Here, a new medium, named intensive soil extract medium (ISEM), based on new soil extract (NSE) using 80% methanol, was used to efficiently isolate previously uncultured bacteria and new taxonomic candidates, which accounted for 49% and 55% of the total isolates examined (n = 258), respectively. The new isolates were affiliated with seven phyla (Proteobacteria, Acidobacteria, Firmicutes, Actinobacteria, Verrucomicrobia, Planctomycetes, and Bacteroidetes). The result of chemical analysis showed that NSE included more diverse components of low-molecular-weight organic substances than two conventional soil extracts made using distilled water. Cultivation of previously uncultured bacteria is expected to extend knowledge through the discovery of new phenotypic, physiological, and functional properties and even roles of unknown genes. IMPORTANCE Both metagenomics and single-cell sequencing can detect unknown genes from uncultured microbial strains in environments, and either method may find the significant potential metabolites and roles of these strains. However, such gene/genome-based techniques do not allow detailed investigations that are possible with cultures. To solve this problem, various approaches for cultivation of uncultured bacteria have been developed, but there are still difficulties in maintaining pure cultures by subculture.

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In order to measure the levels of serotonin (5-hydroxyltryptamine, 5-HT), dopamine (DA), 3,4-hydroxyphenylacetic acid (DOPAC), 3-methoxytyramine (3-MT) and homovanillic acid (HVA) simultaneously, an effective derivatization method followed by high-performance liquid chromatography (HPLC) coupled to electrochemical ionization mass spectrometry was used. The derivatization reaction of biological samples with ethyl chloroformate occurred rapidly at room temperature in aqueous conditions, and the resulting derivatives were isocratically separated with good selectivity using a C18 reversed-phase column within 30 min. The study results showed that the new derivatization procedure offers an excellent means of simultaneous determination of 5-HT, DA and their metabolites in mouse brain homogenates, which are important in a number of physiological and behavioral functions.Key words neurotransmitter; HPLC-MS; ethyl chloroformate; derivatization Dopamine (DA) and serotonin (5-hydroxyltryptamine, 5-HT), which are widely distributed as a monoamine neurotransmitter in the central nervous system (CNS), 1) play important physiological and behavioral roles such as the control and regulation of affective behavior, sleep, reward system, motor and cognitive functions.2) Changes of DA and 5-HT basal levels have been linked to some CNS-related disorders such as depression, panic disorder, schizophrenia and Parkinson's disease (PD).2,3) PD is pathologically characterized by the death of dopaminergic neurons in the substantia nigra pars compacta 4) and caused by neurotoxins such as 1-methyl-4-phenyl-1,2,3,6-tetrahypyridine (MPTP), which produces a clinical syndrome similar to PD. Because the administration of MPTP is known to affect the dopaminergic neurons in the nigrostriatal system with subsequent depletion of DA and its metabolites, 5,6) the measurement of 5-HT, DA and their metabolites is necessary to discover and test new drugs for treating these disorders. 7)Various analytical methods have been widely investigated for determining the level of monoamine neurotransmitters and their metabolites, including high-performance liquid chromatography (HPLC) coupled with electrochemical detection (ECD), dual ECD, fluorescence detection (FLD), FLD/ECD system, chemiluminescence detection and mass spectrometer (MS) and gas chromatography (GC) coupled with MS in biological samples such as plasma, rat brain, and mouse brain and in vitro experiments.8-10) HPLC-ECD, which is the most common analytical technique used to determine monoamines and their metabolites, has the advantages of low cost and fast speed 11) but maintaining its routine use is problematic due to sudden noise problems.12) The HPLC-FLD method is used to measure fluorescent derivatives of monoamine neurotransmitters by a derivatization procedure, 13) which was performed by benzylamine and 1,2-diphenylethylenediamine in a two-step reaction in weakly alkaline media and in the presence of potassium hexacyanoferrate (III).14) However, since this protocol requires the vigorous reaction ...