Comparison of two optical-density-based methods and a plate count method for estimation of growth parameters of Bacillus cereus

Abstract

Quantitative microbiological models predicting proliferation of microorganisms relevant for food safety and/or food stability are useful tools to limit the need for generation of biological data through challenge testing and shelf-life testing. The use of these models requires quick and reliable methods for the generation of growth data and estimation of growth parameters. Growth parameter estimation can be achieved using methods based on plate counting and methods based on measuring the optical density. This research compares the plate count method with two optical density methods, namely, the 2-fold dilution (2FD) method and the relative rate to detection (RRD) method. For model organism Bacillus cereus F4810/72, the plate count method and both optical density methods gave comparable estimates for key growth parameters. Values for the maximum specific growth rate (mu(max)) derived by the 2FD method and by the RRD method were of the same order of magnitude, but some marked differences between the two approaches were apparent. Whereas the 2FD method allowed the derivation of values for lag time (lambda) from the data, this was not possible with the RRD method. However, the RRD method gave many more data points per experiment and also gave more data points close to the growth boundary. This research shows that all three proposed methods can be used for parameter estimation but that the choice of method depends on the objectives of the research.

FIG. 1.

Values for μ_max (h⁻¹) (□) and λ (h) (▴) estimated by the 2FD method as a function of pH for B. cereus F 4810/72 cultured at 30°C in BHI broth acidified with sulfuric acid. The estimation growth curve (dashed line) using equation 1 is based on the model of Presser et al. (30).

FIG. 2.

TTD as a function of the natural logarithm of the inoculum size (ln N_inoc) (▪) in BHI broth at pH 6.93, linear fitting (solid line) of the data points to estimate μ_max, and estimation of λ using the theoretical TTD (dashed line) assuming λ = 0, according to the 2FD method of Cuppers and Smelt (5).

FIG. 3.

Numbers of B. cereus F4810/72 CFU in BHI broth at pH 7 cultured at 30°C, as monitored by plate count (▴) and OD₆₀₀ measurement (▪).

FIG. 4.

Relationship between cell numbers and an OD₆₀₀ value of 0.2 (vertical line) of the Bioscreen C. The pH of BHI broth was set at the following values: 7 (⧫), 6.75 (▪), 6.5 (▴), 6.25 (X), 6 (□), 5.8 (•), 5.6 (+), 5.4 (Δ), and 5.2 (—).

FIG. 5.

Values for μ_max (h⁻¹) as a function of pH for B. cereus F4810/72, as determined by the 2FD method (□) (57 target values), the RRD method (⋄) (59 target values) and the plate count method (▴) (8 target values).