High-Throughput Rapid and Inexpensive Assay for Quantitative Determination of Low Cell-Density Yeast Cultures

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.

How does temperature trigger biofilm adhesion and growth in Candida albicans and two non‐Candida albicans Candida species?

“…Data from the biofilm forming ability test were analysed following the procedure for the determination of low cell‐density yeast cultures already developed in our laboratory. 49 Briefly, kinetic parameters such as the lag phase were used to estimate the size of the inoculum, by referring to the density of the cells that were able to adhere to the surface, after the priming step. The determination of the lag phase is based on the most widespread definition that describes it as the transition period during which the specific growth rate increases to that of the exponential phase.…”

Biofilm Specific Activity: A Measure to Quantify Microbial Biofilm

“…The supernatant was removed, and the pellet was washed twice with Phosphate Buffered Saline (PBS). The washed cells were then resuspended in an RPMI-1640 medium (Sigma Aldrich, Saint Louis, MO, USA, used for yeasts) or TSB (Tryptic Soy Broth, Biolife, Milan, Italy, used for bacteria) in order to obtain a final density of 1.0 × 10 6 cells/mL, by adjusting the density after spectrophotometrical readings at OD 600 and the calculation with the regression equation of the species-specific curves [30]. One hundred µL of this standardized cell suspensions were seeded in each selected well of a 96-well microtiter plate; one unseeded well acted as the negative background control for the subsequent steps.…”

“…After biofilm surface priming, the medium in each well was removed carefully with a multichannel pipette, taking care to not disrupt the biofilm; each well was subsequently washed three times with PBS. At this stage, it was not possible to spectrophotometrically evaluate the biofilm formation because its optical density was under the detection limit of the plate reader [30]. After these washing steps, 100 µL of the appropriate medium was added to the wells.…”

“…One solution to this problem was proposed by Heydorn and colleagues who developed a software to try to calculate several parameters from three dimensional clusters of biofilm images [28]. Another way is the one proposed by Bakke et al or Casagrande et al, which tried to use an optical approach to quantify the biofilm [29,30]. In general, most of the methods can be divided into two categories according to the ability to measure the biofilm biomass as CV or its metabolic activity as XTT.…”

Potential Probiotic Yeasts Sourced from Natural Environmental and Spontaneous Processed Foods

“…It was used as the growth medium. An inoculum of about 10 6 cells/mL of each pre-culture was made in duplicate, and the changes in cell density after 48 and 120 h of incubation at 37 • C were monitored by counting the cells using a Thoma-Zeiss counting chamber, as suggested by Casagrande-Pierantoni et al [34]. Viability of the cells was estimated using methylene blue staining.…”