Multiple symbiotic and free living Vibrio sp. grow as a form of microbial community known as biofilm. In the laboratory, methods to quantify Vibrio biofilm mass include crystal violet staining, direct Colony Forming Unit (CFU) counting, dry biofilm cell mass measurement, and observation of development of wrinkled colonies. Another approach for bacterial biofilms also involves the use of tetrazolium (XTT) assays (used widely in studies of fungi) that are an appropriate measure of metabolic activity and vitality of cells within the biofilm matrix. This study systematically tested five techniques, among which the XTT assay and wrinkled colony measurement provided the most reproducible, accurate, and efficient methods for the quantitative estimation of Vibrionaceae biofilms.

Abiotic variation can influence the evolution of specific phenotypes that contribute to the diversity of bacterial strains observed in the natural environment. Environmentally transmitted symbiotic bacteria are particularly vulnerable to abiotic fluctuations, given that they must accommodate the transition between the free-living state and the host's internal environment. This type of life history strategy can strongly influence the success of a symbiont, and whether adapting to changes outside the host will allow a greater capacity to survive in symbiosis with the host partner. One example of how environmental breadth is advantageous to the symbiosis is the beneficial association between Vibrio fischeri and sepiolid squids (Cephalopoda: Sepiolidae). Since Vibrio bacteria are environmentally transmitted, they are subject to a wide variety of abiotic variables prior to infecting juvenile squids and must be poised to survive in the host light organ. In order to better understand how a changing abiotic factor (e.g., pH) influences the diversification of symbionts and their eventual symbiotic competence, we used an experimental evolution approach to ascertain how pH adaptation affects symbiont fitness. Results show that low pH adapted Vibrio strains have more efficient colonization rates compared to their ancestral strains. In addition, growth rates had significant differences compared to ancestral strains (pH 6.5–6.8, and 7.2). Bioluminescence production (a marker for symbiont competence) of pH evolved strains also improved at pH 6.5–7.2. Results imply that the evolution and diversification of Vibrio strains adapted to low pH outside the squid improves fitness inside the squid by allowing a higher success rate for host colonization and symbiotic competence.