture dish containing ~3 mL of HL medium. Falcon 35-3001/Corning 430165 dishes are coated and treated for tissue culture. Un-coated dishes appear to have a negative impact on the health of de-cocooned leech embryos in culture.
• Helobdella embryos can be cultured in a wide range of temperature (15-30°C) and developmental rate changes with culture temperature. Changing culture temperature is a possible way to manipulate the developmental rate. However, the resulting effect of temperature shifting may be erratic. One should not perform temperature shifting when using morpholino or other technologies based on molecular interactions. We use 25°C as our standard culture temperature, but 23°C culture is more commonly used in literature (presumably reflecting a lower A/C temperature setting in the US).
• Make sure there is no Vorticella (the tiny protozoans colonizing the outer surface of adult leeches) contaminating the embryo culture. These little bugs can get embryos sick rapidly. They are visible under a stereomicroscope. You can avoid this by inspecting the dish carefully under a stereomicroscope. Remove the protozoans by pipetting if necessary. If contamination is serious, replace the dish. If contamination comes back even after the dish is replaced, throw the dishes and embryos away and start anew.
• Antibiotics (tetracycline and/or penicillin-streptomycin) can be added to prevent bacterial contamination, but this does not stop Vorticella. In our lab, we prepare a tetracycline stock (10 mg/mL) and use 10 kU Pen-Strep stock (6 mg/mL penicillin + 10 mg/mL streptomycin). Tetracycline is supplied at 10 μg/mL and Pen-Strep at 50 U/mL in HL medium. Add 100 μL tetracycline stock and/or 500 μL Pen-Strep stock to 100 mL HL medium. HL medium is stored at 4°C.
• HL medium should be replaced daily. Before changing HL medium, make sure that the new HL has returned to room temperature. It should be noted that Helobdella embryos can tolerate occasional lapse in the maintenance schedule. There is no need to throw everything away just because you forget to change medium on one occasion. Just examine the embryo under a microscope and see if they are okay. If they are fine, just return to the regular maintenance schedule and change the culture medium daily.
• For sorting embryos manually, we prefer to use a flame polished micropipette (see the picture below). To make a polished micropipette, pass the sharp end of micropipettes over a flame briefly so the tip is melt and polished but the micropipette remains straight, and place a flag (made from lab tape) at the open end.

Recipes

Classic Htr HL medium (for H. triserialis)

NaCl (4.8 mM), KCl (1.2 mM), MgCl₂ (2 mM), CaCl₂ (8 mM), maleic acid (1 mM), pH 6.6

To make 5 L,

5 M NaCl 4.8 mL

1 M KCl 6.0 mL

1 M MgCl₂ 10.0 mL

1 M CaCl₂ 40.0 mL

1M maleic acid 5.0 mL

fill with ddH₂O to 5 L,

adjust pH with 1 M NaOH (carefully and slowly; do not overshot; remake the solution if overshot the pH).

Hau HL medium (for H. austinensis, 2010 revision)

NaCl (17.3 mM), KCl (1.2 mM), MgCl₂ (2 mM), CaCl₂ (8 mM), maleic acid (1 mM), pH 6.6

To make 5 L,

5 M NaCl 17.3 mL

1 M KCl 6.0 mL

1 M MgCl₂ 10.0 mL

1 M CaCl₂ 40.0 mL

1M maleic acid 5.0 mL

fill with ddH₂O to 5 L,

adjust pH with 1 M NaOH (carefully and slowly; do not overshot; remake the solution if overshot the pH).

Divalent-free HL medium for microinjection

NaCl (27.3 mM), KCl (1.2 mM)

To make 500 mL,

5 M NaCl 2.73 mL

1 M KCl 0.60 mL

fill with ddH₂O to 500 mL.