Chapter 20
PROTOCOL
Porifera
Subprotocol 1
Cells from Gemmules of Fresh Water Sponges (Demospongia)
Materials
- Incubator
Equipment
- Aquarium
- lO-ml syringe
- 6-Well tissue culture plates
- Rasmont's culture medium "M" (Rasmont, 1961; see Appendix 1)
Solutions
- 5% Sodium hypochlorite
- 70% Ethanol
- BME amino acids (no. K0263; 100x stock solution; Biochrom)
- BME non-essential amino acids (no. K0293; Biochrom)
- BME vitamin (no. K0273; Biochrom)
- Chicken embryo extract (no. 061-05115C; Gibco BRL)
- Gemmules of fresh water sponges
Animals
Procedure
1. Collect gem mules (Fig. 1) of fresh water sponges from a river.
Sterilization
2. Keep the gemmules at 4°C in tap water until use.
3. Surface sterilize the gemmules in 5% sodium hypochlorite and 70%
ethanol for 5-10 min.
4. Wash the gem mules with sterile distilled water and leave them in 10 ml
distilled water.
1. Transfer approximately 100 gemmules to 20°C to initiate hatching Dissociation
process. Under the conditions described in the step 4 of Sterilization,
90 % of gemmules will start to hatch after 48 h at 20°e.
2. After 48 h, dissociate the gemmules by squeezing them through a 10ml syringe.