Why use BactoBox®

OD and McFarland drift with cell size. BactoBox® counts cells, so your inoculum hits the specification.

Few things are more frustrating than wasted work. You set an inoculum specification, hit it on the photometer, and start the experiment. Days later the retrospective plate counts say you missed. Results turn inconsistent and the do-over costs you a week.

Optical density (OD) and McFarland standards are the usual tools for standardizing inoculum. Both measure turbidity. Turbidity is not a cell count.

OD and McFarland depend on cell size

There is a simple explanation for why OD and McFarland often fail. Both methods measure scattered light, and scattering depends on object size as well as on concentration. Cells change size throughout a growth curve. So the factor that converts arbitrary turbidity units to colony forming units (CFU) changes with them.

That leaves you guessing. Pick your calibration point early in the curve and every later number is too low. Pick it late and every earlier number is too high. The drift is one-directional, so more replicates do not average it away.

We tracked a single E. coli culture with BactoBox®, plate counts, and OD₆₀₀ at the same time points. The OD-to-CFU factor rose 6.6× across the run. See Drifts in OD to CFU ratios.

Optical methods are strongly dependent on the size of objects. The two samples scatter the same amount of light, but the bacteria in exponential stage (left) are considerably bigger than the cells in stationary stage (right). Same OD but ~5× different cell concentrations.

OD is blind at early growth phases

At low cell concentration early in the growth curve, OD₆₀₀ readings are indistinguishable from a blank. BactoBox® quantifies 50–500× lower, depending on how much you dilute. See OD is blind at low cell concentrations.

The OD blindness problem becomes even more severe with extremely small cells e.g. Mycoplasma ssp and Avibacterium ssp. Sometimes the culture need to reach late exponential before the OD is above the limit of quantification.

Standardize a dilute, early-stage culture by OD and you are reading noise.

BactoBox® is virtually 1:1 with CFU in active cultures

BactoBox® is a label-free flow cytometry in a benchtop format. Cells are detected one at a time, so cell size does not influence the result. You get sensitive and precise cell concentrations in real time.

BactoBox® arrives calibrated. Unlike OD and McFarland you can skip building a standard curve for every strain. As shown in Correlation with plate counts BactoBox® demonstrates great agreement with CFUs for active cultures.

Klebsiella aerogenes growth curve from shake flask with tryptic soy broth (TSB). BactoBox® cells (lavender) is practically superimposed on the plate counts (CFU, yellow).

BactoBox® reveals the growth stage

You get more than a cell concentration. The CIZE metric reports average cell size, and cell size tracks the growth stage. Map out your growth curve once. After that, a CIZE value tells you which stage a new culture is in.

For the practical steps, see Know the growth phase of your culture.

Illustration of cell size during a growth curve.

Summary

With BactoBox® you inoculate with confidence.

The right dose of bacteria.

The right growth stage.