Know the growth phase of your culture

Bacterial cultures behave differently across growth phases, which can affect experimental outcomes of critical experiments like in vivo infectivity studies.

OD and McFarland measure turbidity, but not growth phase. BactoBox® provides both concentration and CIZE in a single measurement, helping you place your culture on the growth curve.

CIZE tells you what your cells are doing

CIZE (Cell-Impedance-derived siZe Estimate) is the average electrical diameter of cells when modeled as a spherical object. It is primarily driven by cell size. Every bacterial measurement on BactoBox® also comes with a CIZE assessment.

The figure below shows a frequently sampled E. coli shake-flask batch experiment using single measurements. CIZE is shown in black on the secondary axis.

Cell size changes before cell count does. During lag phase, cells adapt to the nutrient-rich environment and grow before dividing: cell count stays flat while CIZE increases. CIZE typically peaks just before exponential growth begins, then decreases as nutrients are consumed.

A similar P. fluorescens experiment, with three replicates per time point, shows excellent CIZE repeatability and clear differences between exponential, deceleration, and stationary phases. Despite different growth rates, E. coli and P. fluorescens show similar CIZE patterns—a trend generally observed in batch cultures.

Cell concentration and CIZE across two bacterial batch cultures. Growth phases were assigned based on growth-rate analysis (ACC = acceleration phase). Error bars for P. fluorescens show the standard deviation of three independent dilution and measurement replicates.

Read the two together to determine growth phase

Neither CIZE nor concentration identifies the growth phase on its own. Together, they can.

To demonstrate this, a cryo stock was inoculated 1% (v/v) into growth medium and further diluted in a 1:10 series across individual shake flasks. After 10 hours under optimal growth conditions, each culture was measured with BactoBox®. The resulting cell concentrations and CIZE values are shown in the table.

Cryo stock diluted to five different starting concentrations directly in LB-containing shake flasks.

The measured cell concentrations and CIZE values are compared with the reference growth curve. CIZE is the primary indicator, but it can be ambiguous on its own. For example, a CIZE of 1.3 µm may occur in both lag and late exponential phase. Cell concentration helps resolve this ambiguity.

Culture A, for example, started at ~1 × 10² cells/mL and increased by several orders of magnitude over 10 hours. It therefore cannot be in lag or acceleration phase. Combined with its CIZE, this places culture A in exponential phase.

The same rationale is used to infer the growth stage of the remaining flasks. The plot below maps each culture to the reference growth curve, making it easy to select the desired inoculum—for example, flask A for mid-exponential phase.

Shake flask culture A–E asssigned to their respective growth phases.

What this gives you

BactoBox® combines CIZE and cell concentration to determine the growth state of your culture.

Instead of defining an inoculum as simply “overnight,” you can specify “exponential phase, CIZE 1.4 µm.” This makes inoculum selection more consistent and reproducible across runs and operators.

CIZE is an experimental feature

CIZE may change from BactoBox® to BactoBox®, and from flow cell to flow cell. It may also depend on the electrical conductivity of your sample.

Stick to a single BactoBox® device and a single flow cell, and keep the electrical conductivity of your sample as consistent as possible. Your cells/mL concentration must also be above the limit of detection before you get a CIZE value.