Improvement for isolation of soil bacteria by using common culture media.
Since Grobstein introduced the transfilter culture of metanephric mesenchymal cells in 1953 (2), membrane filters have proven to be an invaluable tool in experimental cell biology. They have been widely used as cell growth substrates to study cellular transport, absorption and secretion; for example porous transwell membranes were used for cultivation of human bronchial epithelial cells at the air/liquid interface (3) and for adhesion, invasion, and migration of SK-Hep1 human hepatoma cells (4). Successful application of transwell membranes in mammalian cell culture prompted their utilization as substrates for growth of bacteria from diverse habitats with the notion that transwell membranes would promote isolation of new microbes recalcitrant to traditional cultivation.
A previous study showed that utilization of soil substrate membrane system (SSMS) allowed for selection of previously uncultured soil bacteria (5). As a result, 8 dominant microcolonies were isolated after 7-day incubation using SSMS; only 1 of them could grow in subculture, confirming that SSMS-microcultivated organisms preferred a slow growth K-strategy (6). This observation was further substantiated in another study showing benefits of a polycarbonate membrane for recovery of microcolony-forming bacteria resistant to traditional growth conditions (7).
Based on these data, the current study presents a novel cultivation method for previously uncultured soil bacteria that is not only very simple but also productive for subsequent subcultivation. This method is based on utilization of a transwell permeable membrane as a growth support, soil as microbial source, and soil extract as the substrate, enabling isolation of previously uncharacterized bacterial species.
MATERIALS AND METHODS
Soil sampling and transwell plates
Soil was collected from the surface around plant roots in the forest at Kyonggi University, Suwon, Gyeonggi-do, South Korea. Samples were dried at room temperature, passed through a 2mm-mesh sieve to remove soil aggregates, gravels, and debris of plant materials, analysed for physic-chemical properties (Table 1), and used as a source of soil nutrients in cultivation medium. A subsample of soil was used as an inoculum, which was added to media in a transwell insert-this strategy ensured recovery of all bacteria present in the environment, as some microorganisms could be lost to sample preparation methods such as sieving. Note that we added medium to each transwell insert when it sank down, which ensured sufficient supply of nutrients for microbial growth, preventing membrane drying and subsequent bacterial death.
Transwell plates (Corning, Lowell, NY, USA) originally designed for cell culture, in this study were used for cultivating soil bacteria, especially strains resistant to traditional cultivation. Each 6-well plate was supplied with 6 24 mm-diameter transwell inserts that at the bottom had a 0.4-pm porous membrane permeable only for water-soluble soil nutrients, but not for soil particles or bacteria (Fig. 1A). Figures 1B and 1C show the schematic diagram and actual setting of transwell experiments, respectively.
Cultivation and isolation of soil bacteria
In the novel cultivation method, 3 g of soil were added to each well of a 6-well plate and covered with an insert; then 3 mL of each medium supplemented with 100 [micro]L of soil suspension (1 g-soil in 10 mL DW) was added to each insert. Plates were incubated in a shaking incubator (Hanbaek Science Co., Bucheon, Korea) at 120 rpm and 28[degrees]C for 2 weeks. During the incubation, the level of medium in each well was monitored every 2 days and restored to original volume as needed. After 2 weeks we collected the culture media and performed a 10-fold serial dilutions up to 10-6 using 15 mL test tubes; 100 [micro]L of each dilution was spread on an agar plate, and incubated at 28[degrees]C in an incubator (Vision Scientific, Daejeon, Korea) until colonies appeared. The number of colonies was counted and all different types of colonies were picked up and streaked on a plate for isolation of pure culture. As a control conventional cultivation method was used in which 3 grams of soil were added to 30 mL of each medium in 50 mL Erlenmeyer fask (the most popular liquid culture method in laboratory); incubation and isolation conditions and processes were the same as for the new method.
For cultivation we used 5 traditional media such as nutrient broth (NB), Luria Bertani (LB), tryptic soy broth (TSB), mineral salts medium (MSM), and R2A; soil extract (SE) and its mixtures MSMSE (500 mL MSM plus 500 mL SE) and R2ASE (500 mL R2A plus 500 mL SE); and distilled water (DW). SE was completed through the following steps: mixing soil (500 g) and DW (1 L) by stirring overnight, filtration, and centrifugation. Chemical composition of each medium is shown in Table 2.
Among isolates, the potential new species were stored as glycerol stocks and freeze-dry amples, resulting in regrowth afterward.
DNA extraction and 16S rRNA gene amplification
Bacterial genomic DNA was extracted from bacterial cells grown on agar plates using an InstaGene[TM] Matrix (BIO-RAD). Primers 518F-52 CCA GCA GCC GCG GTAATA CG-32 and 800R 52-TAC CAG GGT ATC TAA TCC-32 were used for the PCR amplification (EF-Taq, SolGent, Daejeon, Korea). Reactions were performed using 20 ng of genomic DNA as a template in 30-pl reaction mixture in following conditions: activation of Taq polymerase at 95[degrees]C for 2 min; 35 cycles of 95[degrees]C, 55[degrees]C, and 72[degrees]C for 1 min each; and a final elongation step at 72[degrees]C for 10 min.
Amplified products were purified with a multiscreen filter plate (Millipore Corp., Bedford, MA, USA) and sequenced using the PRISM BigDye Terminator v3.1 Cycle sequencing Kit. DNA samples were added to Hi-Di formamide (Applied Biosystems, Foster City, CA), and mixtures were incubated at 95[degrees]C for 5 min, followed by 5 min on ice and analysed using the ABI Prism 3730 XL DNA analyzer (Applied Biosystems, Foster City, CA).
Phylogenetic tree construction
The EzTaxon server was used to identify phylogenetic neighbours and to calculate the pairwise 16S rRNA gene sequence similarities (8). The related 16S rRNA sequences were obtained from GenBank database and edited with the BioEdit program (9). Multiple alignments were performed with the CLUSTAL_X program (10). A phylogenetic tree was constructed by applying evolutionary distance, parsimony, and bootstrapped parsimony using the neighbour-joining algorithm (11), maximum likelihood, and maximum parsimony method (12) in the MEGA5.03 program (13) with bootstrap value on 1000 replications (14).
RESULTS AND DISCUSSION
Isolation properties of the novel cultivation method
In this study, 68 soil-bacterial strains among total 151 bacterial isolates including many same strains were obtained from the same soil through the new and traditional cultivation methods using several conventional media and distilled water. Among the 68 strains (100%), 20 (29.4%) overlapped in both methods, i.e., only 1 strain (NHI-5) (1.5%) was obtained through the traditional while 47 strains (69.1%) through the new method (Table 3 and Fig. 2A). Thus, the new cultivation method appeared to be much more effective in isolation of soil bacteria. However, strain NHI-5 probably grew well in only synthetic media without the addition of soil nutrients as the new method.
Furthermore, only 5 potentially new species were isolated by the conventional method versus 14 new species obtained by the new method (Table 3). The 5 conventionally isolated species were also selected by the new method, indicating that the additional 9 isolates could be obtained only by using the new method. The potentially new species were identified based on the 16S rRNA gene sequence similarity of less than 98.5% with the closest phylogenetic neighbour. Thus, the new method appeared to be over 3 times more efficient for isolation of new microbial species than the traditional cultivation technique.
Among the 39 strains (including overlapped) obtained through the traditional method, the majority (12 strains) were isolated from the R2A cultures, 8 from LB, 7 from NB, 6 from TSB, 4 from MSM, and 1 from each of MSMSE and SE cultures (Fig. 2B). Among the 10 potentially new species (including overlapped) 3 were isolated from R2A, 2 from each of NB and TSB, and 1 from each of LB, MSMSE, and SE media. Among total of 112 strains (including overlapped) detected by the new method, the largest number of isolates, 31, was similarly obtained from the R2A cultures, while 17 were isolated from NB, 15 from DW, 13 from TSB, 12 from LB, 8 from MSM, 7 from MSMSE, 5 from SE, and 4 from R2ASE cultures (Fig. 2B). Among the 24 potentially new species (including overlapped) 7 were isolated from R2A, 4 from each of NB, TSB and DW, 2 from LB, and 1 from each of MSMSE, R2ASE, and SE media. These results indicate that among all the media tested R2A was the best for cultivation of diverse strains including potentially new species, either by the traditional or by the new method. The reason that R2A provides more diverse colonies seems to be the low concentration of carbon and energy sources (Table 2) as mentioned in the previous study regarding cultivation of uncultivable bacteria (1).
Otherwise, the total colony number was not distinguishable between two methods, but it depended on the kinds of media: 50-80 cfu in 100 [micro]L at [10.sup.-4] dilution in TSB, LB, NB and R2A; 30-60 cfu at [10.sup.-2] dilution in MSM, MSMSE and SE. This indicates that the new method influence only the diversity of colonies, not the number.
The modified new method developed in this study differs from the techniques used in previous studies (5,7) in that it promotes adaptation of cultivation-resistant soil bacteria to traditional culture media with continuous supply of soil micronutrients thereby facilitating continuous cultivation. An increased number of different strains successfully isolated by the new method using traditional media proved that this novel cultivation approach can be a useful tool for isolation and characterization of diverse soil bacteria.
Community structure analysis
All 68 strains are shown in a phylogenetic tree with their closest neighbours and are grouped according to phylum or class level (Fig. 3). They are affiliated with 4 phyla: Firmicutes, Actinobacteria, Proteobacteria, and Bacteroidetes. All isolated strains in Proteobacteria fall into three classes: a-Proteobacteria, [alpha]-Proteobacteria, and [??]-Proteobacteria. Most of the strains - 27 out of 68 -belong to Firmicutes, including 18 of 45 strains isolated exclusively by the new cultivation method. Other 24 strains (including 14 isolated by the new method) belong to Proteobacteria as the second most populated phylum; among them [alpha]-Proteobacteria comprises [11.sup.8], [alpha]-Proteobacteria [8.sup.5], and [gamma]-Proteobacteria 51, respectively. Fifteen strains were identified as members of Actinobacteria, among them 13 (87%) were isolated exclusively by the new method constituting the highest proportion among the 4 phyla. The last 2 strains belong to Bacteroidetes; they were isolated by both methods.
The distribution of potentially new species among Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, was 6, 5, 2, and 1, respectively. Among the 6 Proteobacteria strains, 5 belong to [alpha]-Proteobacteria and 1 to [gamma]-Proteobacteria. The 9 potentially new species isolated exclusively by the new method belong to Proteobacteria (4), Firmicutes (4), and Actinobacteria (1). Among the 4 Proteobacteria species, 3 belong to [alpha]-Proteobacteria, and 1 to [gamma]-Proteobacteria. These results indicate that the new method may be particularly useful for isolation of new species of soil bacteria related to Firmicutes and [alpha]-Proteobacteria.
According to distribution of the strains on the genus-level, the new cultivation method appears to be selective for specific genera (Fig. 4). It may improve those members to adapt synthetic media more than other genera by supply of soil nutrients in the beginning. The conventional method detected isolates of the following 16 genera: Bacillus (17.4%), Lysinibacillus (13.0%), Bosea (8.7%), Cupriavidus (8.7%), Achromobacter (4.3%), Arthrobacter (4.3%), Dyella (4.3%), Kitasatospora (4.3%), Mesorhizobium (4.3%), Niabella (4.3%), Pedobacter (4.3%), Pelomonas (4.3%), Pseudomonas (4.3%), Sporosarcina (4.3%), Staphylococcus (4.3%), and Stenotrophomonas (4.3%). The new method identified isolates belonging to Bacillus (19.7%), Lysinibacillus (7.6%), Arthrobacter (6.1%), Paenibacillus (6.1%), Mesorhizobium (4.5%), Pseudomonas (4.5%), Citrobacter (3.0%), Cupriavidus (3.0%), Dyella (3.0%), Methylobacterium (3.0%), Microbacterium (3.0%), Rhodococcus (3.0%), Serratia (3.0%), Staphylococcus (3.0%), Streptomyces (3.0%), Achromobacter (1.5%), Bosea (1.5%), Brevibacillus (1.5%), Burkholderia (1.5%), Enterobacter (1.5%), Kitasatospora (1.5%), Leucobacter (1.5%), Micrococcus (1.5%), Mycobacterium (1.5%), Niabella (1.5%), Nitrobacter (1.5%), Pedobacter (1.5%), Pelomonas (1.5%), Sporosarcina (1.5%), Stenotrophomonas (1.5%), and Tsukamurella (1.5%).
The strains of Bacillus and Lysinibacillus were isolated in relatively high proportion by both methods, while 4 strains of Paenibacillus and 1 or 2 strains in each of other 14 genera: Brevibacillus, Burkholderia, Citrobacter, Enterobacter, Leucobacter, Methylobacterium, Microbacterium, Micrococcus, Mycobacterium, Rhodococcus, Serratia, Streptomyces, Nitrobacter, and Tsukamurella could be obtained exclusively by the new method.
Many microbiologists have successfully cultivated many uncultivable bacteria by using modified or diluted media, and long incubation times; strains thus isolated belonged to 11 phyla such as Acidobacteria, Actinobacteria, Bacteroidetes, Rubrobacteridae, Chloroflexus, Firmucutes, Fusobacteria, Gemmatimonadetes, Planctomydetes, Proteobacteria, and Verrucomicrobia (1). Here, we succeeded in isolation representatives of only 4 of those phyla, suggesting that our new method can be complemented by incorporation of the aforementioned approaches in future investigations of phylogenetic diversity in soil bacterial communities.
This study was based on application of transwell membranes previously used in mammalian cell culture, for isolation and growth of soil bacteria resistant to traditional cultivation. This novel method proved efficient for cultivation of uncultivable and identification of new, bacterial species through adaptation to laboratory culturing conditions. The transwell membrane liquid culture technique provided isolation of higher number of strains including potentially new species than the traditional method suggesting that the novel method can be successfully utilized to study phylogenetic diversity of complex bacterial populations. This study also showed that R2A medium was more suitable for isolation of soil bacteria than other media such as LB, NB, TSB, MSM, and SE whether by the conventional or the new method. We believe that the use of modified and diluted media with the transwell membrane system would pave the way for more diverse bacterial isolates and new species in the future.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2011-0010144), and by the GAIA project (RE201202062) funded by Korea Environmental Industry & Technology Institute and Korean Ministry of Environment.
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Van H. T. Pham and Jaisoo Kim *
Department of Life Science, Graduate School of Kyonggi University, 154-42 Gwanggyosan-Ro, Youngtong-Gu, Suwon, Gyeonggi-Do 443-760, Republic of Korea.
(Received: 13 November 2015; accepted: 19 January 2016)
* To whom all correspondence should be addressed. Tel.: 31-249-9648; fax: 31-253-1165; E-mail: firstname.lastname@example.org
Caption: Fig. 1. Transwell plate system.
Caption: Fig. 3. Evolutionary phylogenetic tree based on 16S rRNA gene sequences showing the phylogenetic distribution of bacteria isolated from forest soil and their closest neighbors. Bootstrap percentages were based on 1000 replications and are shown at the branch points. Bar, 0.05 substitutions per nucleotide.
Caption: Fig. 4. Genetic diversity of microorganisms isolated from forest soil by the old and new cultivation methods.
Table 1. Physico-chemical properties of the soil sample used in this study Anions (mg/kg) Cl S[O.sub.4] Moisture Temperature content (%) ([degrees]C) 19 9 14.5 20 Anions (mg/kg) Soil Texture (%) Cl S[O.sub.4] Sand Silt Clay Texture Class 19 9 76 20 4 Loamy Sand Table 2. Compositions of media used in the new cultivation method Medium type (g/L distilled water-DW) No. Components NB LB TSB R2A 1 Beef extract 3 2 Peptone 3 Proteose Peptone 0.5 4 Tryptone 10 5 Acid digest of Casein 0.5 6 Yeast Extract 5 0.5 7 Enzymatic Digest of Gelatin 5 8 Enzymatic Digest of Casein 17 9 Enzymatic Digest of Soybean Meal 3 10 Soluble Starch 0.5 11 Sodium Pyruvate 0.3 12 Dextrose 2.5 0.5 13 Ca[Cl.sub.2] * 2[H.sub.2]O 14 K[H.sub.2]P[O.sub.4] 15 [K.sub.2]HP[O.sub.4] 2.5 0.3 16 MgS[O.sub.4] * 7[H.sub.2]O 0.05 17 NaCl 10 5 18 KN[O.sub.3] 19 Fe[Cl.sub.3] * 7[H.sub.2]O 20 [(N[H.sub.4]).sub.2]S[O.sub.4] 21 Trace element solution SL-6 (a) 22 Vitamin solution (b) 23 Soil extract Medium type (g/L distilled water-DW) No. MSM SE MSMSE R2ASE 1 500 mL 500 mL of MSM of R2A 2 3 4 5 6 7 8 9 10 11 12 13 0.02 14 2 15 2 16 0.2 17 0.4 18 1 19 0.01 20 2 21 1 mL 22 1 mL 23 1 L 500 500 mL mL (a) ZnS[O.sub.4]*7[H.sub.2]O, 0.1 g; Mn[Cl.sub.2] * 4[H.sub.2]O, 0.03 g; [H.sub.3]B[O.sub.3], 0.3 g; Co[Cl.sub.2]*6[H.sub.2]O, 0.2 g; Cu[Cl.sub.2]*2[H.sub.2]O, 0.01 g; Ni[Cl.sub.2]*6[H.sub.2]O, 0.02 g; [Na.sub.2]Mo[O.sub.4]*2[H.sub.2]O, 0.03 g; DW, 1000 ml. (b) biotin, 10 mg; nicotiamide, 35 mg; thiamine dicloride, 30 mg; p-aminobenzoic acid, 20 mg; pyridoxal chloride, 10 mg; Ca-pantothenate, 10 mg; vitamin B12, 5 mg; DW, 100 ml. Table 3. Bacterial strains isolated from forest soil by using the traditional and new cultivation methods using various traditional media. No Name of Closest type strains isolates 1 TSB11 Achromobacter spanius LMG [5911.sup.T] 2 EU-2 Arthrobacter oxydans DSM [20119.sup.T] 3 NHI-27 Arthrobacter parietis LMG [22281.sup.T] 4 NHI-19 Arthrobacter ramosus CCM [1646.sup.T] 5 EU-8 Arthrobacter ramosus CCM [1646.sup.T] 6 NHI-15 Bacillus anthracis ATCC [14578.sup.T] 7 R2ASE9 Bacillus anthracis ATCC [14578.sup.T] 8 R2A1 Bacillus aryabhattai [B8W22.sup.T] 9 NHI-1 Bacillus cereus ATCC [14579.sup.T] 10 SE2 Bacillus horikoshii DSM [8719.sup.T] 11 TSB1 Bacillus fordii [R-7190.sup.T] 12 Aii-TSB Bacillus fordii [R-7190.sup.T] 13 LB1 Bacillus fortis [R-6514.sup.T] 14 NHI-37 Bacillus licheniformis DSM [13.sup.T] 15 NHI-38 Bacillus methanolicus NCIMB [13113.sup.T] 16 AR-II-1 Bacillus methylotrophicus [CBMB205.sup.T] 17 NHI-10 Bacillus mycoides DSM [12442.sup.T] 18 NHI-16 Bacillus thuringiensis ATCC [10792.sup.T] 19 NHI-5 Bosea robittiae [LMG2638.sup.T] 20 NHI-8 Bosea thiooxidans [AJ25079.sup.T] 21 R2A2 Brevibacillus reuszeri NRRL NRS-[1206.sup.T] 22 EU-1 Burkhoideria stabils LMG [14294.sup.T] 23 NB2 Citrobacter famteri CDC 2991-[81.sup.T] 24 NB1 Citrobacter famteri CDC 2991-[81.sup.T] 25 NHI-6 Cupriavidus basilettsis CCUG [49340.sup.T] 26 NHI-14 Cupriavidus necator [N-1.sup.T] 27 NHI-48 Dyella japonica [XD53.sup.T] 28 NHI-12 Dyella terrae [JS14-6.sup.T] 29 R2A Enterobacter asburiae JCM [6051.sup.T] 30 NHI-20 Kitasatospora saccharophila [SK15.sup.T] 31 TSB3 Leucobacter iaritis [40.sup.T] 32 NHI-46 Lysinibacillus boronitolerans [10a.sup.T] 33 NB6 Lysinibacillus fusiformis NBRC [15717.sup.T] 34 TSB13 Lysinibacillus macroides LMG [18474.sup.T] 35 NB9 Lysinibacillus sphaericus C3-[41.sup.T] 36 NB11 Lysinibacillus xylanilyticus [XDB9.sup.T] 37 TSB12 Lysinibacillus xylanilyticus [XDB9.sup.T] 38 NHI-23 Mesorhizobium chacoense [Pr5.sup.T] 39 NB4 Mesorhizobium robiniae CCNWYC [115.sup.T] 40 LB10 Mesorhizobium shangrilense CCBAU [65327.sup.T] 41 NHI-34 Methylobacterium komagatae 002-[079.sup.T] 42 NHI-33 Methylobacterium oryzae [CBMB20.sup.T] 43 EU-7 Microbacterium natoriense TNJL143-[2.sup.T] 44 SEM-I-3 Microbacterium oleivorans [DSM16091.sup.T] 45 SEM-II-5 Micrococcus yunnanensis YIM [65004.sup.T] 46 SEM-II-6 Mycobacterium obuense ATCC [27023.sup.T] 47 NHI-24 Niabella tibetensis 15-[4.sup.T] 48 ET-1 Nitrobacter alkalicus [AN1.sup.T] 49 NHI-39 Paenibacillus alvei DSM [29.sup.T] 50 NB5 Paenibacillus nanensis MX2-[3.sup.T] 51 NHI-28 Paenibacillus pabuli JCM [9074.sup.T] 52 R2ASE5 Paenibacillus terrae AM141T 53 NHI-13 Pedobacterpanaciterrae Gsoil [042.sup.T] 54 NHI-21 Pelomonas puraquae CCUG [52769.sup.T] 55 TSB5 Pseudomonas beteli ATCC [19861.sup.T] 56 R2A7 Pseudomonas geniculata ATCC [19374.sup.T] 57 NHI-42 Pseudomonas koreensis Ps 9-[14.sup.T] 58 EU-6 Rhodococcus equi DSM [20307.sup.T] 59 NHI-47 Rhodococcus erythropolis [PR4.sup.T] 60 TSB8 Serratia marcescens subsp. sahtensis [KRED.sup.T] 61 TSB7 Serratia tiematodiphila [DZ0503SBS1.sup.T] 62 NHI-3 Sporosarcina koreensis [F73.sup.T] 63 NHI-11 Staphylococcus warneri ATCC [27836.sup.T] 64 II-5-3 Staphylococcus epidemidis ATCC [14990.sup.T] 65 NHI-22 Stenotrophomonas maltophilia ATCC [13637.sup.T] 66 NHI-49 Streptomyces althioticus NRRL B-[3981.sup.T] 67 NHI-35 Streptomyces xanthocidicus NBRC [13469.sup.T] 68 NHI-25 Tsukamurella pulmonis DSM [44142.sup.T] No Similarity Accession (%) number 1 99.93 AY170848 2 99.32 X83408 3 99.93 AJ639830 4 98.86 AM039435 5 99.06 AM039435 6 98.69 AB190217 7 100.00 AB190217 8 100.00 EF114313 9 99.86 AE017333 10 98.99 X76443 11 98.29 AY443039 12 99.48 AY443039 13 98.38 AY443038 14 100.00 AE017333 15 96.15 AB112727 16 99.90 EU194 897 17 99.02 ACMX01000133 18 99.78 ACNF010000156 19 99.64 FR774994 20 93.19 AJ250796 21 99.71 D78464 22 100 AF148554 23 98.45 AF025371 24 99.32 AF025371 25 99.93 FN597608 26 98.89 CP002878 27 99.86 AB110498 28 99.02 EU604273 29 99.78 AB004744 30 99.58 AB278568 31 99.79 AM040493 32 99.53 AB199591 33 99.32 AB271743 34 99.05 AJ628749 35 100 CP000817 36 98.44 FJ477040 37 99.26 FJ477040 38 97.79 AJ278249 39 97.34 EU849582 40 98.06 EU074203 41 100 AB252201 42 99.36 AY683045 43 99.93 AY566291 44 99.85 AJ698725 45 99.76 FJ214355 46 99.76 X55597 47 96.74 GU291295 48 96.66 AF069956 49 99.13 AJ320491 50 96.85 AB265206 51 100.00 AB073191 52 99.73 AF391124 53 98.26 AB245368 54 100.00 AM501439 55 99.79 AB021406 56 99.08 AB021404 57 99.66 AF468452 58 98.88 X80614 59 100.00 AP008957 60 99.87 AB061685 61 99.66 EU036987 62 99.72 DQ073393 63 100.00 L37603 64 99.86 L37605 65 99.29 AB008509 66 98.39 AY999791 67 99.71 AB184427 68 99.65 X92981 No Used Media Traditional New culture culture method method 1 LB, R2A LB. R2A. MSM 2 R2A 3 LB. NB. SE 4 MSM 5 SE. MSM 6 LB.TSB TSB. LB. MSM. NB. R2A 7 R2ASE 8 MSMSE 9 LB.TSB TSB. LB. DW(NA) 10 MSM 11 TSB 12 TSB 13 TSB 14 R2A. MSMSE 15 R2A 16 R2A 17 NB. R2A DW(R2A) 18 R2A R2A 19 LB. NB. TSB. R2A. MSM 20 NB. R2A NB. DW(R2A) 21 LB 22 R2A 23 R2A 24 R2A 25 LB DW(LB). TSB 26 R2A R2A. NB 27 R2A 28 R2A MSM. R2A 29 R2A 30 MSM DW(MSM), SE 31 LB 32 NB. R2A 33 NB. R2A 34 NB 35 NB 36 LB, TSB.R2A.NB TSB. LB. NB. R2A 37 LB. R2A, NB TSB. LB. NB. R2A 38 MSMSE, SE MSMSE. SE. R2A. NBLB 39 R2A 40 TSB 41 DW(MSM). SE 42 DW(MSM). R2A 43 R2A 44 MSMSE 45 MSMSE. R2A 46 MSMSE 47 TSB DW(TSA). DW(R2A) 48 R2A 49 TSB 50 R2A 51 NB. R2A 52 MSM 53 R2A NB. DW(R2A) 54 MSM MSM 55 TSB 56 R2A R2A. NB 57 R2A. NB 58 R2A 59 R2ASE 60 LB 61 LB 62 LB. NB. TSB, R2A TSB. LB. NB. R2A 63 NB TSB. NB. DW(R2A). DW(LB) 64 MSMSE 65 MSM R2ASE 66 R2ASE 67 R2A 68 DW(TSA).DW(LB). DW(MSM) Note: distilled water as a medium was added into one transwell insert, and soil samples were cultured for 2 weeks as described in Materials and Methods. The cultivated inoculum was transferred to agar plates containing TSA (tryptic soy agar), NA (nutrient agar), LB, MSM, and R2A media. Visible colonies obtained on these plates were named as DW(TSA), DW(NA), DW(LB), DW(MSM), and DW(R2A), respectively. The potential new species are bolded to be distinguished. Fig. 2. Comparison of bacteria isolation efficiency between the new and traditional cultivation methods entirely (A) and by each medium (B). Old culture method only 1.5% New culture method only 69.1% Both 29.4% Note: Table made from pie chart.
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|Author:||Pham, Van H.T.; Kim, Jaisoo|
|Publication:||Journal of Pure and Applied Microbiology|
|Date:||Mar 1, 2016|
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