Analysis of two subpopulation of Bacillus subtilis culture in the initial stage of pelicle formation

Biofilmi so ubikvitarne združbe bakterij, ki so med seboj tesno povezane z zunajceličnim matriksom. Bakterija Bacillus subtilis naredi plavajoče biofilme na interfazi voda-zrak, imenovane tudi pelikule. V magistrski nalogi smo po optimizaciji gojitvene metode s konfokalno vrstično lasersko mikroskopijo časovno in situ spremljali nastanek in dinamiko razvoja plavajočega biofilma v nestresanih pogojih. Uporabljali smo modelni sev divjega tipa bakterije B. subtilis in njegovo mutanto, ki ni sposobna proizvajati surfaktina, oba označena s konstitutivno izraženim proteinom Yfp. Časovno smo ju spremljali vzdolž celotne globine gojišča MSgg z dodanim propidijevim jodidom (PI). Z njim smo določevali mrtve celice in hkrati preučevali njegov učinek na razvoj biofilma. Začetni inokulum je bil bodisi iz celic v eksponentni fazi rasti ali direktno iz spor. Ugotovili smo, da se, ne glede na testirane pogoje v populaciji B. subtilis že v začetnih fazah nastanka plavajočega biofilma ločita dve podpopulaciji, ena na spodnjem delu in druga na zgornjem delu preparata. Pri sevu divjega tipa je več celic pomaknjeno v zgornje plasti preparata kot pri sevu mutante. Tudi dinamika nastanka in propada biofilma je bolj izrazita pri sevu divjega tipa. Tekom nastanka plavajočega biofilma v vseh testiranih pogojih del populacije propade. Dodatek propidijevega jodida v gojišče ima dolgoročni vpliv na potek nastanka biofilma in, predvsem v visokih koncentracijah, vodi do manjše končne biomase. Tip inokuluma vpliva na nastajanje plavajočega biofilma. Čas od inokulacije do nastanka zrelega biofilma se pri inokulumu iz spor pričakovano podaljša, povečata se dvig biofilma nad začetno površino gojišča in največja debelina biofilma. V primeru surfaktinske mutante se razlike glede na tip inokuluma še povečajo, vendar v vseh primerih pride do nastanka zrelega plavajočega biofilma.Biofilms are ubiquitous communities of bacteria that are interconnected by the extracellular matrix. The bacterium Bacillus subtilis forms floating biofilms at the water / air interphase, also called pellicles. After optimization and development, we applied the improved cultivation method to monitor the formation and dynamics of floating biofilm in situ in time under a confocal laser-scanning microscope (CLSM) in static growth conditions. We used a model strain of the wild-type bacterium B. subtilis and its mutant incapable of producing surfactin, both labeled with the constitutively expressed Yfp protein. Time-lapse CLSM imaging was perform over the entire depth of MSgg medium. By adding propidium iodide (PI), we identified dead cells and at the same time studied the effect of PI on biofilm development. The inoculum for growing biofilm was either from the cells in the exponential growth phase or from the spores. We found that regardless of the conditions tested, the population of B. subtilis, splits into two subpopulations already at the initial stages of biofilm formation. One subpopulation occupies the lower part and the other the upper part of the samples. Compared to the mutants, the distribution of the cells is shifted to the upper layers along the depth of the growth media in the wild-type strain. In addition, the dynamics of the formation and collapse of pellicle is more pronounced in the wild-type. Part of the population collapses during the formation of a floating biofilm under all tested conditions. The addition of PI to the medium has a long-term effect on the dynamic of the floating biofilm formation, and in higher concentrations leads to increased lysis and decreased final biomass. Type of the inoculum affects the formation of a floating biofilm. By spore inoculation, the time from inoculation to the formation of a mature biofilm prolongs and the rise above the initial growth medium surface and maximum thickness of the biofilm increase. In the case of the surfactin mutant, the difference is amplified, but in all cases, a mature pellicle is formed