Abstract
A procedure for microbial cell density determination with a high-throughput densitometric assay was developed to allow a precise quantification of both free and sessile cells, such as those of a biofilm, with a large range from low to high cell densities. Densitometry was chosen because it allows fast, rapid and cost-effective measures; it is non-disruptive; and has an easy learning curve. The method setup, and the further validation, was carried out with strains of Candida albicans, C. tropicalis and C. parapsilosis. Equations were developed at the level of the single strains, of the three species and finally a general one applicable to all three species. In the cross validation, with strains absent from the training set, the method was shown to be robust and flexible. The best results were obtained with species specific equations, although the global equation performed almost as well in terms of correlation between real and estimated density values. In all cases, a correlation around 0.98 between effective and predicted density was obtained with figures ranging from 102 to 108 cells mL−1. The entire analytical part of the procedure can be accomplished with a MS Excel macro provided free of charge.
Highlights
The determination of the cell number is one of the most venerable microbial techniques dating back to early times of microbiology
When trying to estimate the total cell density, the most obvious rationale is to calculate it with a regression curve based on the optical density of the cell suspension
This is possible as long as a linear, or at least a monotonic relationship, exists between optical density and total cell density. We tried this approach to evaluate the initial cell density in multi-well plates, which is the situation of an initial biofilm adhesion or of any cell culture starting at very low density
Summary
The determination of the cell number is one of the most venerable microbial techniques dating back to early times of microbiology. Indirect total counts are rapid, insensitive to VNC and amenable to high throughput settings [2,5], but suffer in terms of detection and of density range [6]. Some of these techniques are complex and expensive in terms of consumables and dedicated equipment such as Real Time PCR (RT-PCR) [7,8,9,10] or some fluorescent based techniques [11]. One can overcome the problem by serial diluting the cell suspension, but this would make the procedure even more complex and laborious
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