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A new technique for photographing perineal patterns of root-knot nematodes.

Light microscopic examinations of the perineal pattern of female root-knot nematodes remain useful for routine identification of root-knot nematodes, Meloidogyne species (Eisenback and Hunt, 2009; Eisenback et al., 1980; Hartman and Sasser, 1985). Unfortunately, conventional light microscopy often produces images that lack adequate contrast or reveal too much of the underlying tissues as a distraction to viewing the surface (Fig. 1 A). A new technique of illumination, utilizing an annulus in the stage condenser, produces images with increased resolution and enhanced surface morphology that are strikingly similar to those produced with a scanning electron microscope (SEM) (Fig. 1 B). We refer to this as annulus-enhanced illumination (AEI). 
 
 
 
Fig. 1 
 
Light micrographs at the same magnification of a perineal pattern of a female of the southern root-knot nematode, Meloidogyne incognita (Kofoid and White, 1912) Chitwood, 1949. A) Brightfield. B) Brightfield image with the specimen illuminated with an ... 
 
 
 
Although the initial picture obtained by AEI appears similar to a photographic negative (Fig. 1D), through the use of image processing software and a computer, the final image is inverted into a photograph that resembles a positive (Fig. 1B). The colors of the digital photographs presented in Fig. 1. B and C were inverted with Adobe Photoshop® software using the menu lines: “image-adjustments-invert” (“command I” for Macintosh® computers and “control I” for computers utilizing Microsoft Windows®). An image taken with normal brightfield illumination and inverted has little effect on the appearance of the image (Fig. 1C). 
 
Annular rings are most often mounted in the stage condenser of a light microscope for phase microscopy. These rings can be purchased from a microscope distributor and installed in the condenser turret or they can be hand-made and placed in the condenser tray (Walker, 2004). They are necessary for phase and have been shown to increase resolution (Vainrub et al., 2006). This increase in resolution occurs from the coherent illumination (light waves that vibrate with constant phase relationships) produced by the annular aperture that is absent in the normal brightfield condenser. This coherent illumination contributes to the enhanced image of the perineal pattern by increasing the resolving power of the light, and it also increases contrast and enhances surface morphology, making the perineal pattern appear three-dimensional. 
 
Observation of perineal patterns with an annular ring in the stage condenser is useful for increasing the resolution of the image. These enhanced images emphasize the surface morphology and increase the details that can be seen. This new technique of illumination may increase the value of the observation of perineal patterns for identification of Meloidogyne species, as well as other surface structures such as visualizing surface morphology of cyst nematodes, en face views, lateral lines, phasmids and other sensory openings, lateral lines, etc.