In this paper we studied cryopreservation of Cyclamen persicum Mill. callus to avoid variations produced by sub-culture. The callus in the logarithmic phase after sub-culture were used for experiments. Firstly, the callus were pre-cultured in culture-medium containing 4%, 6% or 8% sucrose for different time periods, transferred to different cryoprotectants to directly cryopreserve or incubated for 2 hours at -20 degrees C, then submersed in liquid nitrogen, lastly thawed rapidly in a waterbath at 37 degrees C, and washed with liquid culture-medium containing the corresponding concentration of sucrose. Cell survival rate was computed after stained by Neutral Red, and SPSS 13.0 software was used for statistical analysis. The results showed that sucrose concentration, pre-culture time, cryoprotectants had various impacts on cell survival rate. We have developed a simple but effective protocol for the cryopreservation of callus of C. persicum. Of the different protocols tested, 4%sucrose, pre-culturing for 3 days, No. 9 cryoprotectant and freezing directly after 30 minutes at 0 degrees C results in the highest cell survival rate.
Cryopreservation ; Culture Techniques ; methods ; Cyclamen ; growth & development

Objective To determine the in vitro production of virulence factors for Candida (C.) tropicalis,including aspartyl proteinases,phospholipases and hemolytic activities,describe the regulation of virulence factors varying with time in C.tropicalis,and analyze the differences in aspartyl proteinases and hemolytic activities of C.tropicalis isolated from anatomically distinct sites.Methods A total of 64 C.tropicalis strains were spot-inoculated onto bovine albumin agar,egg yolk agar and sheep blood agar plates,respectively.Then the plates were incubated for 24,48 and 72 hour at 37 ℃,respectively.The aspartyl proteinases,phospholipase and hemolytic activities were determined at each time point,respectively.Results All the C.tropiclais isolates showed positive aspartyl proteinases and hemolytic activities at each time point,but no phospholipases activity was detected in C.tropicalis.On comparison of aspartyl proteinases and hemolytic activities at different time points,aspartyl proteinases activity at 48 and 72 hour was higher than that at 24 hour.During 72 hour,hemolytic activity of C.tropicalis increased.No statistical significant differences in aspartyl proteinases and hemolytic activities of C.tropicalis were observed among different infection sites (P=0.368 and 0.985).Conclusion The C.tropicalis clinical isolates in China have aspartyl proteinases activity,hemolytic activity,but have no phospholipase activity.

OBJECTIVETo understand the species, genotypes and mating types of Cryptococcus neoformans and Cryptococcus gattii isolated from clinical samples in Guigang, Guangxi Zhuang Autonomous Region.

METHODSA total of 20 Cryptococcus strains were isolated from clinical samples in Guigang from 2009 to 2012. The biological identification was conducted by polymerase chain reaction (PCR) to amplify internal transcribed spacer (ITS) sequences. The serotypes and mating types of C. neoformans and C. gattii were identified by PCR with serotype-specific and mating type-specific primers. The genotype was characterized by PCR fingerprinting and URA5 gene restriction fragment length polymorphism (URA5-RFLP). Phenotype study included growth test at 37 °C, melanin production test and urease test.

RESULTSAmong the 20 strains, 19 (95%) were identified as C. neoformans varieties (var.) grubii (serotype A, mating type α, genotype VN I), and only 1 was identified as C. gattii (mating type α, genotype VG I). The results of virulence test showed that all the strains grew well at 37 °C and positive in both urease test and melanin production test.

CONCLUSIONC. neoformans var. grubii (serotype A, genotype VN I and mating type α) was the predominant pathogen causing cryptococcosis in Guigang, and C. gattii strain was also detected.

China ; Cryptococcus gattii ; genetics ; isolation & purification ; pathogenicity ; Cryptococcus neoformans ; genetics ; isolation & purification ; pathogenicity ; Genotype ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Virulence