The utilization of carrier-free ionic18F is a convenient method for studying the effects of very low extracellular fluoride concentrations on the metabolism of the cariogenic bacterium Streptococcus mutans. We have developed a method for the preparation of carrier-free aqueous solutions of ionic18F with high specific activity (about 707 Bq/ml). Fluoride distribution and fluoride binding experiments showed that submicromolar fluoride concentrations have already a negative effect on the metabolism of Streptococcus mutans.
Authors:Sándor Nagy, Tamás Kőszegi, Lajos Botz, and Béla Kocsis
Direct bioautography is a potent means of obtaining information about the antimicrobial activity of a compound separated from a complex mixture. In this process the developed TLC plate is dipped into a broth culture of a test bacterium and the bacterium will grow directly on the plate. Optimum experimental conditions must, however, be used for each test bacterium.The main purpose of this study was to find optimum culture conditions for a Gram-negative test bacterium,
(ATCC 25922) enabling us to establish a direct bioautographic method with the shortest possible performance time. Because the intracellular adenosine-5′-triphosphate (ATP) level is a direct and sensitive measure of bacterial well-being, ATP assay was used for this purpose. As far as we know this is the first report of the use of an ATP method for optimization of direct bioautography with
. Our optimizing experiments on
culture showed that the bacteria had to be in the log phase (optical density,
= 0.1–0.4) in the bacterial suspension used for dipping. TLC plates immersed in the log-phase culture needed a shorter incubation time for bacterial growth on the TLC plate (3 h) than for the original ‘overnight’ culturing suggested in studies by others.In this paper we will show that:
ATP assay is a valid method for optimizing
Bacterial ATP level oscillates during the growth phase in culture media.
TLC plates should be immersed in
dipping suspension with
Dipping a developed TLC plate for 10 s gave acceptable results.
Incubation of the seeded TLC plate at 37°C for 3 h was found to be optimum.
An ATP/protein ratio of 10–15 nmol mg
in dipping culture and ∼5 nmol mg
on seeded TLC plates were the minimum threshold values for visualization of living bacteria by means of the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reaction.
With our optimized coditions the total performance time of
direct bioautography is 9.6 h instead of the originally reported 11.5 h.
Our procedure results in much sharper contrast of the inhibition zone than that without optimization.
Authors:Sándor Nagy, Béla Kocsis, Tamás Kőszegi, and Lajos Botz
The main purpose of this study was to determine optimum conditions for culture of a test microbe
(ATCC 6633) which enabled us to establish its use for direct bioautography. The viability of the bacteria on TLC plates was measured on the basis of their adenosine-5′-triphosphate (ATP) content as determined by bioluminescent luciferin/luciferase assay, the data being referred to values for total bacterial protein. In the first experiments, we used a ‘20-h’ culture of
prepared by dilution of an optical density (
) ≫ 0.4 culture to furnish a culture of
= 0.4 (Method A). Later, on the basis of our optimization experiments we found that a ‘5–9-h’ broth culture of
was suitable. Under these conditions the bacteria remained in the log phase (
= 0.2–0.4) for 5–9 h (Method B) in immersion bacterial suspension. Because the test bacteria were in the log phase a much shorter incubation time (4–8 h) was sufficient for TLC plates instead of the original 18 h in a previous study. One advantage of this method, in addition to the shorter incubation time, is that we can use TLC plates coated with adsorbents other than silica.
Authors:N. Lago, J. L. Legido, M. I. Paz Andrade, I. Arias, and L. M. Casás
metabolism. To date, to our knowledge there are no studies on characterisation of the growth of this bacterium using a calorimetric method. Thus, in this study, a Calvet microcalorimeter was used and several experiments were carried out with concentrations
Authors:Manrong Tan, Yongsheng Ren, Dan Yan, Xianghong Meng, Longhu Chen, Lingling Qiu, Yan Yan, Jianyu Li, and Xiaohe Xiao
0.9% sterile sodium chloride solution. Then, the elution was diluted to the approximate concentration of 10 cfu. Take couple samples of 1.5 mL of the above-mentioned bacterium to 5 mL plastic tube individually, sealed up completely. One pot was
Authors:Wioleta Jesionek, Barbara Majer-Dziedzic, Györgyi Horváth, Ágnes M. Móricz, and Irena M. Choma
Thin-layer chromatography—direct bioautography (TLC—DB) followed by liquid chromatography—tandem mass spectrometry (LC—MS/MS) was used for screening and tentative identification of the antibacterial constituents of Salvia officinalis L. ethanol extract. Seven bacterial strains were used as test organisms, both pathogenic and nonpathogenic, that is, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, luminescence gene-tagged Pseudomonas syringae pv. maculicola, and naturally luminescent marine bacterium Aliivibrio fischeri. Eight fractions with the widest antimicrobial spectrum were detected using TLC—DB, isolated by semi-preparative TLC, and subjected to LC—MS/MS analyses. Finally, five bioactive components were tentatively identified, based on their fragmentation pattern, such as salvigenin, cirsimaritin, rosmanol, carnosic acid, and 12-O-methyl carnosic acid.
Authors:Ágnes M. Móricz, Dániel Krüzselyi, and Péter G. Ott
In this study, the antibacterial profiling of the ethanolic leaf extract of greater burdock (Arctium lappa L.) is demonstrated, applying thin-layer chromatography (TLC) coupled bioassays against the Gram-positive soil bacterium Bacillus subtilis and the Gram-negative pepper pathogen Pseudomonas syringae pv. maculicola. The main active component was isolated by eluting from the adsorbent bed and subjected to a targeted characterization by high-performance liquid chromatography–diode array detection–electrospray ionisation–mass spectrometry. The identification of the germacranolide sesquiterpene lactone onopordopicrin was based on its retardation factor, bioactivity in TLC-based methods, and retention tim as well as ultraviolet (UV) and mass spectra, compared to those of the reference substance isolated earlier in our laboratory from Onopordum acanthium leaf.
Authors:Ágnes Móricz, Györgyi Horváth, and Péter Ott
The antibacterial effect of the components of clary sage (Salvia sclarea L.) and spearmint (Mentha spicata L. var. crispa (Bentham) Danert) was investigated by means of high-performance thin-layer chromatography-direct bioautography against the Gram-positive soil bacterium Bacillus subtilis (Bs) and Gram-negative bacteria such as a pepper pathogen Xanthomonas euvesicatoria (Xe), a luminescence gene-tagged Arabidopsis pathogen Pseudomonas syringae pv. maculicola (Psm) and a luminescent marine Aliivibrio fischeri (Af). Sclareol, linalool, and linalyl acetate were identified as active components of clary sage and carvone as the antibacterial substance in spearmint. Sclareol inhibited all tested bacteria, linalool and carvone showed antibacterial effect against all Gram-negative strains tested, while linalyl acetate only against Xe and Af. Some minor components of the clary sage essential oil also gave a zone of inhibition when tested on Gram-negative bacterium strains.
Authors:N. Tsibakhashvili, T. Kalabegishvili, L. Mosulishvili, E. Kirkesali, S. Kerkenjia, I. Murusidze, H. Holman, M. Frontasyeva, and S. Gundorina
The dose-dependent formation of Cr(III) complexes and uptake of chromium by Arthrobacter oxydans — a Gram-positive bacterium from contaminated Columbian basalt rocks (USA) — were studied along with the testing under aerobic
conditions of two bacterial strains of Arthrobacter genera isolated from the polluted basalts from the Republic of Georgia. Instrumental neutron activation analysis (INAA) was
used to track the accumulation of chromium in the bacterial cells. To monitor and identify Cr(III) complexes in these bacteria,
electron spin resonance (ESR) spectrometry was employed.