Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria

Abstract

Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable 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 not 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 not result in the isolation of members of rarely isolated groups. Newly developed 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 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use 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) as well as members of the subclasses Rubrobacteridae and Acidimicrobidae of the phylum Actinobacteria.

FIG. 1.

(A) Variability in colony numbers of replicates within counting sets relative to mean colony number per plate for each counting set. The variability is expressed as the coefficient of variation (SD/mean) for the replicates (three or five) at any one 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 x axis, and the numbers of colonies on the corresponding plates with a 10-fold smaller inoculum in the same counting set are plotted on the y axis. The diagonal line represents the 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 point on all panels representing the result from three replicate plates at each dilution level; ○, data from experiments performed in 2003, with each point representing the result from five replicate plates at each dilution level.

FIG. 2.

(A) Viable counts at different inoculum sizes (dilution levels) for three different media after 12 weeks of incubation. Symbols: ○, DNBG; □, VXylA; ▵, VXylG. Each point represents the mean of five replicate plates. The thick horizontal line indicates the mean, and the vertical lines indicate one standard deviation from the mean. (B) Increase in viable 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 percentage of the 12-week count for that counting set, and each point represents the mean of three media, with each used for seven 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.

FIG. 3.

Mean viable counts after 12 weeks of growth on VL55 medium with different growth substrates. Each solid bar represents the mean of three counting sets prepared from the March 2001 soil sample and three counting sets prepared from the April 2001 soil sample. The error bars indicate 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.

FIG. 4.

Increases in viable counts on different media with increasing incubation times. Symbols: •, 0.1× TSA; ▪, WSA; ▴, CSEA; ▵, VXylG; □, VXylA; ○, DNBG. Each point represents the mean of seven experiments, each of which included five replicate plates. For clarity, the standard errors are not shown; the mean standard error of all the points was 14.5% of the values plotted.