BactoBox® for Κ

BactoBox® is an Impedance Flow Cytometer (IFC), resembling Fluorescence Flow Cytometry (FFC) in the sense that objects traverse the detector element in a single file. Unlike FFC, which uses lasers, BactoBox® uses an electric field as its detector element. The lipid membrane and conductive cytoplasm of bacteria provides a distinct signature that enables specific detection of bacteria without the need for staining.

BactoBox® measurement principle. The membrane and cytoplasm of cells creates a distinct electrical signature.

Advantages of BactoBox® for K

The main advantage of BactoBox® for determining Κ is that the measured cell concentration remains stable once the carrying capacity is reached at the onset of the stationary phase. In practice, this provides a broad and permissive sampling window for determining Κ. In contrast, CFU measurements typically peak during the stationary phase but decline relatively quickly as the culture enters the death phase. This is demonstrated for five of the species in the peer-reviewed performance qualification, specifically E. coli, K. aerogenes, A. baumannii , L. innocua, and P fluorescens. The Klebsiella data is illustrated in the below figure.

CFU values drop after stationary phase. K. aerogenes in TSB medium. The data points represent BactoBox® (lavender) and plate counts (yellow). Triplicate repeats were done for each method. The red box highlights how CFU values deflate in decline phase whereas BactoBox® cells/mL stays constant.

The below figure illustrates the stable BactoBox® cell concentrations on a log axis (left) and linear axis (right) for E. coli cultivated in four different growth media. The cell concentrations are stable once hitting the max concentration.

E. coli shake flask cultures investigated with single replicates. BactoBox® cell concentrations are constant after reaching the carrying capacity of the medium. Four growth media are presented: Terrific broth (Terrific), Bacto™ CD Supreme fermentation production medium with 4 mL/L glycerol (CD), Luria-Bertani broth (LB), and Tryptic Soy Broth (TSB). Left plot presents data on a log₁₀ axis while the right plot has a linear y-axis.

Each BactoBox® measurement typically corresponds to thousand of detected bacteria. This makes the method precise. The sample workup is simple and typically only requires simple vortexing and dilution. The default gating works for a wide range of species and it is therefore easy to implement, and operate BactoBox®. Measurements are completed within few minutes making BactoBox® results available in real time.

Compared to fluorescence flow cytometry (FFC), the gating process is straightforward. In most cases, the default measurement program is sufficient, and post-acquisition gating is unnecessary. The method is also stain-free, making it easier to standardize while reducing hands-on time and minimizing exposure to potentially carcinogenic compounds such as propidium iodide.

Disadvantages of BactoBox® for K

BactoBox® is slightly more demanding than off-line OD in terms of sample preparation. For BactoBox® care should be taken to get a single-cell suspension - for OD measurements this is not as important. The flow cell measurement channel does not allow objects larger than 5 µm to enter the measurement channel (sphere-equivalent diameter). BactoBox® is therefore not suited for samples with high proportion of clumping and or chaining cells.

Samples with high levels of background particulates can also pose a challenge. When bacteria constitute less than ~1% of the total particles, detection effectively becomes a needle-in-a-haystack problem. BactoBox® is therefore not directly suited for analyzing bacteria in samples such as homogenized milk, where casein micelles are similar in size to bacterial cells.

The BactoBox® setup is relatively budget-friendly compared to microscopes and fluorescence flow cytometers. That said, each BactoBox® measurement incurs a per-sample cost. The cost per measurement is low relative to the operator time and labor expenses associated with microscopy, plate counts, staining, and similar methods.

Practical recommendations

BactoBox® is well suited for determining K of different growth conditions. This is because the sampling window is broad and permissive, precision is high, and the method is easy to implement.

BactoBox® is not recommended for samples containing clumps and chains or samples with very high proportion of non-bacterial objects.