Oocyst culture: the field variant
Culture conditions
This technique works well for individual gametocysts of all kinds. The methods described below are for gametocysts from terrestrial hosts. Gametocysts from aquatic hosts should be incubated using a similar technique that omits the carrier dot and includes a small volume of water in the bottom of the genitalia vial. The field variant of this technique uses small glass insect genitalia vials with silicon stoppers (BioQuip 1133M or similar) as the primary culture container. Individual genitalia vials are stored individually in screw-top gasketed polypropylene microtubes held in a standard microtube carrier box.
Surface sterilization
Stylocephalid gametocysts are protected by an outer epicyst and do not require surface sterilization. All other gametocysts should be surface sterilized by serial pipetting through the wells of a 3-welled dish. The first 2 wells are filled with water and the final well is filled with a 0.1% solution of Methyl Paraben in water.
Culture carriers
Gametocysts are much easier to handle, incubate, and store if they are placed on a carrier. In the field variant technique, we use 4-mm circles of black construction paper cut with a cork borer. Carrier dots are cut en mass and stored in a solution of 0.1% methyl paraben in water until needed. Presoaking the carrier dots leaches any excess dye and saturates the paper, improving capillary flow through the carrier dot. (NB: All brands of construction paper are not suitable. Some use heavy metal mordants in the dye process and these metals prevent gametocyst development. We find that the more expensive, archival papers work best.)
Loading carrier dots
Remove a carrier dot from the storage solution and place it on a piece of filter paper. The filter paper drains excess fluid from the carrier dot. Individual gametocysts are pipetted onto the carrier and excess transfer fluid is drawn through the carrier dot and absorbed by the filter paper.
Incubation
Individual carrier dots are placed in glass insect genitalia vials with silicon stoppers (BioQuip 1133M or similar) as the primary culture container. Individual genitalia vials are stored individually in screw-top gasketed polypropylene microtubes held in a standard microtube carrier box. Gametocysts from aquatic hosts are stored and incubated in a similar manner with the carrier dot omitted and a small volume of water added to the gentialia vial.
