Medical Mycology December 2004, 42, 499Á/504 Evaluation of Microsporum canis in different methods of storage R. S. N. BRILHANTE*$, C. S. P. CAVALCANTE*, F. A. SOARES-JU´ NIOR*, A. J. MONTEIRO%, E. H. S. BRITO*, R. A. CORDEIRO$, J. J. C. SIDRIM$ & M. F. G. ROCHA*$ *School of Veterinary Medicine, Post-Graduation Program in Veterinary Science, State University of Ceara´, Fortaleza; $Department of Pathology and Legal Medicine, School of Medicine, Medical Mycology Specialized Center and %Department of Statistics and Applied Mathematics, Federal University of Ceara´, Fortaleza, Brazil Downloaded from https://academic.oup.com/mmy/article/42/6/499/967874 by guest on 30 September 2021 The main objective of this investigation was to evaluate different methods of storage for Microsporum canis based on materials and equipment that are readily available in developing countries. We tested 32 strains of M. canis at /208Cin potato dextrose agar (PDA) in its plain condition, or amended with 10% dimethyl sulfoxide or with 10% glycerol. In addition, we tested 258C storage of isolates in plain saline (0.9% NaCl) and in saline covered with a mineral-oil layer. After 9 months of storage, none of the M. canis strains frozen in PDA supplemented with glycerol survived, while only 16 and 6%, respectively, of the isolates in plain and DMSO medium lost viability. Nine month’s storage in saline with or without mineral oil increased the amount of pleomorphic development of sterile hyphae; this phenomenon occurred at a significantly higher level than was seen in isolates stored at /208C. The physiological characteristics of M. canis were not affected by the different storage tests. The results suggest that, in order to ensure optimal viability, purity and pristine isolate condition, each M. canis isolate maintained should be held in at least two methods of storage, namely, PDA at /208C and saline with a mineral-oil layer at 258C. Keywords cats, dogs, Microsporum canis, storage Introduction Various methods have been proposed for the high quality preservation of fungal cultures; for example, The preservation of fungal strains is a very important good results have been shown for storage in sterile soil activity in a mycological laboratory. Strains may be [3], storage in sterile distilled water [1,4,5], freezing at preserved as diagnostic reference stocks, or for com- /708C [6], freeze-drying (lyophilization) [7], mainte- parative studies, or for use in training [1]. Some fungi nance under paraffin oil overlays [8] and immersion in are difficult to maintain in good condition, however, vessels held in liquid nitrogen (cryopreservation) [9]. and some dermatophytes in particular mutate rapidly It is recommended that in order to minimize the to produce morphological variants quite unlike the probability of strains being lost, each strain should be parental strain. Changes in physiological, biochemistry, maintained by at least two different procedures, when- pathogenicity and genetic characteristics may ever practical. At least one of these, where possible, also occur, often without obvious morphological should be lyophilization or storage in cryopreservation, changes [2]. as for most strains these are the best methods for minimizing the risk of genetic change [7,9,10]. In many parts of the world, however, such methods are not available and alternative techniques must be found. Received 31 January 2003; Accepted 10 October 2003 The purpose of the present investigation was to assay Correspondence: R. S. N. Brilhante, Rua Bara˜o de Caninde´ 210; Montese, CEP 60.425-540, Fortaleza CE, Brazil. Tel: /55 085 214 which widely available, inexpensive methods would be 2853; Fax: /55 085 295 1736; E-mail: [email protected] suitable for preservation of the macroscopic and – 2004 ISHAM DOI: 10.1080/13693780410001712052 500 Brilhante et al. microscopic features of Microsporum canis isolates in further analysis was done. For strains producing a Brazilian laboratory lacking routine access to lyophi- macroconidia, 10 nonoverlapping /40 microscope lization, ultra-low-temperature freezing and liquid fields were examined. Macroconidial numbers in the nitrogen storage. combined fields was noted as 0Á/10, 11Á/50 or /50. Quantification of microconidia was done similarly. After quantification, macroconidia were checked to Materials and methods determine if they had typical morphology (fusiform, Isolates with thick and roughened walls). Thereafter, macro- conidia were randomly selected and measured with a Between November 2000 and August 2001, 32 strains reticule for length and width. These features were of M. canis were obtained from 22 dogs and 10 cats observed at /40. examined in the Medical Mycology Specialized Center, Physiological characteristics of the revived isolates Downloaded from https://academic.oup.com/mmy/article/42/6/499/967874 by guest on 30 September 2021 Faculty of Medicine, Federal University of Ceara´, were evaluated, as recommended by Sidrim and Brazil. The samples were obtained in collaboration Moreira [11], with nutritional tests (thiamine, nicotinic with five veterinary clinics located in the city of acid, inositol and histidine) and the urease test. In Fortaleza, Ceara´, Brazil. addition, the in-vitro hair perforation test was done. Storage strains Statistical analysis Definitively identified M. canis isolates were main- A test for homogeneity, the Fischer’s exact test, was tained in saline (0.9% NaCl) at room temperature used to compare numbers of surviving M. canis strains (258C). This procedure was intended to establish a in different methods of storage. common zero time for all strains. Inoculum from the saline stocks was then used to inoculate potato dextrose agar (PDA; Difco, Detroit, MI), which was incubated Results for 15 days at room temperature. The strains of M. canis were then subcultured into the following storage In pre-storage micromorphological analysis, Sabour- aud agar was observed not to favor conidiogenesis: 63% media for storage at /208C: (i) PDA, (ii) PDA with 10% dimethyl sulfoxide (DMSO) and (iii) PDA with of the M. canis strains grown on this medium formed 10% glycerol. In addition, each strain was stored at only sterile hyphae (Fig. 1A). Of the 37% strains that 258C in saline (7-ml vol. of 0.9% NaCl) with and produced macroconidia, only 13% produced typical without a 2-ml covering of sterile mineral oil. macroconidia. PDA, rice agar and lactrimel agar were equivalent for macroconidia production; the difference among these media was not significant (P/0.4190). Fungal viability verification for the different storage methods However, the lactrimel agar medium appeared in At intervals of 3, 6 and 9 months, frozen strains were general to be more favorable for the observation of thawed and material was inoculated into Sabouraud conidium formation, since of all media it induced the agar and PDA tubes. Strains stored in saline and saline lowest production of sterile mycelium, a problem with mineral oil were manually agitated and a portion affecting only a single one of the 32 cultures grown of each suspension was transferred to growth media as on it prior to preservation (Fig. 1B). used for the frozen samples. When preserved isolates were examined for sterile Microsporum canis colonies in Sabouraud and PDA mycelium production, it was seen that storage in saline, media were analyzed after 15 days, and the colony with or without mineral oil, increased the amount of surface texture and reverse pigmentation were noted. sterile mycelium seen (Fig. 2). This difference is Micromorphology of the revived isolates was significant (P/0.0015), when compared to M. canis observed in rice agar, lactrimel agar, Sabouraud agar isolates preserved frozen. and PDA as recommended by Sidrim and Moreira [11]. Lactrimel agar stimulated macroconidium produc- Slides were made in lactophenol cotton blue, and for tion in 31 of the 32 test strains in baseline checks prior each culture, 10 microscope fields were scanned at to storage (Fig. 3). In addition, 75% of the strains /40. grown on this medium produced typical macroconidia. The quantification of macroconidial production was The macroconidial sizes, ranging from 50 to 85 mmin as follows. Initially, we noted whether each test strain length, and 7.5 to 20 mm in width, remained stable produced macroconidia or just sterile mycelium on the during the 9 months of preservation irrespective of the test media. If the strain did not sporulate initially, no method used. – 2004 ISHAM, Medical Mycology, 42, 499Á/504 M. canis and different storage methods 501 Downloaded from https://academic.oup.com/mmy/article/42/6/499/967874 by guest on 30 September 2021 Fig. 1 Frequency of sterile mycelial proliferation in Microsporum canis isolates maintained under different preservation methods and then grown for analysis on different media. (A) Sabouraud agar results; (B) lactrimel agar. Isolates stored 9 months in both saline treatments aud as typical colonies (Table 1). Cottony, unpigmented and in plain PDA and PDA with DMSO lost their colonies made up the largest morphological category, capacity to produce microconidia. These results were accounting for 41% of strains pre-storage. These strains analyzed using lactrimel agar, which stimulated micro- showed further attenuation of morphological differen- conidial production in all but one of the strains prior to tiation during the preservation period, independent of preservation (Fig. 4). the method evaluated. Only a single M. canis strain Only 9% of pre-storage strains grown on Sabouraud preserved 6 months in either saline treatment showed a agar had low cottony colonies with fimbriate margins typical fringed, yellow colony. After 9 months frozen and canary yellow pigment, even though these char- on PDA with DMSO, three of the 32 test colonies acteristics are considered typical for M. canis.How- showed these characters. ever, after storage in PDA with DMSO and saline with The tested physiological characteristics of M. canis, mineral oil, 28% of M. canis strains grew on Sabour- as outlined above, were not changed by the different Fig. 2 Isolates of Microsporum canis showing proliferation of sterile hyphae after 9 months of storage in plain saline and in saline overlaid with mineral oil.