Improved staining method for detecting Cryptosporidium oocysts in stools using malachite green.

is recognized as a significant pathogen of vertebrate hosts, including humans and many other mammals, birds, reptiles, and fishes (Fayer et al., 1997). Current laboratory methods for the diagnosis of cryptosporidial infections generally rely on microscopic examination of fecal samples for the presence of Cryptosporidium oocysts. These oocysts are similar in size and shape to other fecal components (especially some yeasts), and therefore staining techniques that differentiate between the two are very desirable to avoid confusion (O’Donoghue, 1995). A safranin-methylene blue stain has been described by Baxby et al. (1984), which stains oocysts red and yeasts and other fecal debris blue. Acid-fast stains such as Kinyoun (Ma and Soave, 1983), Ziehl-Neelsen (Henriksen and Pohlenz, 1981), and DMSO-carbol fuchsin (Pohjola et al., 1984) stain oocysts red with the background counterstained and are commonly used in many laboratories. Differential staining techniques, however, are timeconsuming and vary in their sensitivity and specificity (Baxby et al., 1984; Moodley et al., 1991; Smith et al., 1989). Furthermore, research in our laboratory has shown that unless samples have been fixed overnight in formalin, oocysts may frequently take up the counterstain (unpublished observations). Negative staining techniques using nigrosin (Pohjola, 1984), light green, and merbromide (Chichino et al., 1991), which stain background yeasts and bacteria but not the oocysts, have also been developed. Negative staining methods are faster but are considered by some to be less sensitive than conventional staining techniques (Casemore et al., 1985; Garcia et al., 1983). In this study, 60 fecal samples from human and animal hosts that were microscopically negative for Cryptosporidium were spiked with known concentrations of Cryptosporidium oocysts and examined blind by a microscopist experienced at diagnosing cryptosporidial infections. An additional 20 samples containing several known naturally infected Cryptosporidium positive samples were also screened blind by the microscopist. The details of the samples screened are listed in Table 1. The 80 samples were examined by using 4 different staining techniques outlined below, including a negative malachite green stain, which we have found to be fast and reliable and should improve the microscopic diagnoses of cryptosporidial infections in clinical laboratories. Fecal samples microscopically negative for Cryptosporidium were divided into 1 g aliquots and emulsified in 8 ml of distilled water. Fresh Cryptosporidium oocysts were obtained by passaging oocysts from a naturally infected calf through mice as previously described (Meloni and Thompson, 1996). Fecal samples were seeded at random with 10 or 10 oocysts and tested blind by the microscopist. Known Cryptosporidium positive samples were obtained from a local pathology laboratory and also tested blind by the microscopist. For the malachite green negative stain, feces were spun at 800 g for 5 min and the supernatant removed. A drop of 5% malachite green in distilled water was then placed on a slide and mixed with one drop of the fecal pellet collected from its surface. Slides were examined for the presence of CrypImproved staining method for detecting Cryptosporidium oocysts in stools using malachite green