Team:Evry/Biology/CellCharacterization/Culture

With the help of the 'Molecules of defense and communication in the microbial ecosystems' team of the National museum of natural history of Paris (their web page here), we succeed to cultivate Pseudovibrio denitrificans in two medium, the marine broth (MB) and the minimal medium M9 added 2% of CASAmino Acid and by 3% of NaCl.

Table1: Composition of used media, Marine Broth and M9-CASA+3% NaCl.

Because of the opaqueness of the marine broth, it was impossible to obtain reliable data of the growth of the bacteria. We try to have a curve of growth in filtered MB but even if the bacteria grow, it's not the same condition. Thus, the growth curve has been made in M9-CASA+3%NaCl 1X, with the TECAN infiniteM200 (cf: Figure1).

Figure1: Curve of growth in liquid M9-CASA+3%NaCl 1X and liquid MB, and microscopic photo of Pseudovibrio denitrificans (left) and Pseudovibrio ascidiaceicola (right).

The growth of our bacteria in 200µL of M9-CASA+NaCl is not very good, moreover the ratio culture/air is not optimal in the TECAN wells. In bigger cultures, the growth is faster.
The marine broth is more complete than the M9-CASA+3%NaCl, even if we have adjusted the salinity. As we can see in the Table 2, Pseudovibrio denitrificans grows better on MB medium. The number of colony is higher, and they are significantly bigger (cf: Table2). Moreover, the growth is faster.

Table2: Number of CFU of MB 1X and M9-CASA+3%NaCl 1X plates.
The number of CFU was calculated by inoculated respectively 10µL of pre-cultures of Pseudovibrio denitrificans in MB 1X and in M9-CASA+3%NaCl at OD (600nm) ~ 1.

In the aim to know if we can transfer Pseudovibrio denitrificans from a media, to the other one, we have made some tests and calculate a ratio of survivability. We have made two pre-cultures of Pseudovibrio denitrificans in MB 1X and M9-CASA+3%NaCl 1X, and after a culture overnight, we streaked them on plates of MB 1X and M9-CASA+3%NaCl 1X. The CFU on each plate were counted and ratios of survivability were calculated using the formula Sample/Control (controls being the number of CFU obtained on a plate from a pre-culture in the same medium). (cf: Figure2).

Figure2: Tests of survivability, table of ratio and photos (on the left: M9 plates, on the right: MB plates).

Pseudovibrio denitrificans can survive even if we transfer them from a medium to the other one. But we can see that even cells come from a pre-culture of M9-CASA+3%NaCl, they grow better and faster on MB plates. Moreover, even if they come from a MB pre-culture, they don't grow very well on M9-CASA+3%NaCl plates.