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Abstract:
Cooling to -70 C killed a higher percentage of Acholeplasma laidlawii and Mycoplasma mycoides var. capri cells than cooling to -20 C. However, to preserve cell viability for prolonged periods storage at -70 C was much more preferable. The percentage of cells surviving freezing could be increased by increasing the initial cell concentration or by the addition of dimethyl sulfoxide or glycerol as cryoprotective agents. In the presence of 1.5 M of any one of these agents survival rates of up to 100% could be obtained. The optimal cooling rates for maximal survival of A. laidlawii under the experimental conditions tested were 11 C/min for cooling to -20 C and about 15 C/min for cooling to -70 C. Increasing the warming rate during thawing from 0.6 to 67 C/min increased survival by 3 log. Oleic acid enrichment of A. laidlawii membrane lipids, or reduction in the cholesterol content of M. mycoides var. capri membranes, increased the percentage of organisms surviving freezing. Hence, the composition of membrane lipids appears to have a marked influence on the susceptibility of mycoplasmas to freezing injury.
Insights
Storing mycoplasma cells at -70°C is preferable for long-term viability compared to -20°C. Cryoprotective agents like DMSO or glycerol, optimal cooling/warming rates, and membrane lipid composition significantly enhance cell survival during freezing.
Area of Science:
- Microbiology
- Cell Biology
- Cryobiology
Background:
- Mycoplasmas are cell-wall-less bacteria susceptible to environmental stress.
- Cryopreservation is crucial for maintaining mycoplasma viability for research and diagnostics.
- Understanding factors influencing mycoplasma survival during freezing is essential.
Purpose of the Study:
- To investigate the effects of temperature, cooling rates, cryoprotective agents, and membrane lipid composition on mycoplasma cell viability after freezing.
- To determine optimal conditions for cryopreserving Acholeplasma laidlawii and Mycoplasma mycoides var. capri.
Main Methods:
- Cells were cooled to -20°C and -70°C.
- Cryoprotective agents (dimethyl sulfoxide, glycerol) and varying cell concentrations were tested.
- Optimal cooling and warming rates were determined.
- Membrane lipid composition (oleic acid, cholesterol) was manipulated.
Main Results:
- Storage at -70°C preserved higher cell viability than -20°C for prolonged periods.
- 1.5 M cryoprotective agents achieved up to 100% survival.
- Optimal cooling rates for A. laidlawii were 11°C/min (to -20°C) and 15°C/min (to -70°C).
- Increased warming rates significantly improved survival.
- Oleic acid enrichment and cholesterol reduction enhanced freezing survival.
Conclusions:
- Cryopreservation temperature, cooling/warming rates, cryoprotectants, and cell concentration are critical for mycoplasma survival.
- Membrane lipid composition plays a significant role in susceptibility to freezing injury.
- Optimized cryopreservation protocols can achieve high survival rates for mycoplasma species.