A reliable procedure for decontamination before thawing of human specimens cryostored in liquid nitrogen: three washes with sterile liquid nitrogen (SLN2).
OBJECTIVE
To report a washing procedure, to be performed as frozen specimens are taken out of cryobanks, to minimize the risk of hypothetical culture contamination during thawing.


METHODS
Basic research.


METHODS
Private assisted reproduction center.


METHODS
Two batches of liquid nitrogen (LN(2)) were experimentally contaminated, one with bacteria (Pseudomonas aeruginosa, Escherichia coli, Stenotrophomonas maltophilia) and the other with fungi (Aspergillus niger). Two hundred thirty-two of the most common human gamete/embryo vitrification carriers (Cryotop, Cryoleaf, Cryopette) were immersed in the contaminated LN(2) (117 in the bacteria and 25 in the fungi-contaminated LN(2)). The carriers were tested microbiologically, one group without washing (control) and the other after three subsequent washings in certified ultraviolet sterile liquid nitrogen (SLN(2)). The carriers were randomly allocated to the "three-wash procedure" (three-wash group, 142 carriers) or "no-wash" (control group, 90 carriers) using a specific software tool.


METHODS
Assessment of microorganism growth.


RESULTS
In the no-wash control group, 78.6% of the carriers were contaminated by the bacteria and 100% by the fungi. No carriers were found to be contaminated, either by bacteria or fungi, after the three-wash procedure.


CONCLUSIONS
The three-wash procedure with SLN(2) produced an efficient decontamination of carriers in extreme experimental conditions. For this reason, this procedure could be routinely performed in IVF laboratories for safe thawing of human specimens that are cryostored in nonhermetical cryocontainers, particularly in the case of open or single-straw closed vitrification systems.
Reproductive Medicine Unit, GynePro Medical Centers, Bologna, Italy. l.parmegiani@gynepro.it
Lodovico
L
Parmegiani
Antonio
A
Accorsi
Silvia
S
Bernardi
Alessandra
A
Arnone
Graciela Estela
GE
Cognigni
Marco
M
Filicori
D001228:Aspergillosis / Q000517:prevention & control; D001755:Blastocyst / Q000166:cytology / Q000382:microbiology; D015925:Cryopreservation / Q000592:standards*; D003666:Decontamination / Q000379:methods*; D004866:Equipment Contamination / Q000517:prevention & control; D004927:Escherichia coli Infections / Q000517:prevention & control; D005260:Female; D016905:Gram-Negative Bacterial Infections / Q000517:prevention & control; D006801:Humans; D008297:Male; D009584:Nitrogen; D009865:Oocytes / Q000166:cytology / Q000382:microbiology; D011552:Pseudomonas Infections / Q000517:prevention & control; D015203:Reproducibility of Results; D027724:Reproductive Techniques, Assisted / Q000592:standards*; D020615:Stenotrophomonas maltophilia; D013242:Sterilization / Q000379:methods*; D014015:Tissue Banks / Q000592:standards; D014466:Ultraviolet Rays; D058989:Vitrification
D009584:Nitrogen