Impact of various preservation and storage methods on the viability of mycoplasma field strains isolated in Mali

The survival of five mycoplasma strains was studied in different storage media (mycoplasma complete media without cryopreservative agent, mycoplasma complete media with addition of horse serum, mycoplasma complete media with addition of glycerol and lyophilized cultures without stabilizer) under different temperatures (+37 °C, +4 °C, -20 °C, -85 °C) for 24 months. Five mycoplasma strains, Mycoplasma mycoides mycoides, Mycoplasma bovis, Mycoplasma agalactiae, Mycoplasma gallisepticum and Mycoplasma synoviae were isolated from various parts of the country. The initial titers of the strains determined by the agar plate count before storage were 42.4x107 UFC/ml (8.6 log UFC/ml) for Mycoplasma mycoides mycoides strain; 32.4x108 UFC/ml (9.51 log UFC/ml) for Mycoplasma bovis strain; 12.4x109 UFC/ml (10.09 log UFC/ml) for Mycoplasma agalactiae strains; 2.4x109 UFC/ml (9.38 log UFC/ml) for Mycoplasma gallisepticum and 2.8x109 UFC/ml (9.45 log UFC/ml) for Mycoplasma synoviae strain. After 3 weeks of storage, no viable mycoplasmas were detected in all the conservation media at +37 °C and after 3 months of storage at +4 °C except for the lyophilized cultures in which an average viability rate of 17.81 % was observed. Overall, the mycoplasma strains remained viable at freezing temperatures after 24 months regardless of the storage medium, but with decreasing titers, which was noticeable with mycoplasma complete media, and mycoplasma media with horse serum. Conversely, at -20 °C the average viability rates after 24 months of storage were 84.36 % (with glycerol) and 90.04 % (lyophilized cultures). At -85 °C after 24 months of storage, this was 87.98 % (with glycerol) and 91.44 % (lyophilized cultures). These findings suggest that, in the absence of the lyophilization process, the addition of glycerol may be recommended for long-term storage of frozen mycoplasma isolates.