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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

Frozen and freshly-prepared inocula display similar growth and virulence characteristics, but only frozen stocks diluted directly (not washed) gave consistent survival results. aIn vitro growth curves of subcultures started from fresh overnight cultures and frozen vials are similar. TSB cultures were seeded with equal numbers of fresh or frozen bacteria, and aliquots were removed at the time points indicated. Serial dilutions were plated to measure CFUs. Results are combined from six independent experiments. Median values are plotted with error bars denoting interquartile ranges. b Multiple preparations of bacterial inocula were prepared on different days for intravenous injection into mice, at the doses shown on the x-axis. Freshly-prepared inocula (“Fresh Culture”, squares) were quantitated based on O.D. readings on the day of preparation. Alternatively, frozen stocks were used to prepare infectious inocula, either by washing 3x to remove trace amounts of glycerol (“Washed”, triangles), or by directly diluting the frozen stock into PBS for injection (“Direct”, circles). Frozen stock inocula were based on the known concentrations of bacteria per vial following freezing. Each symbol represents a single preparation, injected into N = 5 mice per group on a different day. Survival percentages are shown on the y-axis
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Fig2: Frozen and freshly-prepared inocula display similar growth and virulence characteristics, but only frozen stocks diluted directly (not washed) gave consistent survival results. aIn vitro growth curves of subcultures started from fresh overnight cultures and frozen vials are similar. TSB cultures were seeded with equal numbers of fresh or frozen bacteria, and aliquots were removed at the time points indicated. Serial dilutions were plated to measure CFUs. Results are combined from six independent experiments. Median values are plotted with error bars denoting interquartile ranges. b Multiple preparations of bacterial inocula were prepared on different days for intravenous injection into mice, at the doses shown on the x-axis. Freshly-prepared inocula (“Fresh Culture”, squares) were quantitated based on O.D. readings on the day of preparation. Alternatively, frozen stocks were used to prepare infectious inocula, either by washing 3x to remove trace amounts of glycerol (“Washed”, triangles), or by directly diluting the frozen stock into PBS for injection (“Direct”, circles). Frozen stock inocula were based on the known concentrations of bacteria per vial following freezing. Each symbol represents a single preparation, injected into N = 5 mice per group on a different day. Survival percentages are shown on the y-axis

Mentions: We next asked whether cryopreservation impacts the growth characteristics of A. baumannii in vitro. We compared two sub-cultures of A. baumannii HUMC1: one started from a fresh overnight culture and the other started from a frozen stock vial of bacteria. Multiple growth curves of each subculture, performed several times under the same conditions on different days, demonstrated that, despite a slight, early lag in growth compared to the freshly inoculated culture, there were no significant differences between the curves following the 1 h time point (Fig. 2a). These results demonstrated that cryopreservation of A. baumannii does not result in a significant change in growth pattern in vitro.Fig. 2


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)

Frozen and freshly-prepared inocula display similar growth and virulence characteristics, but only frozen stocks diluted directly (not washed) gave consistent survival results. aIn vitro growth curves of subcultures started from fresh overnight cultures and frozen vials are similar. TSB cultures were seeded with equal numbers of fresh or frozen bacteria, and aliquots were removed at the time points indicated. Serial dilutions were plated to measure CFUs. Results are combined from six independent experiments. Median values are plotted with error bars denoting interquartile ranges. b Multiple preparations of bacterial inocula were prepared on different days for intravenous injection into mice, at the doses shown on the x-axis. Freshly-prepared inocula (“Fresh Culture”, squares) were quantitated based on O.D. readings on the day of preparation. Alternatively, frozen stocks were used to prepare infectious inocula, either by washing 3x to remove trace amounts of glycerol (“Washed”, triangles), or by directly diluting the frozen stock into PBS for injection (“Direct”, circles). Frozen stock inocula were based on the known concentrations of bacteria per vial following freezing. Each symbol represents a single preparation, injected into N = 5 mice per group on a different day. Survival percentages are shown on the y-axis
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Frozen and freshly-prepared inocula display similar growth and virulence characteristics, but only frozen stocks diluted directly (not washed) gave consistent survival results. aIn vitro growth curves of subcultures started from fresh overnight cultures and frozen vials are similar. TSB cultures were seeded with equal numbers of fresh or frozen bacteria, and aliquots were removed at the time points indicated. Serial dilutions were plated to measure CFUs. Results are combined from six independent experiments. Median values are plotted with error bars denoting interquartile ranges. b Multiple preparations of bacterial inocula were prepared on different days for intravenous injection into mice, at the doses shown on the x-axis. Freshly-prepared inocula (“Fresh Culture”, squares) were quantitated based on O.D. readings on the day of preparation. Alternatively, frozen stocks were used to prepare infectious inocula, either by washing 3x to remove trace amounts of glycerol (“Washed”, triangles), or by directly diluting the frozen stock into PBS for injection (“Direct”, circles). Frozen stock inocula were based on the known concentrations of bacteria per vial following freezing. Each symbol represents a single preparation, injected into N = 5 mice per group on a different day. Survival percentages are shown on the y-axis
Mentions: We next asked whether cryopreservation impacts the growth characteristics of A. baumannii in vitro. We compared two sub-cultures of A. baumannii HUMC1: one started from a fresh overnight culture and the other started from a frozen stock vial of bacteria. Multiple growth curves of each subculture, performed several times under the same conditions on different days, demonstrated that, despite a slight, early lag in growth compared to the freshly inoculated culture, there were no significant differences between the curves following the 1 h time point (Fig. 2a). These results demonstrated that cryopreservation of A. baumannii does not result in a significant change in growth pattern in vitro.Fig. 2

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