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Razvoj metode za časovno spremljanje razvoja biofilmov z mikroskopom
Jeršinovič, Nataša (Author), Mandić Mulec, Ines (Mentor) More about this mentor... This link opens in a new window, Dogša, Iztok (Co-mentor)

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Abstract
Biofilmi so v matriks ujeti večcelični agregati bakterij, ki se pritrjujejo na biološke in nebiološke površine. Bakterijam omogočajo kolonizacijo različnih niš in preživetje neugodnih pogojev. Razvoj biofilma vključuje prehod planktonske oblike življenja v večcelično skupnost. Časovno spremljanje porazdelitve celic pri modelni bakteriji Bacillus subtilis, ki tvori biofilme (pelikle) na stičišču zrak-tekoče gojišče je slabo preučeno. V okviru naloge smo razvili metodo za časovno in prostorsko spremljanje razvoja peliklov modelne bakterije B. subtilis PS-216 s svetlobnim mikroskopom v tekočem gojišču MSgg. Uporabili smo svetlobno-mikroskopski tehniki DIC in konfokalne mikroskopije. S tremi različicami poskusa smo ponazorili delovanje in uporabnost novo razvite metode. Primerjali smo razvoj pelikla pri divjem tipu B. subtilis PS-216 in mutanti Δeps, ki ne proizvaja zunajceličnega polisaharida Eps. Pelikle smo gojili ob dodatku propidijevega jodida, ki obarva DNA in ne vstopa v žive – fiziološko aktivne celice. Ugotovili smo, da so se bakterijske celice divjega tipa med inkubacijo porazdelile bodisi na površino bodisi na dno petrijevke. Barvanje s propidijevim jodidom je pokazalo, da so celice, ki se porazdelijo na dno v slabšem fiziološkem stanju in kmalu po začetku inkubacije odmrejo. Pri gojenju mutante Δeps smo opazili, da celice na površini gojišča začnejo tvoriti pelikel, a se ta ni zmožen obdržati na površini.

Language:Slovenian
Keywords:mikrobne združbe, biofilmi, nastanek biofilmov, Bacillus subtilis, DIC mikroskopija, konfokalna mikroskopija, eps, fluorescentni proteini, porazdelitev celičnih gostot
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:BF - Biotechnical Faculty
Year:2018
Publisher:[N. Jeršinovič]
UDC:579.24+579.26:681.723.26/.27
COBISS.SI-ID:4921208 Link is opened in a new window
Views:413
Downloads:87
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Secondary language

Language:English
Title:The development of the time-lapse microscopy method for observation of the biofilm formation
Abstract:
Biofilms are bacterial aggregates, encased in extracellular matrix and attached to biological or abiological surfaces. They represent an important bacterial lifestyle and protect encased bacterial cells. Biofilms represent a survival strategy that helps bacteria survive unfavourable conditions and/or colonize different ecological niches. In B. subtilis early stage of biofilm formation is associated with transformation of planktonic cells, which lose mobility and align into chains. Transformation stages into sesile mode of growth and especially formation of floating biofilms (pellicles) that form at liquid-air interface are poorly understood at the microscopic level. Here we developed a method to temporally monitor cellular distribution during pellicle formation at different depths of microscopic specimen using the model bacterium B. subtilis PS-216. In addition to the wild type strain we also evaluate its mutant Δeps, lacking matrix polysaccharide Eps. Results show that B. subtilis pellicle formation is a very dynamic process and that biofilm form either on the surface of the growth medium or at the bottom of the petri dish. Propidium iodide staining has further shown that cells at the bottom have impaired membranes and lysed relatively soon after the onset of incubation. A very different scenario was observed for the Δeps mutant that transiently formed but did not retain the pellicle at the surface of the growth medium.

Keywords:microbial aggregation, biofilms, biofilm formation, Bacillus subtilis, DIC microscopy, CLSM microscopy, eps, fluorescent proteins, cell density distribution

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