Topography of Ascaridia galli Eggs Exposed to Low Temperatures or Cryoprotectants as Shown by Scanning Electron Microscopy

Abstract

Eggs cooled to -22 or -93 C were shrunken, and/or depressed. The topography of frozen eggs appeared abraded and mottled compared to the smooth surface of shelled, unfrozen controls. A smooth seam on the roughened lipid surface of deshelled, air-dried eggs appeared as a line of dehiscence. The folded and wrinkled lipid surface of deshelled, freeze-dried eggs had a polar ridge. The only published report of scanning electron microscopy (SEM) of nematode eggs is that of Ubelaker and Allison (1972). They described an elaborate reticular network producing irregular depressions on the surface of Ascaris lumbricoides and A. suum eggs. Also, a round depression was seen at one pole, a possible site of larval egression. In our preliminary cryobiologic studies, more fertilized, shelled eggs (chitinous layer present) survived freezing to -22 C than to -93 C. Also, 5 and 10% glycerol was toxic to fertilized, deshelled eggs (chitinous layer removed), but 5 and 10% dimethyl sulfoxide (DMSO) was not. The SEM was used to delineate effects tfiat may explain these observations. MATERIALS AND METHODS Shelled eggs were recovered from female worms incubated at 38 C in petri dishes containing 0.85% NaCi solution. These eggs were gently dislodged from the bottoms of petri dishes, pipetted into clinical centrifuge tubes, and washed 5 times with distilled water. Shelled eggs were suspended in distilled water and sealed in 1.2-cc glass ampules. Ampules were placed in either a refrigerator freezer (ambient temperature reached -22 C in approximately 30 min) or a REVCO low-temperature freezer (ambient temperature reached -93 C in approximately 9. min) for 24 hr. After thawing at 38 C, the shelled eggs were treated as follows: Fixed for 30 min at room temperature in 12% glutaraldehyde in 0.05 M Sorensen's phosphate buffer, pH 7.2; washed several times in 0.05 M Sorensen's phosphate buffer, pH 7.2; postfixed for 30 min at room temReceived for publication 17 August 1973. * Contribution No. 1208, Division of Biology, Kansas Agricultural Experiment Station, Kansas State University, Manhattan, Kansas 66506. The authors acknowledge technical assistance of Dr. Charles Pitts and Mr. Paul Enos. perature in 1% osmium tetroxide in 0.05 M Sorensen's phosphate buffer, pH 7.2; washed several times with deionized, double-distilled water. Shelled eggs maintained at 30 C for 24 hr (controls) were fixed in like manner. All shelled eggs were then mounted on aluminum stubs and dehydrated as follows: A thin layer of ethyl acetate extract of double-coated tape No. 666 (3M Co.) was applied to the stubs; a small drop of deionized, double-distilled water containing eggs was pipetted at intervals around the edge of the stub; the stubs supporting the specimens were placed in a desiccator and dehydrated by air-drying overnight. Then dehydrated specimens were removed and coated with a thin layer of gold and palladium. Eggs were deshelled to expose the lipid layer according to the method of Elliott (1954). Five groups of deshelled eggs were placed one group each into 5 and 10% concentrations of dimethyl sulfoxide or glycerol and in distilled water (as controls). All tubes of eggs were rotated on a roller drum apparatus at ?/-. rpm for 12 hr at 23 C. The deshelled eggs were fixed, washed, mounted on stubs, and air-dried the same as the frozen shelled eggs were. In addition, a second group of mounted deshelled eggs were freeze-dried according to the method of Weise (1973). Specimens were examined with the ETEC scanning electron microscope (ETEC Corp.) at an accelerating voltage of 20 kv.