ABSTRACT
The ciliate Paramecium bursaria harbors several hundred symbiotic algae in its cell and is widely used as an experimental model for studying symbiosis between eukaryotic cells. Currently, various types of bacteria and eukaryotic microorganisms are used as food for culturing P. bursaria; thus, the cultivation conditions are not uniform among researchers. To unify cultivation conditions, we established cloned, unfed strains that can be cultured using only sterile medium without exogenous food. The proliferation of these unfed strains was suppressed in the presence of antibiotics, suggesting that bacteria are required for the proliferation of the unfed strains. Indeed, several kinds of bacteria, such as Burkholderiales, Rhizobiales, Rhodospirillales, and Sphingomonadales, which are able to fix atmospheric nitrogen and/or degrade chemical pollutants, were detected in the unfed strains. The genetic background of the individually cloned, unfed strains were the same, but the proliferation curves of the individual P. bursaria strains were very diverse. Therefore, we selected multiple actively and poorly proliferating individual strains and compared the bacterial composition among the individual strains using 16S rDNA sequencing. The results showed that the bacterial composition among actively proliferating P. bursaria strains was highly homologous but different to poorly proliferating strains. Using unfed strains, the cultivation conditions applied in different laboratories can be unified, and symbiosis research on P. bursaria will make great progress.
ABSTRACT
To evaluate the reliability of our previously reported antimicrobial susceptibility test by ATP method, we have compared our ATP method to the reference test methods such as Mycobacteria Growth Indicator Tube(MGIT) method, minimum inhibitory concentration(MIC) method, NCCLS M24-T agar proportion method(M24-T method), and Vite spectrum method. The concentrations of drugs used for the assessment were isoniazid(INH) 0.1 microgram/ml, rifampicin(RFP) 2.0 micrograms/ml, ethambutol(EB) 2.5 micrograms/ml, streptomycin (SM) 2.0 micrograms/ml, and kanamycin (KM) 5.0 micrograms/ml. When six M. tuberculosis ATCC strains were subjected to 6 independent experiments by using ATP method, highly reproducible results were obtained on the fifth day of the incubation. We examined correlation among ATP method and reference test methods in drug susceptibility testing for 65 clinical isolates of M. tuberculosis. The correlation between ATP method and MGIT-, MIC-, M24-T method were more than 95% for all drugs. When ATP method and Vite spectrum method was compared, the correlation was 87.7% for INH, 98.5% for RFP, 90.8% for EB, 92.3% for SM, 96.9% for KM. The culture period for determining susceptibility between ATP method and MGIT method was compared by using ATCC reference strains and clinical isolates. Six M. tuberculosis ATCC strains were subjected to 6 independent experiments. By the MGIT method, 8 days were required to obtain the results, whereas 3 days were enough by the ATP method. For 65 clinical isolates, the MGIT method required 9 days for determining susceptibility of all isolates. The ATP method required only 5 days for the same strains. These data demonstrate that the improved ATP method that we reported, is simple, rapid, highly reproducible and nonradiometric, and could be used for the assessment of drug susceptibility for M. tuberculosis with high reliability.
