Prepare a thin smear using approximately 10 µl of 10% formalin fixed stool suspension (unconcentrated) on a glass slide. This staining method was developed at CDC using various components of the trichrome staining method to differentiate microsporidia spores from background fecal elements. The background should stain uniformly green. from a 10% formalin preserved specimen should be included with each staining run. To confirm internal morphology, use 100× oil immersion objective.Ī control slide of Cryptosporidium spp. Examine 200 to 300 fields using 40× or higher objectives.Mount with a coverslip using desired mounting media. Dry on a slide warmer at 60☌ for about 5 minutes.Rinse briefly with distilled water and drain. Counterstain with Malachite green for 2 minutes.Destain with acid alcohol for 2 minutes.Stain with Kinyoun’s carbol fuchsin for one minute.Fix with absolute methanol for 30 seconds.Prepare a smear with 1 to 2 drops of specimen on the slide and dry on a slide warmer at 60☌ until dry.3% Malachite green: dissolve 3 g of malachite green in 100 ml of distilled water.Kinyoun’s Carbol fuchsin: may be purchased commercially.Acid Alcohol: 10 ml Sulfuric Acid + 90 ml Absolute ethanol.There are four steps to this procedure requiring the following solutions: Other types of clinical specimens such as duodenal fluid, bile, pulmonary samples (induced sputum, bronchial wash, biopsies) may also be stained. Specimen:Ĭoncentrated sediment of fresh or formalin-preserved stool may be used. Unlike the Ziehl-Neelsen Modified Acid-Fast Stain, this stain does not require the heating of reagents for staining. This technique is useful for the identification of oocysts of the coccidian species ( Cryptosporidium, Cystoisospora, and Cyclospora), which may be difficult to detect with routine stains such as trichrome.
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