| Summary: | Thesis (MSc)--Stellenbosch University, 2004. === 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.
|
|---|
