Effects of Air Drying and Critical-Point Drying on Morphology of Trichomonas gallinae

Abstract

Upper alimentary tract tissues of ring doves (Streptopelia risoria), experimentally infected with virulent Trichomonas gallinae, were processed for scanning electron microscopy by three procedures. Preparatory schedules which included aldehyde fixation, tannic acid, and guanidine hydrochloride fixation, as well as osmium tetroxide fixation, permitted air drying of large tissue samples from Freon?. Tissue samples prepared by this method revealed improved preservation of surface details and fewer structural artifacts when compared with tissues prepared by standard techniques and critical-point drying. Artifacts in biological tissues that have been prepared for scanning electron microscopy (SEM) may be a result of poor fixation rather than of a specific drying technique (Gamliel, 1985). In studies of megakaryocytes, human myeloblastic leukemia cells, and other soft tissues, Gamliel (1985) indicated that improved preservation of surface details and reduced swelling and shrinkage occurred when tannic acid-guanidine hydrochloride fixation and air drying from Freon? were included in tissue preparation schedules. Gamliel (1985) also indicated that comparative tissues prepared by standard techniques and criticalpoint dried appeared to be in poorer condition. A technique such as this may be beneficial in studies of trichomonad parasites (Protozoa: Sarcomastigophora). Effects of improved fixation and air drying on the morphology of Trichomonas gallinae (Rivolta, 1878) Stabler, 1938 and on avian pharyngeal epithelium were investigated. Modifications to Gamliel's (1985) basic protocol were made, and the results were compared to those obtained by standard methods. These methods were applied to other flagellated protozoa (Buchel et al., 1987; Erlandsen & Bemrick, 1987; Heath, 1981; John & Squires, 1978; Ovcinnikov et al., 1975; Warton & Honigberg, 1979). MATERIALS AND METHODS Ring doves (Streptopelia risoria) known to be specific pathogen-free were each orally intubated with 3.0 x 106 cells of virulent Trichomonas gallinae. ' I thank Dr. Harry T. Horner and Mr. Bruce Wagner, Bessey Electron Microscopy Center, for their help during technical phases of this study. This work was supported in part by a grant from the Iowa State University Graduate College. 2 Present address: Division of Mathematics and Science, Wayne State College, Wayne, Nebraska 68787, U.S.A. TRANS. AM. MICROSC. SOC., 110(2): 172-177. 1991. ? Copyright, 1991, by the American Microscopical Society, Inc. This content downloaded from 207.46.13.103 on Thu, 20 Oct 2016 04:35:14 UTC All use subject to http://about.jstor.org/terms VOL. 110, NO. 2, APRIL 1991 Prior to intubation, parasites were subcultured three times in simplified trypticase-serum (STS) medium (Difco) that had been enriched with 10% fetal bovine serum (Gibco). Birds were killed 72 h post-infection and tissue samples between 5 and 7 mm2 were excised from the pharyngeal region of each bird. Tissues from the six infected birds were processed randomly by one of the following procedures. Procedure 1. Tissue samples were fixed by immersion at 4?C for 24 h in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M Sorensen's dibasic sodium phosphate-monobasic potassium phosphate buffer (Lillie, 1965), pH 7.2. Following primary fixation, tissues were washed in three changes of cold buffer and then placed for 2 h in a solution of freshly filtered 2% tannic acid, 2% guanidine hydrochloride (TA-GuHCI), and buffer. Samples were washed in three changes of cold buffer, fixed in 1% osmium tetroxide (OS04), washed in three changes of cold buffer, dehydrated in ethanol, infiltrated with Freon? 113 (trichlorodifluoromethane) and exposed to air for drying. Gamliel's (1985) procedure differed from this one in that primary fixation was in 2% glutaraldehyde in Dulbecco's phosphate-buffered saline (DPBS) for 4 h or less and that all steps were performed at room temperature. Procedure 2. Tissue samples were treated similarly to those of procedure 1, except that the TA-GuHCI step was eliminated and critical-point drying replaced air drying from Freon?. Procedure 3. Tissues processed by this procedure were fixed for 6 h at 4?C by immersion in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2. Steps following aldehyde fixation were similar to those of procedure 1, except that the TA-GuHC1 and air drying steps were eliminated, and samples were critical-point dried. All dried samples were mounted on brass specimen stubs with colloidal silver paint (Ted Pella, Inc.) and coated with approximately equal amounts of gold palladium. A JEOL JSM-35 scanning electron microscope set at 15 kV was used for all sample examinations. Photomicrographs were taken from approximately the same location on each tissue sample with Polaroid? 665 positive/negative film.