Among Live and Dead Bacteria, the Optimization of Sample Collection and Processing Remains Essential in Recovering Gut Microbiota Components

Abstract

The human gut microbiota plays an important role in human health. Accessing and culturing the gut microbes remains critical in microbiology. The culturomics approach, combined with sequencing strategies, provides new insights in the study of gut microbiota components. However, we are still far from having described all the microbes. Many factors are involved in recovering as much bacteria as possible. One of the most important factors is sample preparation and conservation. The aim of the present work was to evaluate three different types of stool sample conditioning by mainly studying the effect of atmospheric oxygen on bacterial viability and culturability. Quantitative analysis of fecal samples from eight different healthy individuals was carried out using a culture-independent method (flow cytometry) and a culture-dependent method (plating technique). We found that the cultured bacteria reached a 50% yield when the samples were exposed to oxygen for 120 min without any protectant medium, while the percentage of culturability increased to 67% in the presence of antioxidants. More importantly, when samples were exposed to oxygen for less than 2 min, combined with the work under the anaerobic chamber, no discordance was found between the two counting techniques and the culturability increased to 87%. Our study confirmed the importance of sample conditioning to preserve the bacterial viability in samples, especially for oxygen-sensitive intestinal bacteria.

Keywords: culturability; microbiota; protectant medium; sample processing; viability.

FIGURE 1

Schematic representation of the protocols used. The routine protocol (black arrows): the stool was exposed to oxygen for more than 1 h without any protectant medium. The optimized protocol (blue arrows): the stool was exposed to oxygen for more than 1 h and conserved in the protectant medium. The implemented optimized protocol (red arrows): the stool was exposed to oxygen less than 2 min, conserved in the protectant medium and all manipulations were performed inside the anaerobic chamber. The enumeration of bacterial cells was performed by Flow cytometry (FCM) and by the plate count method (CFU).

FIGURE 2

Total cell counts by Flow cytometry (FCM) (dead, live and injured bacteria) and Colony forming unit (CFU) counts (aerobes and anaerobes bacteria) in the three protocols. The percentage of culturability was counted from total cells in fecal samples and from live cells counted by FCM. Significant differences between protocols were highlighted using the Mann–Whitney’s test.

FIGURE 3

(A) Routine protocol, (B) optimized protocol versus (C) implemented optimized protocol. The graphs represent Mann–Whitney’s test performed to assess the significance of the number of viable colonies measured by the CFU method and FCM.