There are questions that need to be answered before I can recommend a buffer or solution. My first comment is that rehydrating, even in water, will activate your cells, but putting them into -20 C will probably kill them, unless you are adding glycerol, e.g., 10-15%. For most prokaryotes and fungi, lyophilized cells can be rehydrated in water, LB broth, YPD, or other suitable medium. This is especially true if you simply plan to streak them after hydration. However adding an aqueous buffer, followed by freezing will rupture many of your cells (many log decrease). I would think twice about that approach.

These pages might help: https://opsdiagnostics.com/notes/ranpri/aguidetobacteriapreservation.htm

https://opsdiagnostics.com/notes/ranpri/bacteria_lyophilization_overview.htm

Dear David Burden

Thank you for your valuable feedback and recommendations. I would like to provide some clarifications regarding our work and seek your further guidance.

We are working with lyophilized cultures containing approximately 600-700 cells per mL. Our objective is to rehydrate these cultures and maintain their viability for at least 2-3 weeks, as this specific cell count is critical for our application.

We have evaluated different buffer systems, including PBS and HEPES, supplemented with cryoprotectants such as glycerol and trehalose. Immediately after rehydration, there was no measurable loss in viability. However, after one week of storage at –20 °C, we observed a significant reduction in viability. In particular, recovery was completely lost on selective media such as MacConkey Agar for E. coli, although colonies could still be obtained on general-purpose media.

We seek your further guidance on optimizing the rehydration and storage strategy to ensure both stability and consistent recovery on selective media.

Thank you,

Uday Kumar