Methoden zur Evaluation von Zytotoxizit¨at und Struktur ... - OPUS

180 7. Summary
determination of the antibacterial activity of potential drugs. Therefore novel methods
were developed based on capillary electrophoresis (CE) (Section 4.2) and nuclear ma-
gnetic resonance (NMR) (Section 4.4), which were suitable for the description of cell
cultures and could be employed for activity screening.
Having developed suitable CE-conditions, and optimized bacterial cultures and sam-
ple preparation a reliable CE-method was established for the bacterial strains Strep-
tococcus vestibularis and Pseudomonas fluorescens. The method was performed by UV
detection and showed good precision, selectivity, specificity, and linearity. It allowed
for separation of microbial agglomerates and chains of different size, and was used to
create characteristic fingerprints.
Staining bacteria by using the LIVE/DEAD BacLight viability kit the constitution of a
bacterial population could be examined by means of LIF-CE. The kit consists of the two
fluorescent nucleic acid stains SYTO9 and propidium iodide, which differ in their abili-
ty to penetrate healthy cells making a distinction between live and dead cells possible:
SYTO9 stains all cells green, propidium iodide stains cells with damaged membrane
red. Monitoring electropherograms of bacterial samples by dual LIF detection, the ratio
of live to dead cells was correlated with the electrophoretic profile. Therefore, the new
LIF-CE method, based on the use of a second unlabeled bacteria injection as a stacking
front, allowed for the direct observation of the tendency of chain formation and al-
terations in the live/dead ratio of the bacterial composition in presence of different
antibiotics. By correlation of the ratio of green and red fluorescence with the percenta-
ge of live cells dose response curves of drug-treated bacterial samples were calculated,
which allowed for the assessment of ED50 values. To verify the results the determinati-
on of the antibacterial activity was repeated by fluorescence microplate reader (FMR)
measurements and broth microdilution method. For ciprofloxacin, ampicillin-Na, and
vancomycin-HCl similar results were obtained for each of the three methods. The fluo-
rometric methods allowed for a rapid differentiation between cytostatic and cytotoxic
efficiency, which was previously not possible with broth microdilution.
Using NMR experiments, it was shown by magnetic resonance imaging (MRI) at 17.6 T
that the transverse relaxation T2 is suitable for the description of anaerobical, bacterial
proliferation. The parameter T2, which is influenced by chemical exchange, is sensitive
to all variations of the bacterial culture. Consumption of the medium, metabolism, and
varying cell density change the composition and the concentration of medium as well
as pH, ionic strength and viscosity. To establish the dependence of T2 on cell prolife-
ration 1 H NMR spectra of Streptococcus vestibularis samples of different growth stages
were correlated with the extracellular parameters T2, OD600, and pH. The consumpti-
on of medium and the pH-variation evoked by the production of metabolites during
cell growth were identified to be the basis for the successful parametrization of cell
growth by T2. MRI allowed for measuring growth curves on the basis of T2 of several