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Background  Microbiological assays require accurate and reproducible preparation of bacterial inocula. Inocula prepared on different days by different individuals can vary significantly from experiment to experiment. This variance is particularly problematic for Gram-negative bacterial infections, for which threshold effects can result in marked variations in host outcome with minor differences in inocula.    Results  We compared the accuracy of traditional methods versus using frozen stocks for preparing  Acinetobacter baumannii  inocula for infection in mice. Standard inoculum preparation resulted in substantial variability, both with respect to the actual inocula achieved compared to the targeted inocula, and with respect to the  in vivo  outcome resulting from similar inocula. Cryopreservation of the bacteria resulted in no significant decrement in growth of the bacteria. Furthermore, preparation of multiple infectious inocula from a frozen stock significantly improved the accuracy of the achieved inocula, and resulted in more reproducible  in vivo  outcomes from infection. Frozen stocks reduced inter-experiment variability associated with inoculum preparation, displayed no significant loss of growth capacity, and maintained virulence, increasing the reliability of infection.    Conclusions  Frozen stocks require considerably less time to prepare and enhance reproducibility of  in vivo  experimental results when infecting with  A. baumannii .

Cryopreservation of virulent Acinetobacter baumannii to reduce variability of in vivo studies