OD is inflated by background

OD₆₀₀ measures how much light your sample blocks. It does not specifically measure cells. Anything that scatters or absorbs light at 600 nm adds to the reading: Insoluble medium components, precipitates, cell debris, antifoam, extracellular polymeric substances, and pigments. OD₆₀₀ counts all of it as signal. The measurement signal is complex and you cannot separate the contributions afterwards.

This study aimed to quantify the OD₆₀₀ signal from two cell-free interferents, and to compare it with BactoBox® results on the same samples.

Take-home messages

• OD₆₀₀ cannot distinguish cells from other particulates.
• BactoBox® reports cells/mL and total/mL separately and the cell concentration is therefore independent of the non-cellular objects.

The complete investigation and supporting data are presented below.

Study design

We investigated two cell-free particulate-containing substances with both methods: Molasses and a reference solution of 2 µm polystyrene beads.

• A working solution of molasses was prepared by diluting 1g liquid molasses in a final volume of 15 mL BactoBox® diluent. Molasses was received as a gift by a company producing probiotics for soil.
• 2 µm polystyrene (PS) particles, a defined non-biological control. A working solution was prepared by diluting the stock solution 500 times. PS was procured from Polysciences.
• OD₆₀₀ measurements were done on an Implen™ diluphotometer with standard cuvettes.
• Both substances were measured across a dilution series within the linear range of each instrument. These ranges corresponded to different concentrations of molasses and PS.

  • OD₆₀₀ measurements were within 0.1 - 0.8 AU.
  • BactoBox® measurements were within 100,000 - 5,000,000 objects/mL for the PS beads. Molasses had high conductivity and therefore it was not possible to go beyond ~1,000,000 total/mL.
• A single measurement was done for each method.

Electrical phenotypes for bacteria and particulates

Bacteria have layers. The interior cytoplasm is rich in ions and therefore conductive, whereas the surrounding phospholipid membranes are hydrophobic and electrically non-conductive. As demonstrated in the below phase diagram, this results in a special electrical phenotype (green distribution). The white ranges of the plot correspond to objects classified as cells when using the default measurement program BacTotal_v2024-10.

Abiotic particulates rarely have layers. With PS beads the distribution is uniform and centered at non-conductive properties from ~0.6–1.0 rad (brown curve). Molasses contain non-conductive objects in the same range, but also some highly conductive objects from ~-2.5 to -1.8 rad (orange curve). The non-cellular objects are found between -2.5 to +1.2 rad (grey region).

This plot explains how BactoBox® discerns bacteria from abiotic objects: The objects are detected one at a time and the electrical properties of cells are clearly different from that of molasses and PS.

BactoBox® discerns E. coli cells (green) from 2 µm polysterene beads (brown) and molasses particulates (orange).

Particulates add background to OD₆₀₀ measurements

Molasses is often used as a an inexpensive carbon source for complex media. Molasses is pitch black, meaning that it practically absorbs all light. For OD₆₀₀ this will likely result in high background contribution from the growth medium and that the culture must reach very high bacterial concentrations before the signal from the cells is significantly above the signal of the background. In addition to absorbing light, molasses also contain light-scattering non-cellular particulates.

2 µm polystyrene beads represents a well-defined particulate object within the bacterial size range. It is white and does therefore not absorb light, but it still scatters light. 2 µm PS represent a model for particulates present in growth media, e.g. cellular debris and extracellular polymeric substances like extracellular DNA (eDNA), alginate and cellulose.

Molasses

The OD₆₀₀ readings showed a strong dependency of the molasses concentration (plot A, R² = 0.9973). In contrast, the lavender data points for BactoBox® (plot B) were below the limit of quantification and therefore cell enumeration by BactoBox® is unaffected by the presence of molasses. Quantification of non-bacterial objects depends linearly on the molasses particulates (slate data points).

Molasses causes a profound increase in OD600 (plot A) while it does not influence the cell concentrations provided by BactoBox measurements (plot B, lavender data points). The image shows pitch black molasses in a blue-cap flask (C).

Polystyrene beads

Similarly, the OD600 readings show a strong dependency of the polystyrene beads concentrations (plot A, R² = 0.9996). BactoBox detects the objects as non-cellular particulates (plot B, black data points) not cells (plot B, lavender data points).

Implications

It is always best to select a method that is specific for the objects of interest. This study demonstrates that BactoBox® discerns cells from the non-cellular compounds.

In contrast, the molasses and PS beads data demonstrate that OD600 is strongly influenced by light-scattering and absorbing compounds. With OD₆₀₀ you don't know if a high signal is merely due to high cell concentration or if it is simply due to presence of other light-scattering or absorbing compounds.

For example, an OD₆₀₀ of 1 AU corresponds to 1×10⁸ to 1×10⁹ E. coli cells/mL. In the present example, a solution of just ~0.1 g/mL molasses leads to a similar increase in OD₆₀₀.

With BactoBox® you gain specificity for cells.