Direct-imaging-based quantification of Bacillus cereus ATCC 14579 population heterogeneity at a low incubation temperature

Abstract

Bacillus cereus ATCC 14579 was cultured in microcolonies on Anopore strips near its minimum growth temperature to directly image and quantify its population heterogeneity at an abusive refrigeration temperature. Eleven percent of the microcolonies failed to grow during low-temperature incubation, and this cold-induced population heterogeneity could be partly attributed to the loss of membrane integrity of individual cells.

FIG. 1.

(a) Growth of Bacillus cereus ATCC 14579 in BHI broth at 12°C. Cells were plated on BHI agar plates (⋄), BHI agar plates plus 2.5% salt (□), and BHI agar plates plus 5% salt (▵). (b) Growth of microcolonies on Anopore strips placed on BHI agar plates at 12°C. The area of each microcolony per imaging time point was measured in pixels and log₁₀ transformed. Data points represent the average microcolony size of the grown population of microcolonies per imaging time point (○). The specific growth rates in broth and on Anopore strips were estimated by linear regression using the time points that represented exponential growth (continuous lines).

FIG. 2.

Examples of images of Bacillus cereus ATCC 14579 cultured on Anopore strips which were placed upon BHI agar plates at 12°C. Observed and fitted frequency distributions of the number of cells per microcolony for the imaging time points (t) 0, 6, 12, 18, and 24 h are shown. Histogram shows observed frequencies of number of cells per microcolony. Continuous curves show fitted normal distributions of the log₁₀-transformed microcolony areas, solid lines represent the distribution of all microcolonies, and dashed lines represent the distribution of grown microcolonies only.

FIG. 3.

The variances of the observed frequency distributions of the log₁₀-transformed microcolony areas versus the imaging time points at 12°C and 30°C. The intervals of imaging were 3 h at 12°C and 15 min at 30°C (see reference 6). ⋄, Distribution at 12°C of all microcolonies; ♦, distribution at 12°C of grown microcolonies only; □, distribution of microcolonies at 30°C.

FIG. 4.

Labeling of Bacillus cereus ATCC 14579 microcolonies with SYTO-9 and propidium iodide after 24 h of culturing on Anopore strips which were placed upon BHI agar plates at 12°C. At this imaging time point, two populations of microcolonies were observed, a nongrown population (a) and a grown population (b).