Characterization of the early stages in biofilm development

Marais, Susara (2004-04)

Thesis (MSc)--Stellenbosch University, 2004.

Thesis

ENGLISH ABSTRACT: Complex biofilm communities have extensively been studied in the past. Less work has been done on the early stages of biofilm formation. This study aimed to assess initial colonization patterns of biofilms on different surfaces and under different environmental conditions with application of novel methods describing biofilm surface profiles. Biofilms were cultivated on glass, polyvinylchloride (PVC) and polished stainless steel. Results from microscopy, followed by mathematical analysis and contact angle measurements proved that glass was the most appropriate substrate for this study. More extensive extracellular polymeric substances (EPS) production and apparently less cell attachment were observed on PVC and polished stainless steel surfaces. Two different series of experiments were conducted where biofilms were cultivated on the glass. Biofilm morphology was analysed under various conditions of temperature and nutrient concentration. Different temperature conditions were 8°C, 22°C and 37°C and different nutrient concentrations were 0.1%, 1% and 10% Tryptic Soy Broth (TSB). After obtaining samples after 1, 2, 3 and 4 days respectively, the biofilm surfaces were visualised using atomic force microscopy (AFM) and epifluorescence microscopy. Less cell attachment was displayed at lower temperatures and nutrient limitations. The roughness profile of the early stages of biofilm development was explored by the novel application of various existing statistical methods. Benoit software was applied for the statistical analysis of various data sets obtained from AFM imaging, using power spectrum, variogram and wavelet methods to determine the Hurst exponent. The variogram method proved to be the most suitable to describe biofilm surface profiles with consistent values of ± 0.9, indicating that biofilm growth behaviour will continue in a similar pattern. Fractal dimension values of images obtained from epifluorescence microscopy was determined by the box dimension method. The values described the self-affine patterns displayed by biofilms. Using the results of these investigations, a series of models concerning the initial stages of biofilm formation was compared to describe the development of colony patterns. This study showed that the AFM and epifluorescence microscopy can be used as analytical tools for raw data assembly. It also demonstrated a novel application of existing statistical methods in order to describe the early stages of biofilm formation. Using this approach it was shown that the early stages of biofilm formation display certain colony patterns that can be described and predicted. Such information may be used in efforts to control biofilm formation.

AFRIKAANSE OPSOMMING: Komplekse biofilmgemeenskappe is reeds breedvoerig m die verlede bestudeer. Minder werk is op vroeë stadiums van biofilmvorming gedoen. Hierdie studie het gepoog om die aanvanklike koloniseringspatrone van biofilmvorming op verskillende substrate en onder verskillende omgewingstoestande kwantitatief te bepaal met nuwe metodes om die oppervlakprofiele van biofilms te beskryf. Biofilms is gekweek op glas, polivinielchloried (PVC) en gepoleerde vlekvrye staal. Resultate van mikroskopie, gevolg deur wiskundige analise en kontakhoek-metings het getoon dat glas die mees geskikte substraat vir hierdie studie is. Die produksie van meer ektrasellulêre polimeriese substanse (EPS) en oënskynlik minder selaanhegting is waargeneem op PVC en gepoleerde vlekvrye staaloppervlaktes. Twee verskillende reekse eksperimente IS uitgevoer waar biofilms op glas gekweek IS. Biofilm-morfologie is geanaliseer onder verskillende toestande van temperatuur en nutriënt-konsentrasie. Verskillende temperatuur-toestande was goC, 22°C en 37°C en verskillende nutriënt-konsentrasies was 0.1%, 1% en 10% Tryptic Soy Broth (TSB). Nadat monsters onderskeidelik na 1, 2, 3 en 4 dae verkry is, is die biofilm oppervlaktes gevisualiseer deur atoomkrag mikroskopie (AFM) en epi-fluoressensie mikroskopie. Minder selaanhegting is waargeneem by laer temperature en nutriënt-beperkinge. Die grofheidsprofiele van die vroeë stadium van biofilm-ontwikkeling is ondersoek deur die nuwe toepassing van verskeie bestaande statistiese metodes. Benoit-sagteware is gebruik om die statistiese analise van verskeie data-stelle van AFM beelde te ondersoek deur power spectrum, variogram en wave/et -metodes te gebruik om die Hurst-eksponent te bepaal. Die variogram metode het voorgekom as die mees geskikte om biofilm oppervlakprofiele te beskryf met konstante waardes van ± 0.9, wat aandui dat biofilm groei sal aanhou in 'n soortgelyke patroon. Fraktale dimensie-waardes van beelde wat met epi-fluoressensie mikroskopie verkry is bepaal deur toepassing van Benoit-sagteware se box dimension metode. Die waardes beskryf die selfherhalende patrone wat deur biofilms gedemonstreer word. Deur die resultate van hierdie ondersoeke te gebruik, is 'n reeks modelle aangaande die aanvanklike stadiums van biofilmvorming vergelyk om die ontwikkeling van koloniepatrone te beskryf. Hierdie studie het getoon dat die AFM en epi-fluoressensie mikroskopie gebruik kan word as analitiese gereedskap vir rou data-versameling. 'n Nuwe toepassing van bestaande statistiese metodes om die vroeë stadiums van biofilmvorming te beskryf, is ook gedemonstreer. Deur hierdie benadering te gebruik, is getoon dat die vroeë stadiums van biofilmvorming sekere koloniepatrone aandui wat beskryf en voorspel kan word. Sulke inligting kan gebruik word in pogings om biofilmvorming te beheer.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/49880
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