Revival of frozen bacterial cultures requires careful handling due to their inherent sensitivity during the thawing process. Twist Bioscience glycerol stocks are manufactured in conjunction with the Clonal Gene delivered, utilizing proprietary cell lines (DH10B-like or Stbl3-like, depending on vector stability). To ensure successful growth, a gentle initial incubation period with a nutrient-rich medium is essential. It is highly recommended to streak, plate, and select single colonies from the glycerol stock to ensure cell recovery and minimize the chances of a mixed population in the final culture. Failure to adhere to these best practices may result in reduced performance. This document outlines the steps required to streak and inoculate glycerol stocks.