Isothermal microcalorimetry for thermal viable count of microorganisms in pure cultures and stabilized formulations

Abstract

Background: Quantification of viable microorganisms is an important step in microbiological research as well as in microbial product formulation to develop biological control products or probiotics. Often, the efficiency of the resulting product is dependent on the microbial cell density and their viability, which may decrease over time. Commonly, the number of viable cells is determined by serial dilution and plating techniques or flow cytometry. In 2017, we developed a mathematical model for isothermal microcalorimetry (IMC) data analysis and showed that the new method allows for a more rapid quantification of viable fresh and freeze-dried anaerobic Lactobacillus reuteri cells than traditional viable count methods.

Results: This study developed the new method further by applying it to well-known aerophilic plant-beneficial microbial species (Pseudomonas brassicacearum, Bacillus amyloliquefaciens subsp. plantarum and Clonostachys rosea) used in biological control products. We utilized IMC to quantify viable cells in microbial pure cultures as well as when coated onto wheat seeds. The results from this study confirmed that thermal viable count methods are more rapid and sensitive than traditional viable count techniques. Most interestingly, a thermal viable count method was able to quantify microbes coated on seeds despite the presence of the natural microbiota of the seeds. Our results also showed that, in contrast to plating techniques for which clustered cells skew the results, IMC does not require single cells for accurate viable counts.

Conclusions: Thermal viable count methods are novel methods for the rapid quantification of divergent bacterial and fungal species and enhance the speed, sensitivity, and accuracy of routine viable counts of pure cultures and controlled microbiomes such as plant seed coatings.

Keywords: Aerophilic; Biological control; Isothermal microcalorimetry; Microbial products; Plant protection; Plant seed coating; Viable count.

Fig. 1

Isothermal microcalorimetry thermal power (μW) curves of microbes in different culturing volumes (3, 6, 12 and 18 ml). a Pseudomonas brassicacearum MA250 in TSB, 25 °C. b Bacillus amyloliquefaciens subsp. plantarum UCMB5113 in LB, 25 °C. c Clonostachys rosea IK726 in PDB, 25 °C. The figures for Pseudomonas and Bacillus represent the averages and standard deviations of three independent experiments with 3–6 replicates per treatment. The figure for C. rosea IK726 represents the averages and standard deviations of six replicates per treatment

Fig. 2

Isothermal microcalorimetry thermal power curves (μW) and respective initial microbial cell densities for each curve (log (cfu/ml)). The pure culture volume was 6 ml, and the seed sample size was 3 g, supplemented with 5.5 ml of media. a Pseudomonas brassicacearum MA250 pure culture in TSB, 25 °C. b P. brassicacearum MA250 coated on wheat seeds, 25 °C. C = surface sterilization control (3.1 log (cfu/ml)). c Bacillus amyloliquefaciens subsp. plantarum UCMB5113 in LB, 25 °C. d Clonostachys rosea IK726 in PDB, 25 °C. Each strain displayed distinct and repeatable thermal power curves. The figures represent the averages and standard deviations of 2–3 independent experiments with 3–6 replicates each

Fig. 3

Regression analyses of cell concentrations (log (cfu/ml)) and detection times (h) at certain thermal power values to determine the correlation and resolution of isothermal microcalorimetry thermal power curves for the quantification of microbial densities. a The Pseudomonas brassicacearum MA250 resolution in pure culture was ~ 0.2–0.4 log (cfu/ml). b The P. brassicacearum MA250 resolution after seed coating was ~ 0.5–1 log (cfu/g seed). c Different thermal power (500 μW and 800 μW) time points of P. brassicacearum MA250 pure cultured samples and coated seed samples fitted into the same regression curve despite of the differences in the resolution per treatment (see Fig. 3a and b). d The Bacillus amyloliquefaciens subsp. plantarum UCMB5113 strain’s resolution in pure culture was ~ 0.6–0.8 log (cfu/ml). e The Clonostachys rosea IK726 resolution in pure culture was ~ 1–1.5 log (cfu/ml). The figures represent the averages and standard deviations of 2–3 independent experiments with 3–6 replicates each