A METHOD FOR PREPARING SINGLE MAMMALIAN OVA FOR CYTOLOGICAL STUDIES USING LIGHT AND ELECTRON MICROSCOPY

P. L. SENGER, R. G. SAACKE
1970 Reproduction  
The inability to define accurately the cytological characteristics associated with fertile mammalian ova is due, at least in part, to difficulty in obtaining and preparing adequate numbers of these cells for critical morphological studies. This communication describes a method which has proved useful in preparing single bovine ova for study using both light and electron microscopy. Bovine ova were recovered by follicular aspiration immediately following slaughter. Aspiration was accomplished
more » ... was accomplished with a 20-gauge hypodermic needle and a 2-ml syringe (PL 1, Fig. 1 ). The follicular fluid was placed in a watch-glass (PL 1, Fig. 2 ) and scanned, using a stereomicroscope in order to locate the ovum. Following location, the ovum was transferred with a micro-pipette from the follicular fluid to a fixation ampoule suspended in an ice water bath (PL 1, Fig. 3 ). The fixation ampoules were prepared from 6to 9-in. disposable Pasteur pipettes which had been scored and broken in order to shorten the narrow portion. The narrow tip of the pipette was sealed by flaming. This design (PL 1, Fig. 4) restricted the ovum to a small volume of fixative after it settled into the narrow tip of the fixation ampoule. The settling facilitated the location of the ovum after the fixation period. In addition, the ampoule could be suspended easily in an ice water bath for fixation at 0°C (PL 1, Fig. 3 ). The ova were fixed in 1 % osmium tetroxide buffered to pH 7-4 with veronal-acetate (Palade, 1952) or in 3% glutaraldehyde buffered to pH 7-4 with 0-1 M-phosphate. Fixation times ranged from 30 min to 1 hr. Ova fixed in glutaraldehyde were washed in the phosphate buffer overnight and subsequently placed in 0-1 M-phosphate buffered osmium tetroxide for 1 hr. Caution should be exer¬ cised in choosing fixatives. The preservation of cytological detail is known to vary with the tissue as well as with the fixatives used. Modifications of fixatives and buffers may be necessary if ova of different developmental stages in the bovine or ova from other species are to be studied. Following fixation, the tip of the ampoule was scored with a diamond stylus (PL 1, Fig. 5 ) and broken. The ovum was removed from the tip of the ampoule and subsequently dehydrated in Syracuse watch-glasses (PL 1, Fig. 6 ) using a graded series of ethanol (80, 95, 100 %). Subsequently, embryological watchglasses (A. H. Thomas, Philadelphia, Cat. No. 9844) have been found to be superior due to their smaller size and spherical concavity. A dehydration period 177
doi:10.1530/jrf.0.0210177 pmid:4905088 fatcat:cjb6bzxbzvc7todtktva4zdl3i