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

Nielsen TB, Bruhn KW, Pantapalangkoor P, Junus JL, Spellberg B - BMC Microbiol. (2015)

Bottom Line: 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.Frozen stocks require considerably less time to prepare and enhance reproducibility of in vivo experimental results when infecting with A. baumannii.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine at the University of Southern California (USC), Los Angeles, CA, USA. tbn960@usc.edu.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus

Multiple inocula preparations from fresh overnight cultures display greater variability than inocula preparations from frozen stocks. The ratio of “Actual” bacterial numbers (as measured by CFU counts one day following preparation) to “Aimed-for” concentration was used as a measure of the cumulative accuracy of each method. For fresh preparations, the “Aimed-for” concentration values of washed subcultures were estimated by determining OD600 readings. For frozen vials, the “Aimed-for” concentration values were calculated by using the known, predetermined concentrations of the batch. We typically aim for < +/−15 % variance from the targeted inoculum (as denoted by the shaded region). Numbers within the shaded region denote the percentages of the 20 measurements per group that fell within the 15 % range
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Fig1: Multiple inocula preparations from fresh overnight cultures display greater variability than inocula preparations from frozen stocks. The ratio of “Actual” bacterial numbers (as measured by CFU counts one day following preparation) to “Aimed-for” concentration was used as a measure of the cumulative accuracy of each method. For fresh preparations, the “Aimed-for” concentration values of washed subcultures were estimated by determining OD600 readings. For frozen vials, the “Aimed-for” concentration values were calculated by using the known, predetermined concentrations of the batch. We typically aim for < +/−15 % variance from the targeted inoculum (as denoted by the shaded region). Numbers within the shaded region denote the percentages of the 20 measurements per group that fell within the 15 % range

Mentions: Over the course of several weeks, different individuals prepared multiple individual inocula of A. baumannii HUMC1, a highly virulent strain that we have used in multiple previous publications [5, 7, 8]. We used a traditional, standard method of culturing a freshly streaked colony overnight in a suitable broth medium, subculturing it until log-phase growth, and estimating the bacterial concentration using an O.D. reading of a washed sample. Bacteria resuspended in PBS were then adjusted to a target (“aimed-for”) concentration based on the O.D. reading, and serial dilutions of these working dilutions were made to allow plating of a countable number of CFUs. “Actual” numbers of CFUs in the working dilutions were determined by counting colonies on the agar plates the following day. Multiple repeats of this protocol resulted in a wide range of “Actual”/“Aimed-for” ratios, demonstrating a high degree of variability from day to day when utilizing fresh cultures (Fig. 1).Fig. 1


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

Nielsen TB, Bruhn KW, Pantapalangkoor P, Junus JL, Spellberg B - BMC Microbiol. (2015)

Multiple inocula preparations from fresh overnight cultures display greater variability than inocula preparations from frozen stocks. The ratio of “Actual” bacterial numbers (as measured by CFU counts one day following preparation) to “Aimed-for” concentration was used as a measure of the cumulative accuracy of each method. For fresh preparations, the “Aimed-for” concentration values of washed subcultures were estimated by determining OD600 readings. For frozen vials, the “Aimed-for” concentration values were calculated by using the known, predetermined concentrations of the batch. We typically aim for < +/−15 % variance from the targeted inoculum (as denoted by the shaded region). Numbers within the shaded region denote the percentages of the 20 measurements per group that fell within the 15 % range
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4630970&req=5

Fig1: Multiple inocula preparations from fresh overnight cultures display greater variability than inocula preparations from frozen stocks. The ratio of “Actual” bacterial numbers (as measured by CFU counts one day following preparation) to “Aimed-for” concentration was used as a measure of the cumulative accuracy of each method. For fresh preparations, the “Aimed-for” concentration values of washed subcultures were estimated by determining OD600 readings. For frozen vials, the “Aimed-for” concentration values were calculated by using the known, predetermined concentrations of the batch. We typically aim for < +/−15 % variance from the targeted inoculum (as denoted by the shaded region). Numbers within the shaded region denote the percentages of the 20 measurements per group that fell within the 15 % range
Mentions: Over the course of several weeks, different individuals prepared multiple individual inocula of A. baumannii HUMC1, a highly virulent strain that we have used in multiple previous publications [5, 7, 8]. We used a traditional, standard method of culturing a freshly streaked colony overnight in a suitable broth medium, subculturing it until log-phase growth, and estimating the bacterial concentration using an O.D. reading of a washed sample. Bacteria resuspended in PBS were then adjusted to a target (“aimed-for”) concentration based on the O.D. reading, and serial dilutions of these working dilutions were made to allow plating of a countable number of CFUs. “Actual” numbers of CFUs in the working dilutions were determined by counting colonies on the agar plates the following day. Multiple repeats of this protocol resulted in a wide range of “Actual”/“Aimed-for” ratios, demonstrating a high degree of variability from day to day when utilizing fresh cultures (Fig. 1).Fig. 1

Bottom Line: 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.Frozen stocks require considerably less time to prepare and enhance reproducibility of in vivo experimental results when infecting with A. baumannii.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Immunology, Keck School of Medicine at the University of Southern California (USC), Los Angeles, CA, USA. tbn960@usc.edu.

ABSTRACT

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.

No MeSH data available.


Related in: MedlinePlus