magistrsko delo
Alja Polšak (Author), Iztok Dogša (Reviewer), Ines Mandić-Mulec (Mentor), Polonca Štefanič (Co-mentor)

Abstract

V magistrski nalogi smo želeli razviti in optimizirati metodo za vrednotenje sorodstvene diskriminacije pri bakteriji Bacillus subtilis na nivoju celice. Osredotočili smo se na razvoj metode z uporabo agarskih blokcev za opazovanje rasti in interakcij sevov bakterije B. subtilis z različno stopnjo sorodnosti s kombinacijo epi-fluorescentne in presevne mikroskopije. Za razvoj metode smo uporabili več vrst gojišč in njihovih različic. Preverjali smo še druge parametre, ki vplivajo na rast bakterij, kot so temperatura, čas inkubacije, načini nacepljanja blokcev. Ugotovili smo, da antagonizma ne opazimo na nivoju posameznih celic in na nivoju mikrokolonij, ki jih še lahko spremljamo z opazovanjem pod mikroskopom, kljub temu da ti »non-kin« sevi tvorijo razmejitveno linijo med roji na agarskih ploščah. Na osnovi teh ugotovitev smo sklepali, da je morda pojav antagonizma, ki se kaže v lizi celic odvisen od specifičnih pogojev in morda vpliva tudi celične gostota na stiku, čeprav slednjega nismo direktno kvantitativno vrednotili. V našem primeru celična gostota pri opazovanju pod mikroskopom morda ni bila dovoljšnja, saj so ob celice enega in drugega seva bile večinoma v enem sloju. Preverili smo tudi vpliv sorodnosti sevov na podvojevalni čas, kjer smo pričakovali, da bo hitrost rasti kin sevov boljša v primerjavi z rastjo non-kin sevov. Rezultati ne nakazujejo na podaljšanje generacijskega časa ob hkratni nacepitvi dveh non-kin sevov v primerjavi s kin sevoma, smo pa pri non-kin sevih opazili večjo variabilnost posameznega seva v hitrosti zasedanja površine agarja.

Keywords

Bacillus subtilis;sorodstvena diskriminacija;flourescentna mikroskopija;agarski blokci;

Data

Language: Slovenian
Year of publishing:
Typology: 2.09 - Master's Thesis
Organization: UL BF - Biotechnical Faculty
Publisher: [A. Polšak]
UDC: 579.22/.26:579.852.11
COBISS: 5135224 Link will open in a new window
Views: 647
Downloads: 79
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Other data

Secondary language: English
Secondary title: Development of a method for assessing kin discrimination of Bacillus subtilis at the cellular level
Secondary abstract: In this master's thesis we aimed to develop and optimise a method that would allow us assessment of kin discrimination for bacterium Bacillus subtilis on the cellular level. We concentrated on developing agar pads method to monitor growth and interactions of B. subtilis cells that are genetically more (»kin«) or less (»non-kin«) related by confocal microscopy. We tested the influence of media composition, temperature, time of incubation, inoculation procedure on colony growth. Results did not confirm antagonism between strain, between individual or at the level of microcolonies that we were able to track over time (up to 9 hours) by confocal microscopy. Still both colonies formed a demarcated line and cells mostly did not mix. In the occasions when cells of PS-196 were trapped within the colony of PS-216 we noticed morphological changes of the trapped cells (SMM+ 0,5 % glc). The tested strains still preserved the ability to form visually dramatic boundary at the meeting point of two swarms on agar plates. It is possible that antagonism previously confirmed on agar plates depends on cell density as in microcolonies cells where still at the single cell layer and thus exposed to different conditions than those in swarms on agar paltes. We also examined the influence of relatedness on doubling time. The doubling times of kin strain pairs were more homogeneous in comparison to non-kin strains; however, this conclusion is preliminary as we would need to test higher number of biological replicates to claim this with confidence.
Secondary keywords: Bacillus subtilis;kin discrimination;flourescent microscopy;agar pads;
Type (COBISS): Master's thesis/paper
Study programme: 0
Embargo end date (OpenAIRE): 2020-12-09
Thesis comment: Univ. v Ljubljani, Biotehniška fak., Študij mikrobiologije
Pages: X, 54 f..
ID: 11334323