Microbial contamination analysis of Liquid Nitrogen produced by the Elan2Digital:

Microbiological analysis were performed collaborating with the sector of animal diseases and microbiology and the sector of microbiology for food control of the Department of Veterinary Science of the University of Sassari, Italy.
Tests were performed following the reference standard ISO 11737-1: 2006 Sterilization of medical devices – Microbiological methods – Part 1: Determination of a population of micro organisms on products. https://www.iso.org/obp/ui/#iso:std:iso:11737:-1:ed-2:v1:en

We evaluated the microbial load both in the liquid nitrogen of the dewar, before and after the execution of the sterilization procedure, and in the output of liquid nitrogen of Elan2  avoiding the contact with the dewar (for this procedures we installed provisionally a sterile container at the end of liquid nitrogen dispense tube into the dewar).
Sterilization was performed according to G.A. Pessoa paper, with 70% EtOH solution.

Methods:

• Collection of swabs from liquid nitrogen output and from liquid nitrogen accumulated in the dewar, they were smeared on blood agar and incubated for 72 hours at 37°C and 5% CO2. Results: no contamination was detected.

• After liquid evaporation in the dewar, a sterile saline solution (0,9% NaCl) was added to touch the walls of the dewar. Saline solution was recovered, filtered through membranes, placed over TSA (Trypticase Soy Agar) medium and incubate for 72 hours at 37°C and 5% CO2. This procedures was carried out in dewar both before and after sterilization. Results: no contamination was detected.

• Other membrane, after washing saline solution filtration, was placed in BPW (buffered peptone water) and incubate for 48 hours at 37°C, then seeded on TSA at 37°C 72 hours. Results: no contamination was detected.

• Direct seeding of washing saline solution sample in TSA, 37°C, 5% CO2. Results: no contamination was detected.

Our analytic procedures show that microbial load, in liquid nitrogen produced by Elan2Digital machine if present, is in such quantity to be not detected by the standardized methods applied for microbial load determination. We are confident that liquid nitrogen produced should be free from microbiology contaminants. We must also say that spores and viruses cannot be detected by this methodology, the discovery is complicated and expensive.

References:

• Decontamination of naturally contaminated liquid nitrogen storage tanks, G. A. Pessoa et al., R. Bras. Zootec. vol.43 no.5 Viçosa May 2014 http://dx.doi.org/10.1590/S1516-35982014000500004;
• Sterilization of liquid nitrogen with ultraviolet irradiation for safe vitrification of human oocytes or embryos, L. Parmegiani et al., Fertility and Sterility_ Vol. 94, No. 4, September 2010, doi:10.1016/j.fertnstert.2009.05.089;
• A review of the risk of contamination of semen and embryos during cryopreservation and measures to limit cross-contamination during banking to prevent disease transmission in ET practices, A. Bielanski, Theriogenology 77 (2012) 467–482, doi:10.1016/j.theriogenology.2011.07.043;
• Risk of contamination of germplasm during cryopreservation and
• cryobanking in IVF units, A. Bielanski and G. Vajta, Human Reproduction, Vol.24, No.10 pp. 2457–2467, 2009, doi:10.1093/humrep/dep117.