The choice of a suitable preservation method is critical for long-term microorganisms' viability. The strains should be preserved for long periods using reliable and reproducible methods that minimize genotypic and phenotypic variations and viability losses. The methodologies are usually designed for a better performance in isolated microorganisms. However, atypical primary isolates of Cryptococcus neoformans or Cryptococcus gattii, such as mixed species or even different species of a species complex, are a challenge for long-term preservation and taxonomic review studies. The aim of this study was to evaluate which of the four preservation methods tested presented better performance in the preservation of simulated coexistence strains of C. neoformans and C. gattii. Two environmental strains, one C. gattii and one C. neoformans, were mixed in vitro to test four different preservation methods (freezing at −20°C, −80°C, −196°C, and freeze-drying). The colony-forming units from each preservation method were evaluated, and colonies were randomly selected and cultivated in canavanine glycine bromothymol blue (CGB) agar to evaluate the amounts of CGB-positive (C. gattii) and CGB-negative (C. neoformans) colonies resulting from each preservation method after 1 week, 15 days, 1 month, 6 months, and 1 year. According to our results, cryopreservation at −20°C demonstrated was preferable for C. neoformans species, and further studies after long-term storage are necessary. Recovery of yeast cells after freeze-dried preservation in skim milk is better for both species. Ultrafreezing methods evaluated (−80°C and −196°C) also showed better results in the maintenance of C. gattii. Freeze-drying should be preferred for the maintenance of multilineage isolates from the C. neoformans and C. gattii species complexes.

中文翻译：

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加蒂隐球菌和新隐球菌的混合主要分离株的基因型和表型稳定性：四种保存方法的比较分析。

选择合适的保存方法对于长期微生物的生存至关重要。应使用可靠且可重现的方法将菌株长期保存，以最大程度地减少基因型和表型变异以及生存力损失。通常将这些方法设计为在分离的微生物中具有更好的性能。然而，新型隐球菌或加氏隐球菌的非典型初级分离株，例如混合物种或什至物种复合体的不同物种，对于长期保存和分类学研究是一个挑战。这项研究的目的是评估测试的四种保存方法中哪一种在保存新形成的拟南芥和拟南芥的共存菌株中表现出更好的性能。隐球菌。在体外混合两种环境菌株，一种是加迪梭菌（C. gattii），另一种是新形成梭菌（C. neoformans），以测试四种不同的保存方法（在-20°C，-80°C，-196°C冷冻和冷冻干燥）。评估每种保存方法的菌落形成单位，随机选择菌落，并在黄花甘氨酸甘氨酸溴百里酚蓝（CGB）琼脂中培养，以评估CGB阳性（C. gattii）和CGB阴性（C. neoformans）的量。每种保存方法在1周，15天，1个月，6个月和1年后产生的菌落。根据我们的结果，在-20°C的低温保存证明对新孢梭菌更可取。种类，长期储存后需要进一步研究。对于两种而言，冷冻干燥保存在脱脂奶中后酵母细胞的回收率都更高。评估的超冷冻方法（-80°C和-196°C）在维持加蒂梭菌中也显示出更好的结果。冷冻干燥应优选用于维持来自新隐梭菌和加迪梭菌物种复合体的多谱系分离物。