Abstract
Mycetoma is a lifelong granulomatous disease of subcutaneous tissues and bones. Histopathology is a substantiated indicative method based on the assumption of a definitive diagnosis of mycetoma. It requires efficient processing of tissues including bone decalcification. The decalcification process must ensure complete removal of calcium and also a proper preservation of tissue and microorganisms' staining ability. Objectives. To compare the conventional method used in decalcification with the microwave method using different decalcification solutions. Different characteristics were tested, including the speed of decalcification and morphological and fungal preservation in bone tissue affected with mycetoma. Materials and Methods. Three decalcification solutions were employed to remove calcium from 50 bone tissue samples affected with mycetoma, including 10% neutral buffered EDTA (pH 7.4), 5% nitric acid, and 5% hydrochloric acid. Conventional and microwave methods were used. Haematoxylin-eosin (HE) stain, Gridley's stain, and Grocott hexamine-silver stain were employed to evaluate the bone and fungi morphologies. Results. The decalcification time of the conventional method compared with the microwave method with 10% EDTA (pH 7.4) took 120 hours and 29 hours, while 5% hydrochloric acid and 5% nitric acid took 8 hours and 3 hours, separately. Also, 10% EDTA is the best decalcifying agent for HE staining and fungal stains. 5% hydrochloric acid and 5% nitric acid can be used for fungal staining. Conclusion. The current study investigated the effects of different decalcifying agents as well as two decalcification procedures on the preservation of the bone structure and fungal staining, which will help to develop suitable protocols for the analyses of the bone tissue affected with mycetoma infection.
Highlights
Mycetoma is a lifelong granulomatous, gradually deleterious epidemic disease of the skin and subcutaneous tissues that can progress to deeper structures like muscles and bones and lead to extensive destruction, mainly of the feet, requiring wide local surgical excisions or limb amputation [1]
Histopathology is a fast indicative method as well as advantageous process on the assumption of a definitive diagnosis of mycetoma disease and includes a report that describes the morphological presentation of the causative agents. e causative agent could still be isolated from the bone tissue involved [5]
Materials and Methods is is an experimental descriptive study aimed to compare the conventional decalcifying procedure with microwaveenhanced decalcification with respect to tissue morphology affected by mycetoma infection using haematoxylin and eosin, Gridley, and Grocott hexamine-silver stains for complete identification of the Madurella mycetomatis causal organisms. is study was conducted at the Mycetoma Research Center in Soba University Hospital and the Faculty of Medical Laboratory Science, University of Al Neelain
Summary
Mycetoma is a lifelong granulomatous, gradually deleterious epidemic disease of the skin and subcutaneous tissues that can progress to deeper structures like muscles and bones and lead to extensive destruction, mainly of the feet, requiring wide local surgical excisions or limb amputation [1]. 2. Materials and Methods is is an experimental descriptive study aimed to compare the conventional decalcifying procedure with microwaveenhanced decalcification with respect to tissue morphology affected by mycetoma infection using haematoxylin and eosin, Gridley, and Grocott hexamine-silver stains for complete identification of the Madurella mycetomatis causal organisms. The Grocott hexamine-silver method for fungi as described by Grocott in 1955 was used in which sections were oxidized with 4% aqueous chromic acid for one hour, washed in water for a few seconds, treated with 1% sodium metabisulphite for one minute, washed in running tap water for 3 minutes, rinsed thoroughly in distilled water, placed in preheated working silver solution in a water bath at 60°C for 20 minutes, rinsed well in distilled water, washed with running tap water for 5 minutes, counterstained in working light green for 15 seconds, dehydrated and cleared in xylene and mounted in DPX [16], and examined under a microscope. Differences with P < 0.05 were interpreted as being statistically significant
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