Enhancement by p-Phenylenediamine of Osmium for Staining Coccidia in Cell Culture

Abstract

Most of the procedures used to fix and stain cultured cells infected with coccidia for light microscopy are laborious and time consuming. For instance, 1.5 to 2 hr are required to fix and then stain cultures with iron hematoxylin, which is one of the better stains for coccidia. Staining with Giemsa requires 25 to 30 min and usually does not adequately stain coccidian parasites. Also, procedures routinely used for fixing and staining coccidia in cell cultures usually cause shrinkage and distortion of the parasite. By using a modification of the p-phenylenediamine-osmium method of Ledingham and Simpson (1970, Stain Technol. 45: 255-260) we were able to rapidly fix and stain asexual stages of Eimeria pragensis in cultured cells with essentially no production of artifacts. Monolayers of human embryonic kidney (HEK) cells were grown in minimal essential medium with 5 or 10% fetal calf serum on 13mm-diameter glass or plastic coverslips in Costar culture plates (Bellco Glass, Inc., Vineland, New Jersey) with 24 16-mm-diameter wells. At various intervals after inoculation of each well with 5 X 104 sporozoites of E. pragensis, monolayers on coverslips were fixed in 2% (v/v) glutaraldehyde in Millonig's phosphate buffer for 2 to 5 min, rinsed in buffer for 15 sec, stained in 1% (w/v) Os04 in Millonig's phosphate buffer for 8 to 10 min (pH 7.2), rinsed in buffer for 5 sec, and partially dehydrated by 10-sec rinses in 50 and 70% ethanol. Monolayers were then exposed to freshly-prepared 0.8 to 1.0% (w/v) p-phenylenediamine (Fisher Scientific Co., Fair Lawn, New Jersey) in 70% ethanol for 30 sec, dehydrated by 10-sec rinses in 95 and 100% ethanol, placed in toluene for 20 sec, and mounted in permount (Fisher Scientific Co., Fair Lawn, New Jersey) on a glass microscope slide. Some monolayers were prepared as described above except