Avaliação da viabilidade de leveduras do gênero Cryptococcus armazenadas pela técnica de criopreservação há 10 anos

• DOI: https://doi.org/10.22533/at.ed.630112526028

• Palavras-chave: Cryptococus spp., Estocagem de micro-organismos, Preservação.

• Keywords: Cryptococcus spp., Microorganism storage, Preservation.

• Abstract: Introduction: With scientific and biotechnological advances, the need for the preservation of microorganisms in laboratories for educational, diagnostic, and research purposes has become increasingly evident. Objective: To evaluate the viability of Cryptococcus spp. isolates stored under cryopreservation conditions for a period of 10 years. Methodology: The viability of 166 isolates stored in cryogenic tubes containing Brain Heart Infusion (BHI) broth supplemented with 20% glycerol and maintained in a freezer at -20°C between 2014 and 2023 was assessed. For reactivation, the tubes containing yeast suspensions were completely thawed in a 35°C water bath. Subsequently, 10 µL of the suspension were inoculated onto Sabouraud Dextrose Agar (SDA) plates supplemented with chloramphenicol, and the remaining volume was transferred to tubes containing BHI broth, followed by incubation in a microbiological oven at 35°C for up to 72 hours. Fungal growth was confirmed by the presence of colonies on SDA plates or turbidity in BHI broth, after which the cultures were re-inoculated onto fresh SDA. Micromorphological analysis was performed using India ink staining. Sample purity was confirmed through biochemical tests for species identification. Results: Of the 166 isolates, 15.1% (25) were viable, the majority being C. neoformans (20), followed by C. gattii (4) and C. albidus (1). In light of these findings, it can be concluded that although cryopreservation is an effective technique, it may not be suitable for long-term storage of Cryptococcus spp. isolates, highlighting the need for periodic reactivation within a shorter time frame.