The interesting case of the 1950 specimen-
An outstanding question of this work is why we were able to culture from a 73-year-old specimen but not from specimens from intervening years (1974 and 1986). Because of our copious controls and rigorous experimental conditions, we believe that these isolates are not from environmental contamination. We suspect the reason we could culture from the 1950 specimen was one of two factors: 1) We had much higher sample sizes for the 1950 nodule. Initially we thought the historic crushed nodule would provide enough material to streak 10 plates per media type, however it seemed there was not enough liquid so after streaking 10 plates of each media type for the 1950 nodule and its associated leaf control, we scaled the experiment back to 3 plates of each media type. This resulted in over three times as many plates for our 1950 specimen, this alone may have increased the probability that we could capture historic microbes. Additionally, 2) historic specimens are handled very differently depending on the collection conditions and/or the collector/curator at the herbarium (Forrest et al. 2019). Specimens are dried and frozen for variable amounts of time using different methods resulting in increased probability of microbial survival. We don’t have information on how the 1950 specimen was handled but it is possible that it was handled in a way that allowed spore forming bacteria such asBacillus (the dominant genus found in our collection) to persist.