A Simple Method for Examination of the Vulva Area of Mature Cysts of Heterodera spp.

To correctly speciate members of the genus Heterodera, the vulva area of cysts must be examined to assess the morphology of the underbridge, bullae, and fenestrae. The usual procedure (1) is to soak the cyst in water for about an hour, after which a scalpel is used to cut away the posterior part of the cyst containing the vulva area. The severed part is cleaned and trimmed, placed in absolute alcohol for a few minutes, transferred to clove oil, and finally mounted in balsam. A new me thod t he totomount water agar method--was developed serendipitiously while examining cysts in water agar for fungal infections. To initiate the totomount water agar method, 3-6 cysts, preferably fresh from soil or roots, or fixed specimens are placed on the surface of a 1.5-cm ~, 2-mm-thick block of 1.7% water agar lying on a microscope slide or on the bottom of a petri dish lid. Using a fine dissecting needle with an offset tip, a small 1-mm-deep cavity is made in the agar for each cyst. The cavity should be slightly smaller than the cyst diameter. Using the dissecting needle, each cyst is gently pushed into a cavity with the anterior end down until the posterior apex of the cyst is level with the agar surface and pointing straight up. A small drop of water is added to a 1215-mm cover glass, after which the cover glass is inverted and gently dropped over the embedded cysts. Immersion oil is added to the cover glass for microscopic examination. Cysts are now ready for examination with a compound microscope. Normally 50% or more of the embedded cysts provide an excellent vulva area presentation (Fig. 1). If the vulva areas should be out of alignment or focus, correction is made by removing the cover glass, orienting the cysts correctly, and adding a new cover slip with a fresh drop of water. The entire procedure takes about 10-15 minutes. Even though it seems improbable that one can provide an excellent vulva area presentation with the light from below passing through or around the lengthwise orientation of the cyst, this method has been employed successfully for 5 years in Florida for numerous cyst identifications. Identification has been achieved with only a single cyst from a soil sample. The vulva area of young white Heterodera females have also been clearly observed using this