Plate counts for Κ

This page reviews the benefits and pitfalls of using plate counts (CFUs) as method to determine carrying capacity, Κ.

Main benefit of using CFU is that it is a well-established method. In fact it is the currrent de facto gold standard. The method is also extremely sensitive and background from other objects than bacteria can be excluded, because debris and particles do not form colonies on plates.

Nonetheless it is impractical to use CFU as a screening method to find the optimal growth conditions. Throughput is low, colonies take days or weeks to appear on a plate. Furthermore the method has relatively low precision which necessitates several replicates on different dilutions for precise results.

But there is an additional pitfall of the CFU method as a screening tool for Κ determination: It is difficult to hit the right window for max CFUs.

Challenges of finding the true stationary phase

First reason is that cultures may show somewhat unpredictable growth patterns before they reach stationary phase. This is illustrated in the P. fluorescens example below, which the transient plateau at ~13 hours followed by regrowth to twice the concentration of the first plateau. This phenomenon is likely caused by diauxic growth where the cells have depleted the preferred substrate at the first plateau. After a lag phase of adaptation the cells switch to a different substrate and continue cell divisions.

Unpredictable growth patterns before reaching stationary phase. P. fluorescens in TSB medium as an example of a culture that hits a transient pseudo-stationary phase and then continues cell divisions. BactoBox® cells/mL (lavender) and plate counts measurements were done in triplicate repeats.

Plate counts decline after reaching the stationary phase

Second reason is that the stationary stage may be a relatively short window in time before the culture proceeds to the decline phase where CFUs will deflate from the peak concentrations. At ~12 hours of incubation, practically all cells are culturable by plate counts. After ~24 hours only roughly 10% of the cells are culturable.

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 the cell concentration stays relatively constant.

Practical recommendations

Unpredictable growth patterns and decline phase makes it tricky to use plate counts to determine Κ. You need to sample frequently and ensure that you hit the true stationary phase before the culturability decreases in the decline phase. This is tremendous, tedious work for the P. fluorescens example where plate counts must be done in the period from ~9 to 24 hours. The true concentration is not known and therefore overkill dilution and plating is usually done "in the blind". For a single data point a typical number of dilutions is 5 with triplicate plating. This amounts to 15 agar plates. With the 10 sampling points in the Klebsiella example you would be doing 150 agar plates and still not know if you hit the right window for maximal CFUs.

CFU is therefore not suitable for screening purposes. Instead you should use direct cell counts from microscopic or flow cytometric methods. Once the optimal growth conditions are found, CFU is well suited as an orthogonal method for finding the best harvest window with max CFUs.