Identification of Rickettsia rickettsii in formalin-fixed, paraffin-embedded tissues by immunofluorescence

Abstract

With slight modification of a trypsin digestion technique, Rickettsia rickettsii were demonstrated specifically by immunofluorescence staining in Formalin-fixed, paraffin-embedded tissue sections from a human, rhesus monkey, and guinea pig with Rocky Mountain spotted fever and in infected membranes from a chicken embryo. Tissues were cut at 4 micron and, using geltain as a tissue adhesive, were hydrated in a routine manner. Sections were then digested in refrigerated 0.1% trypsin for 16 h, washed, and stained specifically for R. rickettsii by direct or indirect immunofluorescence. Rickettsial organisms were localized in affected vessels of the mammalian species and within the yolk sac epithelium of the chicken embryo. Specificity was confirmed by adsorbing antibody conjugates with R. rickettsii organisms. Trypsin digestion probably decreased tissue proteins which interfered with immunochemical attachment of antibody to the rickettsiae. The technique is valuable in that a diagnosis of Rocky Mountain spotted fever can be confirmed from Formalin-fixed tissues processed in a routine manner.