Transmission electron microscopy of human saliva

Abstract

Globular, or micelle-like, structures have been demonstrated in human parotid saliva by photon correlation spectroscopy and transmission electron microscopy (TEM, negative staining). However, preparation of the TEM specimens may lead to artefact formations. To verify the presence of micelle-like structures in human parotid saliva, the application of cryo-transmission electron microscopy may prove valuable. The aim of this study was therefore to examine human parotid saliva using cryo-TEM and to correlate these results with parallel TEM examinations of parotid saliva prepared with negative staining. Whole saliva was also examined for the presence of these structures, as they may be of importance in oral physiology. Human and bovine milk were included as the presence of casein micelles in these fluids is well established. Freshly collected parotid saliva was prepared for cryo-TEM by instantaneous vitrification in liquid nitrogen. Specimens were also prepared with negative staining (2% ammonium molybdate) from freshly collected parotid saliva, from 0.45 μm filtered parotid saliva, from glutaraldehyde fixed parotid saliva, and from clarified whole saliva. Specimens of human and bovine milk were prepared from freshly obtained milk as well as from glutaraldehyde fixed milk samples. Cryo-TEM demonstrated globular structures in parotid saliva, mostly in the size range about 100–350 nm, appearing as single structures or in clusters. Similar globular structures were observed in negatively stained samples of parotid and whole saliva, and appeared to be multi-globular. TEM of human and bovine milk showed numerous multi-globular casein micelles with a structure similar to the globular structures observed in human saliva. The study demonstrates globular structures in human parotid and whole saliva similar to the casein micelles of milk, indicating the presence of salivary micelle-like structures as an important fraction of human saliva and that the negative staining technique may be suitable for TEM examinations of ultra-structural phenomena in saliva.