How Would Changing The Temperature At Which The Bags Were Incubated Affect The Results?
Appl Environ Microbiol. 2005 Feb; 71(2): 826–834.
Furnishings of Growth Medium, Inoculum Size, and Incubation Time on Culturability and Isolation of Soil Bacteria
Kathryn East. R. Davis
Section of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Commonwealth of australia
Shayne J. Joseph
Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia
Peter H. Janssen
Section of Microbiology and Immunology, University of Melbourne, Parkville, Victoria, Australia
Received 2004 Jul 9; Accustomed 2004 Sep seven.
Abstract
Soils are inhabited by many leaner from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Office of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil course visible colonies when inoculated onto standard microbiological media, resulting in low feasible counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did non appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did non consequence in the isolation of members of rarely isolated groups. Newly adult media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to three months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the utilize of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla Acidobacteria (subdivisions 1, 2, 3, and 4), Gemmatimonadetes, Chloroflexi, and Planctomycetes (including representatives of the previously uncultured WPS-1 lineage) too as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.
Soils contain phylogenetic groups of bacteria that are globally distributed and abundant in terms of the contributions of individuals of those groups to total soil bacterial communities (3, ten, 23). However, until recently, no representatives of many of these groups were available for detailed study due to their apparent inability to grow in or on laboratory media. Part of the reason for this is that simply a few (often just most 1%) of the >xix bacterial cells in each gram of soil seem able to form colonies on laboratory media (5, fourteen, 28). This ways that many groups of soil leaner cannot be easily studied due to the inability of microbiologists to grow representatives in the laboratory. Some isolates of these groups take recently been cultured by the utilise of new culture media and extended incubation periods to increment the numbers of colonies formed and by the choice of isolates from plates receiving just small inocula and yielding only pocket-sized numbers of colonies (12, 15, 24). These approaches were chosen empirically in previous studies. Among the isolates obtained were many members of the phylum Acidobacteria as well every bit some members of the phyla Verrucomicrobia and Gemmatimonadetes and of the subclasses Acidimicrobidae and Rubrobacteridae of the phylum Actinobacteria (12, 15, 24). These groups are very poorly studied due to the paucity of cultured representatives from soils.
The aim of the present study was to investigate the effects of growth medium, inoculum size, colony density, and incubation fourth dimension on the appearance of colonies of members of these poorly studied groups of soil leaner on plates of solid growth media inoculated with soil.
MATERIALS AND METHODS
Bacterial strains.
Pseudomonas aeruginosa 185 (ATCC 10145), Pseudomonas fluorescens 192 (ATCC 13525), Bacillus megaterium 4R6259 (ATCC 9885), Bacillus subtilis (ATCC 11774), Sphingomonas paucimobilis CL1/70 (ATCC 29837), Xanthomonas campestris GB296, Chromobacterium violaceum MK (ATCC 12472), and Kocuria rhizophila PCI 1001 (ATCC 9341) were obtained from the collection of the Section of Microbiology and Immunology, Academy of Melbourne. These cultures were maintained on food agar (run across beneath) and used to exam the quality of civilisation media. Their identities were confirmed past comparisons of parts of their 16S rRNA genes, after amplification and sequencing (run into below), to sequences in GenBank (www.ncbi.nlm.nih.gov) by the utilise of BLAST web-based software (1).
50 isolates from our laboratory collection were used in experiments to determine the power of members of different groups to grow on dissimilar culture media. These were isolated from soil from the same sample site during several earlier investigations (12, xv, 24; C. A. Osborne and P. H. Janssen, unpublished data; P. Sangwan and P. H. Janssen, unpublished information) or were isolated during this study. These isolates were affiliated with nine unlike bacterial phyla and were selected so that, at most, ii came from any single family unit-level group (Table 1).
Tabular array i.
Isolates used for experiments to determine the ability of members of dissimilar groups to grow on different culture media
| Phylum | Form, subclass, or subdivision | Family | Isolates (GenBank accession no.) |
|---|---|---|---|
| Acidobacteriaa | Subdivision one | Ellin345/WD217 grouping | Ellin345 ({"type":"entrez-nucleotide","attrs":{"text":"AF498727","term_id":"24021226"}}AF498727) |
| Subdivision 1 | Ellin323/DA038 group | Ellin7137 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673303","term_id":"56683160"}}AY673303) | |
| Subdivision 1 | Ellin337/P18 group | Ellin337 ({"type":"entrez-nucleotide","attrs":{"text":"AF498719","term_id":"24021218"}}AF498719), Ellin5236 ({"type":"entrez-nucleotide","attrs":{"text":"AY234587","term_id":"37961744"}}AY234587) | |
| Subdivision 1 | Ellin351/TM2 grouping | Ellin408 ({"type":"entrez-nucleotide","attrs":{"text":"AF432237","term_id":"20530611"}}AF432237) | |
| Subdivision 1 | UA1/UA2 grouping | Ellin5413 ({"type":"entrez-nucleotide","attrs":{"text":"AY673140","term_id":"56683294"}}AY673140), Ellin7225 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673391","term_id":"56683248"}}AY673391) | |
| Subdivision 3 | Ellin342/MC9 group | Ellin6076 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY234728","term_id":"37961885"}}AY234728) | |
| Actinobacteria | Acidimicrobidaea | "Microthrixaceae" | Ellin5247 ({"type":"entrez-nucleotide","attrs":{"text":"AY234598","term_id":"37961755"}}AY234598), Ellin5273 ({"type":"entrez-nucleotide","attrs":{"text":"AY234624","term_id":"37961781"}}AY234624) |
| Actinobacteridae | Ellin306/WR160 group | Ellin5004 ({"type":"entrez-nucleotide","attrs":{"text":"AY234421","term_id":"37961578"}}AY234421), Ellin5284 ({"type":"entrez-nucleotide","attrs":{"text":"AY234635","term_id":"37961792"}}AY234635) | |
| Actinobacteridae | Ellin5034 group | Ellin5423 ({"type":"entrez-nucleotide","attrs":{"text":"AY673150","term_id":"56683304"}}AY673150) | |
| Actinobacteridae | Geodermatophilaceae | Ellin5211 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY234562","term_id":"37961719"}}AY234562) | |
| Actinobacteridae | Intrasporangiaceae | Ellin5432 ({"type":"entrez-nucleotide","attrs":{"text":"AY673159","term_id":"56683313"}}AY673159) | |
| Actinobacteridae | Microbacteriaceae | Ellin5407 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673134","term_id":"56683288"}}AY673134) | |
| Actinobacteridae | Micromonosporaceae | Ellin5430 ({"type":"entrez-nucleotide","attrs":{"text":"AY673157","term_id":"56683311"}}AY673157), Ellin6203 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673162","term_id":"56683316"}}AY673162) | |
| Actinobacteridae | Mycobacteriaceae | Ellin5292 ({"type":"entrez-nucleotide","attrs":{"text":"AY234643","term_id":"37961800"}}AY234643), Ellin5409 ({"type":"entrez-nucleotide","attrs":{"text":"AY673136","term_id":"56683290"}}AY673136) | |
| Actinobacteridae | Nocardiaceae | Ellin5404 ({"type":"entrez-nucleotide","attrs":{"text":"AY673131","term_id":"56683285"}}AY673131) | |
| Actinobacteridae | Nocardioidaceae | Ellin5426 ({"type":"entrez-nucleotide","attrs":{"text":"AY673153","term_id":"56683307"}}AY673153), Ellin6206 ({"type":"entrez-nucleotide","attrs":{"text":"AY673165","term_id":"56683319"}}AY673165) | |
| Actinobacteridae | Nocardiopsaceae | Ellin5291 ({"type":"entrez-nucleotide","attrs":{"text":"AY234642","term_id":"37961799"}}AY234642) | |
| Actinobacteridae | Pseudonocardiaceae | Ellin5419 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673146","term_id":"56683300"}}AY673146) | |
| Rubrobacteridaea | "Thermoleophilaceae" | Ellin7179 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673345","term_id":"56683202"}}AY673345), Ellin7228 ({"type":"entrez-nucleotide","attrs":{"text":"AY673394","term_id":"56683251"}}AY673394) | |
| Bacteroidetes | Sphingobacteria | Flexibacteraceae | Ellin5402 ({"type":"entrez-nucleotide","attrs":{"text":"AY673129","term_id":"56683283"}}AY673129), Ellin5420 ({"type":"entrez-nucleotide","attrs":{"text":"AY673147","term_id":"56683301"}}AY673147) |
| Sphingobacteria | Sphingobacteriaceae | Ellin6201 ({"type":"entrez-nucleotide","attrs":{"text":"AY673160","term_id":"56683314"}}AY673160), Ellin6205 ({"type":"entrez-nucleotide","attrs":{"text":"AY673164","term_id":"56683318"}}AY673164) | |
| "Deinococcus-Thermus" a | Deinococci | Deinococcaceae | Ellin6033 ({"type":"entrez-nucleotide","attrs":{"text":"AY234685","term_id":"37961842"}}AY234685) |
| Firmicutes | "Bacilli" | Bacillaceae | Ellin5411 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673138","term_id":"56683292"}}AY673138) |
| Gemmatimonadetesa | Subdivision 1 | Gemmatimonadaceae | Ellin5290 ({"type":"entrez-nucleotide","attrs":{"text":"AY234641","term_id":"37961798"}}AY234641) |
| Planctomycetesa | "Isosphaerae" | "Isosphaeraceae" | Ellin6059 ({"type":"entrez-nucleotide","attrs":{"text":"AY234711","term_id":"37961868"}}AY234711) |
| WPS-one | Ellin7224/WD2101 group | Ellin6207 ({"type":"entrez-nucleotide","attrs":{"text":"AY673166","term_id":"56683320"}}AY673166), Ellin7224 ({"type":"entrez-nucleotide","attrs":{"text":"AY673390","term_id":"56683247"}}AY673390) | |
| Proteobacteria | Alphaproteobacteria | Ellin314/wr0007 group | Ellin5002 ({"type":"entrez-nucleotide","attrs":{"text":"AY234419","term_id":"37961576"}}AY234419), Ellin5238 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY237589","term_id":"37931288"}}AY237589) |
| Alphaproteobacteria | Beijerinckiaceae | Ellin5427 ({"type":"entrez-nucleotide","attrs":{"text":"AY673154","term_id":"56683308"}}AY673154) | |
| Alphaproteobacteria | Bradyrhizobiaceae | Ellin5405 ({"type":"entrez-nucleotide","attrs":{"text":"AY673132","term_id":"56683286"}}AY673132), Ellin5431 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673158","term_id":"56683312"}}AY673158) | |
| Alphaproteobacteria | Phyllobacteriaceae | Ellin5412 ({"type":"entrez-nucleotide","attrs":{"text":"AY673139","term_id":"56683293"}}AY673139) | |
| Betaproteobacteria | Burkholderiaceae | Ellin5403 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673130","term_id":"56683284"}}AY673130), Ellin5406 ({"type":"entrez-nucleotide","attrs":{"text":"AY673133","term_id":"56683287"}}AY673133) | |
| Betaproteobacteria | Comamonadaceae | Ellin5418 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673145","term_id":"56683299"}}AY673145), Ellin5428 ({"type":"entrez-nucleotide","attrs":{"text":"AY673155","term_id":"56683309"}}AY673155) | |
| Gammaproteobacteria | Xanthomonadaceae | Ellin5401 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY673128","term_id":"56683282"}}AY673128), Ellin5421 ({"type":"entrez-nucleotide","attrs":{"text":"AY673148","term_id":"56683302"}}AY673148) | |
| Verrucomicrobiaa | "Spartobacteria" | "Chthoniobacteraceae" | Ellin428 ({"blazon":"entrez-nucleotide","attrs":{"text":"AY388649","term_id":"38679256"}}AY388649) |
Soil sampling.
Soil cores (100-mm long, 25-mm diameter) were nerveless past use of a soil corer from control paddock L2 at the Dairy Research Institute, Ellinbank, Victoria, Australia (38°14.55′Due south, 145°56.11′E). The soil characteristics and direction regimen of the paddock accept been described elsewhere (25, 27). Intact soil cores were transported to the laboratory in aluminum trays enclosed in polyethylene bags at the ambient temperature and were processed within 3 h of collection. The collection dates were 2 March 2001, v April 2001, and 29 May 2003. Unless noted otherwise, experiments were performed with soil from the last sampling time. The upper 30 mm of each cadre was discarded, and large roots and stones were removed from the remainder, which was then sieved through an autoclave-sterilized contumely sieve with a 2-mm discontinuity size (Endecotts Ltd., London, United kingdom of great britain and northern ireland), mixed, and used immediately for microscopic investigations, dry weight determinations, or cultivation experiments. The numbers of cells in mixed soil samples were determined by microscopic counts of preparations stained with iv′,half dozen′-diamidino-2-phenylindole plus acridine orange (South. Northward. Cairnduff and P. H. Janssen, unpublished information). Aliquots of freshly sieved soil were accurately weighed then dried at 105°C for three days. The samples were then reweighed later on first being allowed to cool to room temperature in a desiccator. The conversion factor of fresh to dry out weight for soil was calculated, and all results are expressed per gram of dry out soil.
Media.
VL55 medium solidified with gellan and containing different growth substrates was prepared every bit described past Joseph et al. (15). These growth substrates and their final concentrations in the solidified medium were as follows: two mM N-acetyl-glucosamine; a mix of d-glucose, d-galactose, d-xylose, and l-arabinose (0.5 mM [each]) (15); a mix of d-galacturonate, d-glucuronate, l-ascorbate, and d-gluconate (0.5 mM [each]) (xv); a mix of acetate, benzoate, l-lactate, and methanol (0.5 mM [each]) (15); an amino acid mix (nine) with an addition of 0.08 g of l-tryptophan per 100 ml of stock solution, added at 10 ml of stock solution per liter of medium; 0.05% (wt/vol) sodium alginate; 0.05% (wt/vol) xanthan; 0.05% (wt/vol) pectin; 0.05% (wt/vol) xylan (VXylG); and 0.05% (wt/vol) carboxymethylcellulose. VL55 medium with agar as the solidifying agent and with either 0.05% (wt/vol) xylan (medium VXylA) or x mM glucose (VGluA) as the growth substrate was prepared as described by Sait et al. (24). Glucose was added from a 1 Yard stock solution that was sterilized by filtration (0.22-ÎĽm-pore-size filter). Dilute nutrient goop, solidified with agar (DNBA) or with gellan (DNBG), was prepared as described by Janssen et al. (12). Cold-extracted soil excerpt agar (CSEA), Winogradsky'due south salt-solution agar (WSA), and 10-fold-diluted tryptone soy agar (0.1× TSA) were prepared equally described by Joseph et al. (15). Nutrient agar was prepared with 8 yard of Difco nutrient broth (BD Diagnostic Systems, Sparks, Md.) and fifteen one thousand of bacteriological agar no. 1 (Oxoid) per liter of distilled water and had a final pH of approximately 6.0.
All media were used in xc-mm-diameter polystyrene petri dishes.
Cultivation experiments.
An accurately weighed sample of freshly sieved soil (approximately 1 g) was dispersed in 100 ml of sterile distilled water before one-ml aliquots were treated by sonication as described elsewhere (12). These x−two-diluted aliquots were serially diluted to 10−3, 10−4, x−five, x−half-dozen, and 10−7 (12), and 200 ÎĽl each of the last three dilutions was used to inoculate each of 3 or five replicate plates at each dilution level to constitute a counting set of ix or 15 plates. Inocula were spread over the surface of the agar- or gellan-containing medium by the use of sterile glass spreading rods. Three or seven counting sets were prepared on each medium for each soil sample. All one,170 plates were incubated at 25°C in the night for xvi weeks in sealed polyethylene bags (40-ÎĽm film thickness).
Viable counts are expressed relative to the dry weight of the soil. Expected inoculum sizes (expressed as numbers of cells per plate) were calculated from the microscopically determined total counts of cells in the soil, the dilutions made, and the volumes of diluted inocula spread onto the plates. Culturability was defined as the viable count expressed as a percentage of the microscopically determined total count of cells for the sample used in that detail cultivation experiment.
Colony formation was monitored by examining plates later on 3 days, after 7 days, and then at farther 7-twenty-four hour period intervals by using a magnifying lens with a magnification of ×2. When the rate of visible colony formation was being assessed, the midpoint of the week was deemed to be the time of colony germination (1.5 days if colonies appeared within iii days). However, almost identical results were obtained if the start or end of these fourth dimension intervals was used for the calculations.
Student's t exam (two-tailed), analysis of variance (ANOVA), and the χ2 test were performed with Excel 2001 software (Microsoft Corp., Redmond, Launder.).
Identification of isolates.
Colonies were selected randomly and subcultured on VGluA, 0.1× TSA, WSA, CSEA, or DNBA. Partial 16S rRNA cistron sequences (≥400 nucleotides [nt]) were adamant as described by Joseph et al. (15). Isolates were identified by obtaining fractional 16S rRNA gene sequences (401 to 1,452 nt) and then using BLAST to compare these sequences to those in the GenBank database (24). The identification criteria used were those described by Joseph et al. (xv). The nomenclature of phylogenetic and taxonomic groupings by and large follows that of Garrity et al. (8), except for the subdivisions of the phyla Acidobacteria and Gemmatimonadetes, which follow the schemes of Hugenholtz et al. (10) and Zhang et al. (29), respectively, and the class-level groupings (subdivisions) of the phylum Planctomycetes, which are based on the scheme of Fuerst et al. (7). The WPS-ane subdivision of the phylum Planctomycetes was named by Nogales et al. (twenty). For simplicity in grouping isolates, we assumed an estimate subphylum rank equivalence of classes and subdivisions and used the subclasses of the phylum Actinobacteria as similar subphylum groupings. Family unit-level groupings follow the scheme used past Joseph et al. (15).
RESULTS
Minimum colony number per plate.
Nosotros performed counts of CFU of bacteria that were able to class visible colonies within 12 weeks of inoculation. Soil samples were diluted in water, and aliquots from different dilutions were plated onto various media in counting sets with either three (2001 soil samples) or five (2003 soil sample) replicate plates at each inoculum size (dilution level). As expected, the amount of variation in colony numbers between replicate plates in a counting prepare increased when the hateful number of colonies on those three or five plates decreased (Fig. 1A). The coefficient of variation (standard deviation divided by the mean) was relatively constant, with a hateful of 19.7% for dilution levels in counting sets in which the mean number of colonies per plate was ≥40 (north = 57 sets of three or five plates). Dilution levels with mean colony numbers of <40 per plate had larger coefficients of variation (hateful = 52%; maximum = 173%; n = 160) which were also more variable between unlike counting sets.
(A) Variability in colony numbers of replicates inside counting sets relative to mean colony number per plate for each counting ready. The variability is expressed as the coefficient of variation (SD/mean) for the replicates (3 or five) at whatsoever i inoculum size within any one counting set. (B) Comparison of colony numbers on plates inoculated with soil suspensions with 10-fold differences in theinoculum size. The numbers of colonies on plates with the larger inoculum are plotted on the ten axis, and the numbers of colonies on the corresponding plates with a x-fold smaller inoculum in the same counting set are plotted on the y axis. The diagonal line represents the human relationship expected between the numbers of colonies forming on the plates, assuming a 10-fold reduction in colony number with a 10-fold smaller inoculum. (C) Enlargement of the data from the blank lower left section of panel B. Symbols for all panels: •, data from experiments performed in 2001, with each indicate on all panels representing the consequence from three replicate plates at each dilution level; ○, data from experiments performed in 2003, with each signal representing the upshot from 5 replicate plates at each dilution level.
Consequence of inoculum size.
The expected 10-fold reduction in colony number after 12 weeks of incubation did not occur when 10-fold smaller inocula were used. Instead, there was an approximately 5-fold reduction (5.two-fold for 2001 experiments and 5.7-fold for 2003 experiments) in colony number for each ten-fold reduction in inoculum size. The mean colony number was therefore ii-fold higher than that expected from the numbers of colonies on plates in the same counting set, simply with a 10-fold larger inoculum (Fig. 1B). Only at very low colony counts was this effect less apparent (Fig. 1C). As a upshot, viable counts on all media were larger when a more dilute inoculum was used to calculate the viable count, and this result was statistically significant (P ≤ iii × 10−4 for dissimilar inocula in paired t tests and n = 60 counting sets of three dilution levels each, with three replicates per dilution level, for data from 2001; P ≤ 4 × 10−4 and n = 42 counting sets of three dilution levels each, with five replicates per dilution level, for data from 2003).
Figure 2A shows the increased viable counts (P ≤ 5 × ten−half-dozen for comparisons of successive dilution levels) with decreasing inoculum sizes for 3 media that resulted in large counts. Straight epifluorescence microscopic counting of cells (at least xxx fields for each of fifteen subsamples of sieved and mixed soil) showed that there were 1.28 × 109 (standard deviation [SD] = 5.03 × 108) cells per g of dry soil in the soil sample used for this experiment. This allowed u.s. to calculate a mean expected inoculum size for each dilution level. The mean culturability, which is the number of CFU expressed equally a percentage of the number of cells in the inoculum, increased as the inoculum size decreased (Table two). The kinetics of colony development, yet, were not different for the unlike inoculum sizes (Fig. 2B).
(A) Viable counts at different inoculum sizes (dilution levels) for three dissimilar media after 12 weeks of incubation. Symbols: ○, DNBG; □, VXylA; ▵, VXylG. Each point represents the mean of v replicate plates. The thick horizontal line indicates the mean, and the vertical lines indicate one standard deviation from the mean. (B) Increase in feasible counts with incubation time at three different inoculum sizes. •, inoculum of 1,780 cells per plate; ▪, inoculum of 178 cells per plate; ▴, inoculum of 17.8 cells per plate. Data are pooled results obtained with DNBG, VXylA, and VXylG. The results from each counting set were calculated as a per centum of the 12-week count for that counting fix, and each point represents the mean of three media, with each used for vii counting sets, each of which in turn was made of five replicate plates. For clarity, standard errors are not shown; the mean standard error for all points was 12.3% of the values plotted.
Tabular array two.
Effect of inoculum size on culturability after incubation for 12 weeks and on the number of bacteria affiliated with rarely isolated groups that formed colonies in week 8 or after plates of DNBG, VXylA, and VxylG
| Expected inoculum size (cells/plate) | Mean no. of colonies/plate | Mean culturability (%) | No. of isolates identified | % of isolates affiliated with rarely isolated groups |
|---|---|---|---|---|
| 1,780 | 140.3 | 7.ix | 37 | 43 |
| 178 | 24.vii | 13.9 | xvi | 25 |
| 17.8 | 4.2 | 23.vi | xvi | 31 |
Based on these results, we decided to summate viable counts from plates at the near dilute inoculum that yielded a minimum of ten colonies per plate, averaged over three or 5 replicate plates at that dilution level. To overcome the increased variability with these colony numbers, we prepared multiple counting sets for each blazon of medium.
Selection of growth substrate.
The viable counts obtained with VL55 medium, with gellan equally the solidifying amanuensis and with each of x different additional growth substrates or substrate mixes, ranged from 5.0 × 107 to 6.3 × 108 CFU per k of dry soil in individual counting sets later incubation for 12 weeks at 25°C. The viable counts obtained from two different soil cores (in March and Apr 2001) were not significantly dissimilar (P = 0.13 by a paired t examination). The choice of growth medium had a detectable consequence on the viable counts obtained (P = 7 × 10−4 past single-nomenclature ANOVA). The largest mean count was obtained with xylan as the growth substrate (Fig. three), and xylan was therefore called for further experiments.
Mean viable counts after 12 weeks of growth on VL55 medium with different growth substrates. Each solid bar represents the mean of iii counting sets prepared from the March 2001 soil sample and three counting sets prepared from the April 2001 soil sample. The error bars betoken standard errors. Abbreviations: CMC, carboxymethylcellulose; NAG, N-acetyl-glucosamine; AA, amino acid mix; GGAG, mix of d-galacturonate, d-glucuronate acid, l-ascorbate, and d-gluconate; GGXA, mix of d-glucose, d-galactose, d-xylose, and l-arabinose; ABLM, mix of acetate, benzoate, l-lactate, and methanol.
Effect of incubation time and medium on viable count.
The effects of incubation time and the choice of medium were investigated by determining the numbers of colonies that were visible on plates of half-dozen dissimilar media at weekly intervals. The viable counts were significantly different betwixt media, even after only 1 week of incubation (P = 7 × 10−7 by single-nomenclature ANOVA), and this continued for the entire 12 weeks (at 12 weeks, P = 4 × 10−8). Counts with 0.1× TSA reached their maximum afterwards 2 weeks of incubation (Fig. 4), and beyond ane week, the increase in viable counts with time was not statistically significant (P = 0.75 by repeated-measures ANOVA). The use of CSEA and WSA resulted in increasing colony counts with increasing incubation times (P = 3 × 10−3 and 7 × 10−iv, respectively, by repeated-measures ANOVA for weeks 1 to 12), and the final counts were higher than those obtained with 0.ane× TSA (P = 2 × 10−iii and 0.016, respectively, by Student's t test). The media based on VL55 with xylan as the growth substrate (VXylA and VXylG) and DNBG resulted in fifty-fifty higher colony counts (Fig. 4), and these continued to increment over the 12-week incubation period (P < 6 × 10−5 past repeated-measures ANOVA). Fifty-fifty subsequently 12 weeks, the numbers of colonies on the 2 media based on VL55 medium connected to increase. The analyses that follow were performed with colonies appearing in the first 12 weeks. There was no statistical support for differences between the counts obtained on DNBG, VXylA, and VXylG (P > 0.48 by Educatee's t test). Still, VXylG (solidified with gellan) resulted in counts that were 15% college than when the same medium was used with agar as the solidifying agent (VXylA). Culturability after incubation for 12 weeks ranged from a hateful of one.5% of the microscopically adamant total cell count on 0.1× TSA to a mean of 15% on DNBG or VXylG. The media DNBG, VXylA, and VXylG all resulted in higher feasible counts than did the media 0.1× TSA, CSEA, and WSA (P = 3 × 10−four to 0.027 by t-test comparisons between counts on each medium). The continued increment in colony numbers with extended incubation times over the 12-week incubation period was also observed with all of the media shown in Fig. 3 that were based on medium VL55 with gellan as the solidifying agent but with different growth substrates (information non shown).
Increases in feasible counts on dissimilar media with increasing incubation times. Symbols: •, 0.1× TSA; ▪, WSA; ▴, CSEA; ▵, VXylG; □, VXylA; ○, DNBG. Each indicate represents the mean of seven experiments, each of which included five replicate plates. For clarity, the standard errors are not shown; the hateful standard error of all the points was 14.5% of the values plotted.
Identities of isolates.
A total of 250 colony-forming bacteria that grew on plates inoculated with the soil sample from May 2003 were identified on the basis of their 16S rRNA gene sequences. Of these, 212 appeared during the outset week, the fourth and fifth weeks, and the eighth week or after on plates that received an expected inoculum of 17.8 or 178 cells each. Twelve of these were derived from six mixed colonies that were successfully separated into two pure cultures each. The other 38 colonies were randomly selected from those that appeared during week 8 or later on plates receiving an expected inoculum of 1,780 cells each. Of these 250 isolates, 249 were affiliated with eight bacterial phyla (Table 3). The remaining isolate, which was phylogenetically affiliated with the eukaryotic algal family Trebouxiophyceae, was identified on the basis of the sequence of a 16S rRNA gene from its plastids. This isolate was not included in subsequent analyses. A list of all of the isolates obtained in this study detailing their phylogenetic affiliations, GenBank accession numbers of their 16S rRNA cistron sequences, isolation media, and times of colony appearance is available upon request.
TABLE iii.
Phylogenetic affiliations of 250 isolates cultured from soil for this study
| Phylum | Grade, subclass, or subdivision | No. of isolates (on all six media) | No. of isolates appearing on indicated medium | ||
|---|---|---|---|---|---|
| DNBG | VXylA | VXylG | |||
| Acidobacteriaa | Subdivision 1 | ix | 1 | 5 | three |
| Subdivision 2 | 1 | i | |||
| Subdivision 3 | 5 | 4 | ane | ||
| Subdivision 4 | ane | ane | |||
| Actinobacteria | Acidimicrobidaea | two | 2 | ||
| Actinobacteridae | 119 | 19 | 16 | 22 | |
| Rubrobacteridaea | vii | 6 | one | ||
| Bacteroidetes | Flavobacteria | 1 | |||
| Sphingobacteria | 5 | two | 3 | ||
| Chloroflexia | Ellin7237 lineage | 1 | 1 | ||
| Firmicutes | "Bacilli" | vii | |||
| Gemmatimonadetesa | Subdivision 1 | 1 | 1 | ||
| Planctomycetesa | "Gemmatae" | one | 1 | ||
| "Isosphaerae" | one | one | |||
| WPS-i | 2 | 2 | |||
| Proteobacteria | Alphaproteobacteria | 64 | 17 | 15 | 12 |
| Betaproteobacteria | 19 | 3 | 1 | 3 | |
| Gammaproteobacteria | 3 | one | 1 | ||
| Viridiplantae | Chlorophyta | ane | |||
Some colonies were observed to spread rapidly over the entire plate within 1 week of inoculation with diluted aliquots of soil. These occurred mainly on plates containing 0.1× TSA (on 29 of 105 plates) just were besides observed on DNBG (on 2 of 105 plates) and VXylG (on ane of 105 plates). Two of these colonies were part of the collection of 250 isolates, and both were members of the family unit Bacillaceae. A farther eight spreading colonies that were not role of the primary isolate drove were selected from the plates. Five of these were members of the family Bacillaceae, two were members of the family unit Flexibacteriaceae, and ane was a member of the family Paenibacillaceae. These x spreading isolates consistently displayed this phenotype when subcultured on 0.i× TSA (all 10), DNBA (8 of 10), CSEA (5 of 10), or WSA (iv of 9; one could non grow on this medium). However, only 2 of ix displayed a spreading phenotype when grown on a medium (VGluA) based on VL55 (ane isolate could not grow on this medium).
Effect of incubation time and medium on cultured groups.
We compared the appearances of isolates from unlike phylogenetic groups at different time points. To practice this comparison, we divided the 212 isolates from the terminal growth-positive plates of counting sets into the following ii categories: (i) isolates affiliated with commonly isolated groups that are well represented by cultured representatives, i.due east., members of the subclass Actinobacteridae of the phylum Actinobacteria and members of the phyla Proteobacteria (classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria), Bacteroidetes, and Firmicutes, and (ii) isolates affiliated with other groups that are simply rarely isolated from soil (Tabular array 3). This was a very conservative separation, as many members of the commonly isolated category were affiliated with poorly studied families with few reported isolates. For example, 14 (12%) of the 119 members of the Actinobacteridae and 11 (13%) of the 86 members of the Proteobacteria were members of as yet undescribed families (three and four families in the Actinobacteridae and Proteobacteria, respectively).
The number of isolates that belonged to rarely isolated groups increased from 0 to 16% of the isolates with increasing incubation times (Table four), and a χtwo test with the pooled data from all six media suggested that incubation time was a meaning factor for obtaining isolates from these groups (P = vii × x−3 past the χ2 test). The medium also influenced the numbers of members of rarely isolated groups that were isolated (Table five) (P = 2 × ten−three by the χ2 test). None appeared on the 0.ane× TSA, WSA, and CSEA media. When but the results from the three media on which members of rarely isolated groups appeared were analyzed, there was no observable issue of medium (P = 0.19 by the χ2 examination), but the incubation time however had a significant effect (P = 5 × 10−3 by the χtwo test). Members of rarely isolated groups were isolated from all three of these media in the 4- to five-week and 8- to 12-week periods (Tables 4 and 5). Ten of the eleven isolates of the phylum Proteobacteria that represented new families appeared at week 8 or subsequently. This was not the case for the xiv isolates of the bracket Actinobacteridae that represented new families, with just one appearing after week eight.
TABLE four.
Isolates of leaner that formed visible colonies at dissimilar times on terminal plates of counting sets inoculated with soil
| Time of colony advent [wk(s)] | No. of isolates identified | % of isolates affiliated with rarely isolated groups | Groups represented (no. of isolates on all media) | ||
|---|---|---|---|---|---|
| All media | DNBG VXylA, and VXylG | All media | DNBG, VXylA, and VXylG | ||
| i | 71 | 36 | 0 | 0 | Actinobacteridae (39), Bacteroidetes (one), Firmicutes (3), Proteobacteria (28) |
| four and 5 | 77 | 41 | 6 | 12 | Acidobacteria (3), Actinobacteridae (47), Firmicutes (1), Planctomycetes (2), Proteobacteria (24) |
| eight to 12 | 64 | 32 | 16 | 44 | Acidobacteria (two), Acidimicrobidae (1), Actinobacteridae (31), Chloroflexi (1), Firmicutes (3), Proteobacteria (20), Rubrobacteridae (6) |
TABLE v.
Isolates of bacteria that formed visible colonies on terminal plates of counting sets with different media inoculated with soil
| Medium | No. of isolates identified | % of isolates affiliated with rarely isolated groups | Groups represented (no. of isolates) |
|---|---|---|---|
| 0.1× TSA | 33 | 0 | Actinobacteridae (xviii), Bacteroidetes (1), Firmicutes (5), Proteobacteria (9) |
| WSA | 37 | 0 | Actinobacteridae (24), Firmicutes (2), Proteobacteria (11) |
| CSEA | 33 | 0 | Actinobacteridae (xx), Proteobacteria (13) |
| DNBG | 36 | 8 | Acidimicrobidae (1), Acidobacteria (1), Actinobacteridae (19), Planctomycetes (1), Proteobacteria (fourteen) |
| VXylA | 33 | 21 | Acidobacteria (2), Actinobacteridae (fourteen), Proteobacteria (12), Rubrobacteridae (v) |
| VXylG | 40 | 13 | Acidobacteria (two), Actinobacteridae (22), Chloroflexi (i), Planctomycetes (ane), Proteobacteria (xiii), Rubrobacteridae (1) |
Consequence of inoculum size on phylogenetic groups.
We compared the identities of late appearing colonies (actualization in week 8 or subsequently) on plates with different inoculum sizes, i.e., unlike dilution levels (Table 2). Because some media did not yield whatsoever members of rarely isolated groups, this analysis was limited to counting sets created with DNBG, VXylA, and VXylG. Inoculum size did not announced to take an consequence on the isolation of members of rarely isolated groups (P = 0.56 by the χ2 exam). Fifty-fifty on the plates receiving the largest inoculum of soil (an expected one,780 cells/plate), nosotros isolated members of the Acidobacteria (eleven isolates in iv subdivisions), Acidimicrobidae (i isolate), Rubrobacteridae (i isolate), Gemmatimonadetes (1 isolate), and Planctomycetes (two isolates). In total, 36% of all isolates appearing on these media in week 8 or afterward belonged to rarely isolated groups.
Upshot of medium type and incubation time on development of colonies by pure cultures.
Fifty isolates were selected for comparisons of their ability to grow on different media. Thirty-2 of these belonged to the wide category of commonly isolated groups, and the other 18 were members of rarely isolated groups (Table ane). These were selected to cover several different phyla so that no more than than two members of any one family-level grouping were included. A larger proportion of bacteria affiliated with ordinarily isolated groups was able to abound on 0.1× TSA, WSA, and CSEA than that for members of rarely isolated groups (Table half dozen). Members of both groups grew well on VGluA and DNBA. However, members of rarely isolated groups grew significantly more slowly on all media than did members of normally isolated groups (Table vi).
Table 6.
Ability of cultures to abound and time until visible colony advent on dissimilar media for pure cultures of soil bacteria from usually isolated and rarely isolated groups a
| Medium | % of cultures with ability to grow | Hateful time to colony advent (days) | Statistical significance (P value in t exam) b | ||
|---|---|---|---|---|---|
| Members of usually isolated groups | Members of rarely isolated groups | Members of unremarkably isolated groups | Members of rarely isolated groups | ||
| 0.ane× TSA | 97 | 33 | five | 23 | 0.03 |
| WSA | 100 | 44 | 15 | 43 | 0.02 |
| CSEA | 91 | l | x | 35 | 5 × x−4 |
| DNBA | 100 | 100 | seven | xxx | 4 × 10−5 |
| VGluA | 100 | 94 | iv | xix | 1 × 10−four |
To examination the quality of these media, we tested eight strains from the drove of the Department of Microbiology and Immunology, University of Melbourne, for the power to abound on the five media used for these experiments. All eight isolates produced visible colonies on all v media afterwards a mean 2.7 (SD = 1.9) days of incubation at 25°C.
Give-and-take
Choice of media.
A wide range of unlike media have been used to estimate the size of the bacterial community of soil and to isolate representatives of this community (2, 14, 21). However, it has been known for a long time that the number of bacteria that are able to grade colonies on microbiological media is mostly only a small part of the total number of bacteria in soil (5, 14, 28). In addition, the advent of molecular ecological technologies has revealed the presence of many novel groups of bacteria in soil, highlighting the inadequacy of cultivation methods for the full general study of soil bacteria (three, 10, 23). Recently, the use of nontraditional media has allowed the isolation of members of some of these previously uncultured groups (12, 15, 24). One of these media, VL55 medium, was formulated to mimic the low concentrations of inorganic ions in soils, with increased concentrations of ammonium and phosphate ions to allow sufficient biomass formation to produce visible colonies. The pH was adjusted to the pH of the soil at the site beingness studied (27). For this study, we tested the effect of a range of growth substrates in this basal medium. Based on the results, nosotros chose xylan as the growth substrate for further experiments, as it yielded the highest mean feasible counts in this study. Xylan was also used successfully as a growth substrate for the isolation of representatives of poorly studied groups of bacteria in before studies (15, 24). We compared this medium and DNBG, which was also successfully used in an earlier report (12), with three media that are more unremarkably used to abound soil bacteria, i.e., CSEA, WSA, and 0.ane× TSA.
Result of inoculum size.
Earlier studies accept repeatedly reported that smaller inocula outcome in higher feasible counts (4, 11, thirteen, 14, 21). In our experiments, diluting the inoculum resulted in a ii-fold increase in the final viable count for each 10-fold decrease in inoculum size. We observed an increment in the variability, measured as the coefficient of variation, as the inoculum size decreased, which is to exist expected. Thus, the precision of the viable counts decreased as the number of colonies on each plate decreased. Nosotros believe, however, that the full general increase in viable counts with decreasing inoculum sizes was real because it occurred consistently beyond a big number of counting sets. It is clear that the standard acceptable ranges of colony numbers on a plate that are generally used for determining viable counts (20 to 200, 25 to 250, or 30 to 300 [6, 16, 22]) are inappropriate for experiments with soil, every bit counts in these ranges are clearly underestimates due to the depressed counts obtained with large inocula. This is probably due to growth inhibition of some species by others when the colonies are too close together or the depletion of nutrients by fast-growing colonies so that slow-growing ones practise not achieve a detectable size. It has been observed that the employ of larger plates partly overcomes this crowding effect (4). The variability between replicates increased dramatically when smaller colony numbers were used to summate feasible counts. The selection of what number of colonies to employ to determine the feasible count became a compromise betwixt increased reproducibility at >40 colonies per plate (90-mm bore) and increased viable counts when plates with <40 colonies were used. We decided to summate viable counts from plates at the smallest inoculum size that yielded a minimum of 10 colonies per plate, averaged over all replicate plates at that inoculum size (dilution level) within a counting set. To overcome the associated increase in variability, we repeated each counting experiment upwards to seven times (with three or five replicate plates for each of iii inoculum sizes).
We had expected to encounter differences in the kinetics of colony formation for the different inoculum sizes, only this was not the case. It was anticipated that the rapid development of larger numbers of fast-growing colonies on plates with larger inocula would prevent slower growing colonies from actualization due to inhibitory or competitive effects. The crowding effect on plates with larger numbers of colonies also did not affect the proportion of members of rarely isolated groups. These organisms formed visible colonies belatedly in the incubation menstruation (run into beneath). If they were prevented from forming visible colonies on crowded plates, so equally slow-growing members of commonly isolated groups were inhibited to a similar extent. This would result in members of both groups beingness isolated in similar ratios, regardless of the inoculum size. Indeed, at that place is no reason to assume that slow growth is restricted to members of the rarely isolated groups, and we establish that members of new families of Proteobacteria are besides slow growing (unpublished data).
Effect of medium.
Nosotros found that 0.i× TSA was the poorest of the six media that nosotros investigated in particular for obtaining rarely isolated bacterial groups. No isolates belonging to rarely isolated groups were obtained on this medium in this study, although a member of the phylum Acidobacteria has been isolated on this medium (eighteen). This medium likewise immune the expression of a spreading phenotype and so that a few unmarried colonies, mainly members of the family Bacillaceae, rapidly covered most or all of the area of the plate. CSEA and WSA were also poor medium choices, resulting in low culturabilities and no isolates affiliated with rarely isolated groups. The media 0.1× TSA, CSEA, and WSA also did non support the growth of as many pure culture isolates of members of rarely isolated groups as the other media did, but they were very good media for growing pure cultures of members of commonly isolated groups.
DNBG and the media based on VL55 medium resulted in higher feasible counts and immune the development of visible colonies of members of rarely isolated groups. Very few spreading colonies were noted on these plates, and the spreading phenotype was not expressed on media based on VL55 past nearly isolates that spread on other media. We found that the use of gellan as a solidifying agent with VL55 medium resulted in higher feasible counts than did the use of agar as the solidifying agent, in agreement with an earlier finding comparing these ii gelling agents in experiments with DNBG and DNBA (12). This finding was not supported by statistical tests of the data, just that may take been the effect of our attempting to detect small differences in data sets with high variabilities. The utilize of agar equally the solidifying agent did not announced to effect in an inhibition of growth of members of rarely isolated groups, in agreement with findings of an earlier study (24).
Jensen (thirteen) stated that for a medium to exist suitable for plate counts, it must fulfill the following four requirements equally much as possible. Firstly, its composition must be standardized and then that it tin can be reproduced with sufficient accuracy anywhere and at whatsoever time. In this study, only CSEA did not fulfill this criterion, since information technology independent a site-derived soil extract. Olsen and Bakken (21), however, showed that CSEA media prepared with soil extracts from unlike soils gave practically identical colony counts, so this may not eliminate CSEA equally a useful medium. Secondly, the medium must permit the development of as large a range every bit possible of the bacteria present, which was true for VXylA, VXylG, and DNBG. Thirdly, the medium must allow the least possible growth of unwanted nonbacterial microorganisms, such as fungi. The number of fungal colonies was low and about the aforementioned for all six media (data not shown). Finally, the growth of spreading colonies must exist suppressed. Overall, media based on VL55 medium or DNB seem to best fulfill Jensen's criteria.
Effect of incubation time.
Information technology is well known that increasing the incubation time results in increased viable counts (12, 13, 14, 26, 28), particularly on media with depression nutrient concentrations (14). However, incubation times on the order of months are only rarely used, and incubation times are by and large in the range of ane week to 1 month (14, 22, 28). Extended incubation periods seem to exist important, equally members of rarely isolated groups appeared predominantly after incubation for 2 months on suitable media inoculated with soil. This suggests that members of these groups are particularly slow growing or have very long lag periods. Pure cultures of members of these groups were similarly deadening growing and took significantly longer to produce visible colonies than did pure cultures of members of normally isolated groups. Members of rarely isolated groups may exist able to grow more rapidly once media and growth atmospheric condition accept been optimized, but we suggest that they volition not, in full general, be every bit rapid growing every bit ordinarily studied soil leaner.
Identities of isolates.
Many of the isolates from commonly isolated groups were affiliated with family unit-level groups that have few cultured representatives but have been detected in soils as 16S rRNA genes. In improver, it was possible to isolate members of bacterial groups that were previously labeled unculturable, as nosotros constitute in earlier investigations (12, 15, 24). In this report, isolates of the phylum Acidobacteria representing four of the 8 subdivisions defined by Hugenholtz et al. (x) were cultured. Some of these belong to new families distinct from those that were previously isolated (15). Nosotros also isolated members of the poorly studied subclasses Acidimicrobidae and Rubrobacteridae of the phylum Actinobacteria.
Three of the isolates of the phylum Planctomycetes were but distantly related to cultured representatives of this grouping. One was affiliated with the "Gemmatae" subdivision but was just distantly related to members of the genus Gemmata. Instead, information technology was related to a group of bacteria that were previously merely known from 16S rRNA genes detected in soil (17). The other 2 were members of the WPS-i lineage of the phylum Planctomycetes, a course-level group that was previously simply known from 16S rRNA genes from soil (20; Fifty. Schoenborn and P. H. Janssen, unpublished data) and other habitats. These two isolates, together with a third from another study performed in our laboratory (P. Sangwan and P. H. Janssen, unpublished information), are the first known cultured representatives of this group.
Ane member of the phylum Gemmatimonadetes was cultured. To date, this phylum is represented by 1 isolate of a named species, Gemmatimonas aurantiaca (29), and by three isolates from the Ellinbank soil site (15). We too cultured an isolate that represents the offset cultured member of a new subdivision of the phylum Chloroflexi which is unaffiliated with any of the recognized subdivisions (10; P. Hugenholtz, personal communication).
Conclusions.
The system we have been studying is a krasnozem dirt loam soil under a mixed rye grass and clover pasture which is managed under a fertilization and grazing regimen that can be considered to exist close to the district norm (19). Nosotros aspect our success in isolating members of rarely isolated groups to the methods used rather than to any unusual properties of this soil arrangement. The successful isolation of members of groups of bacteria that are widely distributed and common in soils worldwide seems to be a result of using appropriate media and extended incubation times. We have empirically used this approach in previous studies (12, fifteen, 24). This study demonstrates the significance of medium pick and incubation time on the successful isolation of representatives of groups of numerically arable but rarely isolated soil bacteria.
Acknowledgments
We give thanks Cameron Gourley and Sharon Aarons (Dairy Research Constitute, Ellinbank, Australia) for their assistance with access to the sampling site; Michelle Sait, Parveen Sangwan, and Catherine A. Osborne for supplying isolates; and Philip Hugenholtz for help with some of the taxonomic assignments.
This work was supported by a grant from the Australian Enquiry Council.
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