Standard Bacteriophage Purification Procedures Cause Loss in Numbers and Activity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Bacteria and Bacteriophage
2.2. Growth and Isolation of Phage
2.3. PEG Precipitation
2.4. CsCl Gradient Centrifugation
2.5. Epifluorescent Imaging of Phage DNA
2.6. Detection of Phage Activity
2.7. Isolation of Phage DNA and qPCR
2.8. Transmission Electron Microscopy (TEM)
2.9. Phage Preparation Workflow
3. Results
3.1. TEM Shows Concentration of Phage Samples by Both PEG and CsCl Gradient
3.2. PEG Precipitation and CsCl Gradient Purification Result in Loss of Phage Activity
3.3. CsCl Gradient Purification Results in Loss of Phage Numbers
3.4. qPCR Quantitation Confirms Loss of Phage Numbers with Processing
4. Discussion
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Phage | Optimal Simple * Purification | Optimal Complex * Purification | Recommendation (Based on Compiled Findings) | |
---|---|---|---|---|
ACTIVITY | NUMBER | NUMBER | ||
M13 | PEG6K | PEG10K | PEG6K | If using phage for activity or attempting to maintain within a complex population, use PEG6K when concentration is necessary. If isolating for highest numbers, use PEG10K. Avoid CsCl. |
T4 | PEG6K | PEG6K | PEG8K † | If concentration is necessary for analysis of phage activity or to maintain highest numbers from bacterial culture, use PEG6K. If isolating for highest numbers in complex phage population, use PEG8K. Avoid CsCl. |
ΦX | PEG6K | PEG6/8/10K | PEG8K † | If concentration is necessary during phage purification from bacterial culture for activity, use PEG6K. If isolating for highest numbers in complex phage population, use PEG8K. Any PEG can be used to retain numbers in purifications from bacterial culture. Avoid CsCl. |
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Carroll-Portillo, A.; Coffman, C.N.; Varga, M.G.; Alcock, J.; Singh, S.B.; Lin, H.C. Standard Bacteriophage Purification Procedures Cause Loss in Numbers and Activity. Viruses 2021, 13, 328. https://doi.org/10.3390/v13020328
Carroll-Portillo A, Coffman CN, Varga MG, Alcock J, Singh SB, Lin HC. Standard Bacteriophage Purification Procedures Cause Loss in Numbers and Activity. Viruses. 2021; 13(2):328. https://doi.org/10.3390/v13020328
Chicago/Turabian StyleCarroll-Portillo, Amanda, Cristina N. Coffman, Matthew G. Varga, Joe Alcock, Sudha B. Singh, and Henry C. Lin. 2021. "Standard Bacteriophage Purification Procedures Cause Loss in Numbers and Activity" Viruses 13, no. 2: 328. https://doi.org/10.3390/v13020328