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

Abstract

The survival of five mycoplasma strains was studied in different storage media (Mycoplasma complet 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) during 24 months. Five Mycoplasma strains, Mycoplasma mycoides subsp mycoides (Mmm), Mycoplasma bovis (Mb), Mycoplasma agalactiae (Ma), Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) were isolated from various parts of the country. The initial titers of the strains determined by the agar plate count before storage were 42.4x10 7 UFC/ml (8.6 log UFC/ml) for Mmm strain; 32.4x10 8 UFC/ml (9.51 log UFC/ml) for M.bovis strain; 12.4x10 9 UFC/ml (10.09 log UFC/ml) for Ma strain; 2.4x10 9 UFC/ml (9.38 log UFC/ml) for Mg and 2.8x10 9 UFC/ml (9.45 log UFC/ml) for Ms 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 lysophylisation process, the addition of glycerol may be recommended for long-term storage of frozen mycoplasma isolates.