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The genus Malassezia comprises lipophilic yeasts that belong to the normal cutaneous microbiota of humans and warm-blooded animals (10), but these yeasts are also associated with a variety of diseases in humans and animals (4, 9, 11).

The taxonomy of this genus has been a matter of controversy since its creation by Baillon in 1889. The great micromorphological polymorphism and the lack of suitable methods for isolation and preservation of these yeasts were the main reasons that made their study difficult. The genus has recently been revised by means of morphology, ultrastructure, physiology, and molecular biology and enlarged to seven species: M. pachydermatis, M. furfur, M. sympodialis, M. globosa, M. obtusa, M. restricta, and M. slooffiae (5, 6). Except for M. pachydermatis, the remaining six species are lipid-dependent organisms, because they require long-chain fatty acids for in vitro growth. The isolation and identification of Malassezia spp., mainly lipid-dependent species, continue to be difficult due to low viability, especially with some of the species.

The purpose of this study was to assay different methods for preservation of microorganisms in order to find a suitable method that allowed a proper and long maintenance of Malassezia spp.

Twenty-four strains of M. pachydermatis and 11 lipid-dependent strains were used in this study. All strains were isolated from different animals except for six lipid-dependent type strains (kindly provided by E. Gricho and J. Guillot) (Table 1).

The strains of M. pachydermatis were grown on Sabouraud glucose agar (SGA) for 3 days at 35°C and the lipid-dependent strains on modified Dixon agar (MD) (5) for 5 days at 32°C.

After incubation, cells were harvested and suspended in 3 ml of 10% skim milk. The number of Malassezia cells was determined microscopically in a Bürker chamber, and serial dilutions in skim milk were performed to obtain final suspensions of approximately 10⁷ and 10⁴ cells per ml. Preservation of lipid-dependent strains was assayed only for the ~10⁷-cells/ml suspension.

Four different preservation methods with some variables included were studied: freezing at 80°C, freeze-drying and storage at room temperature and at 80°C, preservation in distilled water, and use of different culture media stored at 4°C, room temperature, and 28°C. All of these assays were performed in duplicate. In freezing at 80°C, glycerol and dimethyl sulfoxide (DMSO) were employed as cryoprotective agents at concentrations of 10% (vol/vol) and 5% (vol/vol), respectively (2). The solid media used to maintain M. pachydermatis strains were SGA and a medium described by Lorenzini and de Bernardis (LDB) (8). For lipid-dependent strains, the media tested were MD, Leeming's medium (LM) (7), and SGA supplemented with olive oil (10 ml per liter). The preparation of inoculum was performed in distilled water to obtain final suspensions of approximately 10⁷ cells per ml.

Colony counts before freezing at 80°C, lyophilization, and preservation in distilled water were determined on solid media using the surface-spread method. Aliquots of 0.1 ml of the appropriate dilutions were inoculated on three plates of SGA for M. pachydermatis strains and MD and LM for lipid-dependent strains. SGA plates were incubated at 35°C for 5 days, whereas both MD and LM plates were incubated at 32°C for 7 days. In the lyophilization method, colony counts were also determined immediately after the end of the process. Colony counts were determined monthly for a year and a final check at the 18th month when the methods of freezing at 80°C, lyophilization, and preservation in distilled water were used. During the preservation of different culture media stored, the viability was first checked at day 15 and then was checked monthly for a year.

Data obtained were analyzed statistically by means of one-way analysis of variance test, Student's t test, and chi-square test. All statistical analyses were performed using SPSS software (version 8.0).

The effect of freezing at 80°C with glycerol and DMSO on the viability of Malassezia spp. is detailed in Table 2. Results are expressed as mean percentages of recovery from mean colony counts obtained before freezing for all strains tested. Mean percentages of recovery of viable M. pachydermatis cells from the suspensions of inocula estimated in a Bürker chamber of approximately 10⁷ and 10⁴ cells per ml, were 3.2 and 19.5%, respectively, for glycerol and 3 and 46.5%, respectively, for DMSO. Mean percentages of recovery of viable lipid-dependent species from the approximately 10⁷-cells/ml suspension were 4.3% on MD and 3% on LM for glycerol and 4.3% on MD and 3.5% on LM for DMSO.

View this table: [in this window] [in a new window]   TABLE 2.   Effect of freezing and storage at 80°C with glycerol and DMSO on viability of Malassezia spp.a

The effect of lyophilization and storage at 80°C and at room temperature on the viability of Malassezia spp. is detailed in Table 3. Results are expressed as mean percentages of recovery from mean colony counts obtained immediately after lyophilization for all strains tested. Mean percentages of recovery of viable M. pachydermatis cells from the suspensions of inocula estimated in a Bürker chamber of approximately 10⁷ and 10⁴ cells per ml were 8.8 and 37%, respectively. On the other hand, mean percentages of recovery of these yeasts immediately after the lyophilization process were 43.2% (~10⁷-cells/ml suspension) and 64.9% (~10⁴-cells/ml suspension). Mean percentages of recovery of viable lipid-dependent species from the ~10⁷-cells/ml suspension were 4.8% on MD and 4% on LM. Mean percentages of recovery of these yeasts immediately after the lyophilization process were 31.2% on MD and 27.5% on LM from the mean counts obtained before lyophilization.

View this table: [in this window] [in a new window]   TABLE 3.   Effect of lyophilization and storage at 80°C and at room temperature on the viability of Malassezia spp.a

The effect of preservation in distilled water on the viability of Malassezia spp. is shown in Table 4. Results are expressed as mean percentages of recovery from mean counts obtained at time zero. Mean percentages of recovery of viable M. pachydermatis cells from the suspensions of inocula estimated in a Bürker chamber of ~10⁷ and 10⁴ cells per ml were 3.6 and 23%, respectively. Mean percentages of recovery of viable lipid-dependent species from the ~10⁷-cells/ml suspension were 0.9% on MD and 1% on LM.

The number of viable M. pachydermatis strains stored in different solid media at 4°C, room temperature, and 28°C are detailed in Table 5. This method of preservation was also unsuccessful for maintaining viable M. pachydermatis. Although SGA and LDB were very inefficient for maintaining M. pachydermatis strains, SGA recovered a higher number of strains than LDB and particularly in the first months at 28°C.

The number of viable lipid-dependent strains stored in different solid media at 4°C, room temperature, and 28°C are detailed in Table 6. M. globosa CBS 7966^T and M. globosa CCFVB 76 did not grow on MD at the concentration of inoculum assayed in this method. Therefore, the maintenance on this culture medium was not performed for these strains. Maintenance of lipid-dependent species on different solid media was not a good method of preservation. Similar results were obtained for the different culture media assayed and for both media utilized for the recovery of viable yeasts.

The low viability of the yeasts of the genus Malassezia and their difficult maintenance in vitro, especially with some species, constitute the main difficulties that affect the study of this genus (1, 5, 8, 10). In all cases a sharp decrease resulted from the suspensions of inocula estimated in the Bürker chamber in our study.

The methods of preservation assayed in this study are classic methods for maintenance of bacteria and fungi (2, 12, 13). The majority of yeasts can be stored at temperatures between 4 and 12°C and subcultured at intervals of 6 to 8 months (13). However, Malassezia spp. do not fit this pattern.

In our study, freezing at 80°C was the only successful method to maintain viable all Malassezia spp., particularly M. globosa, M. restricta, and M. obtusa, which were reported as difficult species to maintain in vitro (5). However, the best results were obtained when glycerol was used as the cryoprotective agent. It has been reported that the use of skim milk in combination with glycerol is an excellent cryoprotective agent for the preservation of bacteria (3). In this method, mean percentages of recovery were >100% in many cases, especially with the lipid-dependent yeasts.

Homogeneous suspensions are very difficult to obtain with these yeasts and in particular with the lipid-dependent species due to clustering. The cryoprotective agents would facilitate the dispersal of the yeasts, yielding mean recoveries of >100%. The highest mean percentages of recovery were obtained with the lowest suspension of inoculum assayed in M. pachydermatis freezing. In this method, LM improved the mean percentages of recovery of lipid-dependent species. LM was described (7) as the best culture medium for isolation and enumeration of M. furfur in comparison to MD and Faergemann and Fredriksson's medium.

In our study, lyophilization and storage of the freeze-dried cultures at room temperature was an unsuitable method for preservation of Malassezia spp. However, when these freeze-dried cultures were stored at 80°C, all strains tested were recovered except for M. globosa CCFVB 76. Other authors observed that this species does not survive lyophilization (5, 10). Some authors recommend the storage of the freeze-dried cultures between 15 and 18°C, and others recommend storage between 4 and 18°C (12). However, Malassezia spp. only survived when the freeze-dried cultures were stored at 80°C. The best results for the lyophilization and storage at 80°C of M. pachydermatis were obtained with the suspension of approximately of 10⁴ cells per ml.

Among the different methods used to preserve fungi, the distilled water method and preservation on different culture media are two of the more-utilized methods, because they are technically simple and easy to perform. Nevertheless, in our study they were unsuccessful methods for maintaining the yeasts of the genus Malassezia.

As has been mentioned previously, the low viability of Malassezia spp. constitutes one of the main difficulties in the study this genus. Due to the reclassification of the Malassezia genus into seven species and the isolation of new strains from different origins, these results might be considered as preliminary in nature and should be substantiated with further studies on the preservation of Malassezia isolates. According to our findings, we recommend the method of freezing at 80°C immediately after the first isolation in order to avoid the loss of isolates.