Visualizing aquatic bacteria by light and transmission electron microscopy

Abstract

The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.

TEM as an Important Tool to Study Aquatic Microorganisms and their Relationships with Ecological Processes

“…Our quantitative TEM analyses revealed a significant proportion of bacteria with a limiting capsule ( Figure 2B and 2Bi). Our data showed that 31 % of freshwater bacteria had capsular structures [20]. This frequency is higher than that found in marine bacteria (7-27 %) [21][22][23].…”

“…Our data from samples collected from Batata Lake suggest that OMV-mediated secretion is an important cell process of freshwater bacteria (Figure 3). Although the function of OMV remains to be defined, these secretory vesicles, observed for the first time by us in aquatic bacteria from a tropical ecosystem [20], may be important for bacterial survival and inhibition of lysis induced by viral infection that is relevant in this ecosystem [44], as mentioned before. Moreover, OMVs may be relevant in the transport of products involved in the formation of the cell wall, inhibition of toxic components present in the surrounding environment, and formation of biofilms.…”

“…By studying microorganisms from freshwater ecosystems in both natural environmental and cultures through TEM, we have identified a consistent production of OMVs by bacteria [20]. These vesicles were round, delimited by classical membrane with trilaminar appearance, and exhibited morphology similar to those described on the surfaces of other bacterial species [52] ( Figure 3).…”

“…Bacteria also show the cytoplasm with distinct electron-density. Reprinted from ref [20]. with permission.…”

“…(B and Bi) Substratum particles (highlighted in red in Bi) are seen as an adhered coating localized externally to the capsular structure (highlighted in green in Bi) of a bacterium collected from a natural environment. Typical bacterial structures such as nucleoid (N) and granule (Gr) are observed in the cytoplasm (Cy).Figure 2Bwas reprinted from ref [20]. with permission.…”