定期转种法和低温冷冻法保藏致病真菌活性的能力比较

Comparison of periodic subculture and cryopreservation in preserving viabilities of pathogenic fungi

定期转种法和低温冷冻保存法是临床实验室最常用的两种真菌保存方法,为比较两种方法保藏致病真菌活性的能力,本研究使用两种保藏方法对实验室689株致病真菌保藏5年后进行检测。定期转种法是将菌落接种于马铃薯斜面培养基并将其储存在4℃冰箱,每6个月转种1次。低温冷冻法是挑取马铃薯斜面培养基上生长良好的菌落于无菌10%甘油中,放置在-80℃储存。保藏5年后,将两种方法保藏的菌株转种复苏,比较菌株的复活率。对于念珠菌属Candida、新生隐球菌Cryptococcus neoformans、毛癣菌属Trichophyton、曲霉属Aspergillus和孢子丝菌属Sporothirix真菌,两种方法的菌株复活率无统计学差异;对于小孢子菌属Microsporum真菌和马尔尼菲蓝状菌Talaromyces marneffei,使用低温冷冻法保藏的菌株复活率高于定期转种法保藏的菌株复活率;对于着色霉属Fonsecaea真菌,低温冷冻法保藏的菌株复活率低于定期转种法保藏的菌株复活率。因此,我们认为对于常见致病真菌的长期保藏,使用10%甘油作为保护剂的低温冷冻法优于定期转种法,但其不适用于着色霉属Fonsecaea真菌的长期保藏。

Periodic subculture and cryopreservation are the most usually used methods for fungal storage.To compare the keeping effectiveness of periodic subculture and cryopreservation in preserving pathogenic fungi,689 strains of five-year preservation using these two methods were detected.Periodic subculture was that cultures on potato dextrose agar were stored in refrigerator at 4°C and subculture was made every 6 months.As for cryopreserva on,fungal cultures on potato dextrose agar were stored in freezer at-80°C in tubes containing 1 mL sterile 10%glycerol.After 5-year period preservation,the strains were removed and subcultured on potato dextrose agar to detect the survivability.Comparison between the methods of periodic subculture and cryopreservation indicated that the survival rates of Candida spp.,Cryptococcus neoformans,Trichophyton spp.,Aspergillus spp.and Sporothirix spp.showed no statistical difference.For Microsporum spp.and Talaromyces marneffei,the survival rate of the cultures cryopreserved was higher than that of the cultures periodically subcultured.For Fonsecaea spp.,the survival rate of the cultures cryopreserved was lower than that of the cultures periodically subcultured.Therefore,cryopreservation is a better storage method for most fungi isolated in clinic using 10%glycerol as cryoprotectant as compared with periodic subculture.However,cryopreservation is not suitable for Fonsecaea spp.