Saline was added to make the volume to 11 ml to which 2 ml diethyl ether was added and centrifuged at 10,000 rpm for 10 minutes. This procedure was repeated twice after which the supernatant was discarded and the sediment preserved in sterile containers in normal saline. Ether is classed as an extremely flammable reagent requiring storage in suitable flammable-liquid storage cabinets; therefore, we used ethyl acetate
as an alternative. Formalin was not used as it leads to a reduction in the fluorescence intensity of stained spores and being a Polymerase Chain Reaction (PCR) inhibitor, it may interfere with the molecular study of the parasites to be conducted later [4]. After concentration the saline and iodine find more selleckchem preparations of the samples were microscopically observed as above. Staining The concentrated samples were used for staining. Thin smears from all the samples were prepared on two different slides. The first slide was stained by Modified safranin technique [3]. In this method 3% acid alcohol was used for fixation. Safranin was used for staining and counterstaining was done by Malachite green. Kinyoun’s staining was used to stain the second slide [3]. The smear was fixed with absolute methanol and stained with Kinyoun’s carbol fuschin. Destaining was done by
10% alcohol and the smear was counterstained by Malachite green. At least 200 oil immersion fields of the above smears were examined for the parasites. Fluorescence microscopy A wet mount preparation of the concentrated samples was made and checked for autoflourescence of Cyclospora cayetanensis at 200× magnification with a 330 to 380 nm UV filter. The use of Calcoflour White (Sigma, USA) for fluorescent labeling of Microsporidia spores based on the presence of α-chitin in the inner endospore layer of the spore wall was first introduced by Vávra et al [5]. Calcoflour White stain (10 μl)
was added to the same amount of concentrated samples taken on clean, dry slides. The working solution was prepared by making 1:10 dilution of the stock (1%) and adding 0.05% Evan’s Blue dye. Slides were examined with the help crotamiton of UV fluorescence microscope at an excitation wavelength of 405 nm. A modification of the above method was performed by using a fluorescent probe 4, 6-diamidine-2-phenylindole (DAPI). Equal quantities (10 μl) of stool sample and DAPI (Sigma, USA) were put on a slide and left for 5 minutes. Thereafter, 10 μl of Calcoflour White was added and the slides were air dried. The slides were screened with the help of a fluorescence microscope using 435-485 BA filter. Antigen detection The third part of the unconcentrated stool samples was subjected to sandwich ELISA for Cryptosporidium parvum antigen detection. The procedure was performed as per the instructions given in the commercially available kit (IVD Research Inc. CA, USA).