Application of Neutral Red Staining for the Evaluation ofthe Viability of Dermatophytes

Park, Jang Kyu; Lee, Woo Jae; Suhr, Ki Beom; Lee, Jeung Hoon

Abstract

BACKGROUND: Microsopic examination of potassium hydroxide(KOH) preparation and fungus culture is required for the diagnosis of fungal infection. Sometimes dermatophytes fail to grow on culture medium, although these are observed on microscopic examination of KOH preparation. Recently this discrepancy between microscopic examination and fungus culture can be explained by the hyphothesis that some of the fungal elements are non-viable. OBJECT: This study was made to evaluate the viability of dermatophytes using neutral red(NR) staining for the explaination of discrepancies between microscopic examination and fungus culture.
METHODS
After identification of fungus by culture from dermatophytic lesion the hyphae was collected for this study. In order to confirm whether the NR staining is suitable for check the viability of hypae or not, we designed to prepare the preparations of hyphaes by 2 ways. One was killed hyphae by autoclave, the other was kept as viable hyphae. And then we compared the stainability of NR staining and autoradiographic study using (3)H-thymidine. And we compared the results of NR staining and the subsequent culture using the scales which were collected from the lesions 30 dermatophytic patients.
RESULTS
The structure inside of cell wall of hyphae stained red color only in case of viable hyphae preparations, but not stained in killed hyphae preparation. Autoradiographic study using (3)H-thymidine confirmed that grain-positive cells(viable cells) were stained with NR, whereas grain negative cells (non viable cells) were not stained. Among the 30 cases with dermatophytosis 27(90.0%) cases showed NR positive and 14(46.6%) cases showed culture-positive. Except the tinea unguium cases which have shown low culture positive rate, 9(75.0%) cases of the 12 NR positive samples were positive on culture. All 14 cases of the culture positive samples were positive on NR staining. And all 3 cases of the NR negative samples were negative on culture.
CONCLUSION
NR staining can be a useful method for the evaluation of viability of the fungal elements.