Effect of storage media, temperature, and time on preservation of Cryptosporidium parvum oocysts for PCR analysis

Abstract

The effect of storage media, temperature, and time on suitability of oocysts for use in subsequent molecular studies was examined. Cryptosporidium parvum oocysts were stored for 3, 6, 9, or 12 months in sterile dH 2O, 70 or 95% ethanol, (room temperature [RT], 4, −20, and −70 °C), 10% formalin (RT and 4 °C), PBS, TE buffer, antibiotic–antimycotic (A–A) solution (4, −20 and −70 °C), 2% sulphuric acid, 2.5% potassium dichromate (4 °C), and gDNA from 10 4 oocysts was extracted in triplicate and subjected to PCR. To determine the effect of storage media on PCR sensitivity, gDNA from 10 4, 10 2, and 10 0 oocysts stored for 15 months in the media listed above at RT or 4 °C was also extracted in triplicate and subjected to PCR. At RT, ethanol was suitable for up to 15 months, while gDNA from oocysts stored in dH 2O amplified inconsistently after 3 months. At 4 °C, all tested media except dH 2O and formalin were suitable for storage of 10 4 oocysts up to 15 months, but only 70% ethanol, A–A solution, 2% sulphuric acid and 2.5% potassium dichromate supported amplification of gDNA from fewer than 100 oocysts. At −20 °C, 95% ethanol, PBS, or TE were suitable for up to 9 months, while 70% ethanol and A–A solution were effective up to 12 months, and gDNA from oocysts stored in dH 2O was inconsistently amplified after 6 months. Storage at −70 °C for up to 12 months was effective regardless of media type. Oocysts stored in formalin at RT or 4 °C could not be amplified by PCR despite washing prior to gDNA extraction. To maintain gDNA quality suitable for PCR, it is recommended that coccidian oocysts be stored at −70 °C in dH 2O, ethanol, PBS, TE or A–A solution, at 4 °C in A–A or ethanol, or at RT in ethanol where refrigerated storage is unavailable.