A simplified diphenylamine colorimetric method for growth quantification

Cell growth needs to be monitored in biological studies and bioprocess optimization. In special circumstances, such as microbial fermentations in media containing insoluble particles, accurate cell growth quantification is a challenge with current methods. Only the Burton method is applicable in such circumstances. The original Burton method was previously simplified by adopting a two-step sample pretreatment in perchloric acid procedure to eliminate the need for DNA extraction. Here, we further simplified the Burton method by replacing the previous two-step perchloric acid pretreatment with a new and one-step diphenylamine reagent pretreatment. The reliability and accuracy of this simplified method were assessed by measuring the biomass of four model microorganisms: Escherichia coli, Streptomyces clavuligerus, Saccharomyces cerevisiae, and Trichoderma reesei grown in normal media or those containing solid particles. The results demonstrate that this new simplified method performs comparably to the conventional methods, such as OD₆₀₀ or the previously modified Burton method, and is much more sensitive than the dry weight method. Overall, the new method is simple, reliable, easy to perform, and generally applicable in most circumstances, and it reduces the operation time from more than 12 h (for the previously simplified Burton method) to about 2 h.