Primo star iled fluorescence microscope

Serological testing on the serum samples was performed in the Department of Biomedical Science at the Polytechnic of Namibia using the following IgG (Immunoglobulin G) antibody kits from Fuller Laboratories (Fullerton, California, USA): 1) MIF IFA (micro-immunofluorescence assay) IgG antibody (gamma chain specific) kits for C. burnetii (Phase 1 and 2 in the same IFA spot); 2) ELISA (enzyme-linked immunosorbent assay) IgG antibody (gamma chain specific) kits for rickettsiae groups (spotted fever and typhus group rickettsiae); 3) MIF IFA IgG antibody (gamma chain specific) kits for Rickettsia africae/R. typhi (in same IFA spot); and 4) IFA (immunofluorescence assay) IgG antibody (gamma chain specific) kits for B. henselae. Testing protocols followed manufacturer’s instructions, including the cutoff calibrator instructions for the rickettsiae ELISA. All IFA slides were screened by two trained persons using a Primo Star iLED fluorescence microscope (Zeiss), recommended for developing countries by the WHO [47] . Only samples that had a questionnaire were included in the study. Methods for each test kit include the following:

One of the slides containing SSS was dried for 15 min at room temperature and fixed from below by passing slowly through the flame of a spirit burner three times. The slide was then stained with 1% carbolfuchsin solution, heated as above until vapor begins to rise for 5 min. The slide was then washed with running tap water and destained with 1% acid-alcohol for 10–20 s, rinsed with tap water gently, counterstained with 0.2% methylene blue for 1min, washed again with tap water and air dried. Finally, it was examined under a 100X objective of a conventional light microscope [26 ].
For AO staining, the slide was flooded with 0.1% AO (MERCK, Germany, prepared locally) solution for 20 min, destained with 0.5% acid-alcohol for 2 min, counterstained with locally prepared 0.5% potassium permanganate (Riedel-deHaen, Germany) for 4 min, then air dried. The slide was rinsed with sterile water between each step. Then, bacilli examination was carried out using a light-emitting diode fluorescence microscopy (ZEISS Primo Star iLED fluorescence microscope, Germany) with a 40X objective [17 ,19 ].