6.2.1. Method for collecting haemolymph
From Stephens (1962):
- Larvae can be field-collected (see Section 6.1.) or reared in vitro (Section 3).
- Anaesthetize the larvae with CO2 until visible movement ceases. This makes it easier to handle larvae since they are otherwise quite active.
- Surface sterilize the larvae per Section 6.6. or with a hypochlorite solution (24 ml Millendo bleach in 1 l distilled water) for 5 minutes.
- Rinse the larvae twice with distilled water.
- Dry the larvae on sterile blotters at 30ºC until normal movement resumes.
- Wax moth larvae can be bled by cutting a proleg from the body or puncturing the proleg with a sterile needle and collecting the haemolymph that pools at the wound.
- Collect haemolymph by capillary action into sterile
capillary tubes. Larvae from which only a small amount of haemolymph is
collected can survive, complete their development, and reproduce normally.
- The haemolymph can be transferred to pre-cooled Eppendorf tubes containing a few crystals of phenylthiourea. This prevents melanization (Vilcinskas et al., 1997, Wedde et al., 1998).
- 1 ml aliquots can be centrifuged twice at 10,000 g for 5 min to remove haemocytes (Wedde et al., 1998).