104
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 3
Parasitized Embryos (Endoparasitoid Wasp)
Lynn and Hung (1986) developed a unique method in which irradiation-killed host eggs were used to initiate primary cultures of egg parasitoids. Their method is as follows.
Materials
Equipment - y-Radiation facility
- Centrifuge and centrifuge tubes
- Beakers
- Microscalpel
- Pasteur pipettes
- Tissue culture plates
- Petri dish (Falcon)
Solutions - Culture medium, especially primary cell culture medium (PCM; see
Appendix 1)
- Ultracer G (LKB)
- Nuserum (Collaborative Research)
Chemicals - Sodium hypochlorite
- Triton X-lOO
- FBS albumin (KC Biological)
- Gentamicin (HEM Research)
Animals - Female adults of host insects
- Female adults of parasitoids ready to oviposit
Others - Cheesecloth
- Parafilm (American Can)
Procedure
Irradiation l. Allow the female adults of the host insect to oviposit onto the cheesecloth.
2. Irradiate the host eggs with a y-ray at 30 krad.
reparation of l. Allow the parasitoids to oviposit onto the y-irradiated host eggs.
'asitized host 2. Keep the eggs at 27°C under 70%-80% relative humidity for 32-48 h.
eggs
Sterilization 1. Dip the egg-carrying cheesecloth into a solution of 0.25% sodium hypochlorite and 1 % Triton X-I 00 for 5 min. This results in the release
of the eggs from the cloth.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 3
Parasitized Embryos (Endoparasitoid Wasp)
Lynn and Hung (1986) developed a unique method in which irradiation-killed host eggs were used to initiate primary cultures of egg parasitoids. Their method is as follows.
Materials
Equipment - y-Radiation facility
- Centrifuge and centrifuge tubes
- Beakers
- Microscalpel
- Pasteur pipettes
- Tissue culture plates
- Petri dish (Falcon)
Solutions - Culture medium, especially primary cell culture medium (PCM; see
Appendix 1)
- Ultracer G (LKB)
- Nuserum (Collaborative Research)
Chemicals - Sodium hypochlorite
- Triton X-lOO
- FBS albumin (KC Biological)
- Gentamicin (HEM Research)
Animals - Female adults of host insects
- Female adults of parasitoids ready to oviposit
Others - Cheesecloth
- Parafilm (American Can)
Procedure
Irradiation l. Allow the female adults of the host insect to oviposit onto the cheesecloth.
2. Irradiate the host eggs with a y-ray at 30 krad.
reparation of l. Allow the parasitoids to oviposit onto the y-irradiated host eggs.
'asitized host 2. Keep the eggs at 27°C under 70%-80% relative humidity for 32-48 h.
eggs
Sterilization 1. Dip the egg-carrying cheesecloth into a solution of 0.25% sodium hypochlorite and 1 % Triton X-I 00 for 5 min. This results in the release
of the eggs from the cloth.