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Characterization of injury incurred by Escherichia coli upon freeze-drying
Abstract:
When cells of Escherichia coli ML30 were suspended in 2% gelatin and frozen at -40 C, no appreciable metabolic damage or death occurred. After freeze-drying for 8 hr at a platen temperature of 49 C and rehydration with a mineral salts medium, survival of the cells was 0.6%. Metabolic damage of the survivors was found to be 23%. Permeability alterations were detected by several criteria. Freeze-dried cells were susceptible to antibiotics normally ineffective against E. coli and leakage of ribonucleic acid (RNA) occurred. Analysis of ribosomal extracts of rehydrated freeze-dried cells demonstrated the presence of appreciable degradation products. Permeability alterations were shown to be reversible by the observation that antibiotic susceptibility was a time-dependent process and that the gratuitous inducer of beta-galactosidase was not concentrated by freeze-dried cells until the injured cells had been incubated in a nutrient medium for 300 min or more. At approximately the same time, metabolic damage was repaired. RNA synthesis preceded protein synthesis by about 150 min, and deoxyribonucleic acid synthesis occurred with the resumption of normal growth. This was interpreted to be the result of repair of RNA taking place before protein synthesis and growth could resume. A pronounced increase in the lag time of freeze-dried cells was also observed. Peptides and Casamino Acids shortened the lag time for freeze-dried cells but not for the controls. Glycerol and glucose were found to be better carbon sources for growth of freeze-dried cells than sodium lactate or sodium succinate.
Insights
Freeze-drying Escherichia coli causes significant cell damage, including permeability alterations and metabolic injury. However, these effects are reversible upon rehydration, with cells gradually repairing damage and resuming normal growth.
Area of Science:
- Microbiology
- Cell Biology
- Biotechnology
Background:
- Freeze-drying is a common preservation method for microorganisms.
- Understanding the impact of freeze-drying on bacterial cells is crucial for optimizing preservation techniques.
- Escherichia coli is a model organism for studying cellular responses to stress.
Purpose of the Study:
- To investigate the metabolic damage and permeability alterations in freeze-dried Escherichia coli cells.
- To determine the reversibility of these changes and the conditions required for cell recovery.
- To identify factors influencing the lag phase and growth resumption of freeze-dried cells.
Main Methods:
- Cells of Escherichia coli ML30 were freeze-dried and rehydrated.
- Survival rates, metabolic damage, and permeability changes were assessed.
- Antibiotic susceptibility, RNA leakage, and ribosomal integrity were analyzed.
- Cellular recovery was monitored by observing beta-galactosidase induction, RNA and DNA synthesis, and growth resumption.
- The effect of different carbon sources and supplements on lag time was evaluated.
Main Results:
- Freeze-drying resulted in low cell survival (0.6%) and significant metabolic damage (23%).
- Permeability alterations included increased antibiotic susceptibility and RNA leakage.
- Ribosomal extracts showed degradation products.
- Damage was reversible; antibiotic susceptibility decreased, and beta-galactosidase induction occurred after prolonged incubation.
- RNA synthesis preceded protein and DNA synthesis, indicating repair.
- Lag time increased significantly but was shortened by peptides, Casamino Acids, glycerol, and glucose.
Conclusions:
- Freeze-drying induces reversible damage to Escherichia coli cells, affecting permeability and metabolism.
- Cellular recovery involves a sequential repair process, with RNA repair preceding protein synthesis and growth.
- Specific nutrients like peptides, Casamino Acids, glycerol, and glucose can mitigate the extended lag phase in freeze-dried cells.