FACS-iChip culturing was achieved using microorganisms from paddy soils sorted by a flow cytometer and membranesealed iChips incubated in root boxes. For preliminary verification of the method, sterile Luria Broth (LB) medium and dyed Escherichia coli were loaded into each iChip well using a flow cytometer and incubated for two days. Visible colonies were observed in 99% of wells after incubation, indicating that the influences of fluorescence and dyes on microorganisms were negligible, and cell loading was successful.
For method validation, living cell sorting was performed with a flow cytometer (Fig. 1b) and a single living cell was loaded into each well of the iChip plate. An iChip is a sealed device consisting of two membranes and a 384-well culture plate with LB medium in every well (Fig. 1c). iChips were then incubated in root boxes with liquid media for 30 days or solid media for 30 and 50 days of culturing (Fig. 1d). Visible colonies were counted after incubation (Table 1). In the iChips that used solid medium and were cultured for 30 days, there were on average 250 wells (n = 2) with obvious colonies; this method achieved a 65% culture recovery rate (number of visible colony wells/total wells). For the same medium at 50 days, 85 wells (average; n = 3) had visible colonies after incubation; only a 22% culture recovery rate was achieved. Subsequent colony enrichment of solid medium wells with LB medium produced an average of 52% (30 days; n = 2) and 34% of iChip wells (50 days; n = 3) that could be subcultured. Liquid-medium iChips incubated for 50 days had a 35% culture recovery rate and 44% culture retrieval rate (average; n = 3; number of wells that can be subcultured/number of visible colony wells). In total, FACSiChip achieved a culture recovery rate of almost 40% and a culture retrieval rate of 25%.
Figure 1. Schematic diagram of iChip operation. A Sample preparation. Cells were eluted from soil and dyed by TO (thiazole orange) and PI (propidium iodide). B Cell sorting. Dyed microbes were sorted (flow cytometer) into alive and dead cells. C Cell loading. Alive single cells were loaded into the drilled iChip and the whole device was sealed by membranes. D IChip culturing. The device was incubated in a root box for several days and was taken out to purify colonies
Number Media type Culture time Wells with visible colonies Culture recovery rate (%) Colony count Culture retrieval rate (%) 1 Solid medium (LB) 30 days 257 66.93 139 54.09 2 Solid medium (LB) 30 days 242 63.02 123 50.83 3 Liquid medium (LB) 50 days 114 29.69 16 14.04 4 Liquid medium (LB) 50 days 152 39.58 57 37.50 5 Liquid medium (LB) 50 days 133 34.64 60 45.11 6 Solid medium (LB) 50 days 84 21.88 31 36.90 7 Solid medium (LB) 50 days 95 24.74 31 32.63 8 Solid medium (LB) 50 days 74 19.27 26 35.13 Culture recovery rate means the ratio of number of visible colony wells to total wells. Culture retrieval rate means the ratio of number of wells that can be subcultured to number of visible colony wells. The LB medium was used in both preliminary culture and subculture. And the cloudy liquid was regarded as visible colonies in liquid medium
Table 1. Recovery rate of FACS-iChip
To compare the culture results against actual soil samples, five replicate DNA extraction samples from the test soil were analysed with 16S rRNA gene sequencing (Fig. 2). There was a large difference between culturing and soil at the genus level analysis with a total of 790 genera obtained in the test soils, dominated by Thermomarinilinea, Longilinea, Nitrososphaera, Ornatilinea and Geobacter.
In the culture results, a total of 19 genera (46 species) were observed from the cultivation of well samples (Table S1). Microorganisms were dominated by Bacillales, Aeromonas, Pseudomonas and Achromobacter; of these, only six genera were detected with 16S rRNA (Table 2). Interestingly, the ten most abundant genera detected by 16S rRNA gene sequencing were not observed in culture results.
Number Kingdom Phylum Class Order Family Genus Presence in sequencing resultsa 1 Bacteria Proteobacteria Gammaproteobacteria Aeromonadales Aeromonadaceae Aeromonas Yes 2 Bacteria Actinobacteria Actinomycetales Micrococcineae Microbacteriaceae Microbacterium No 3 Prokaryota Bacteria Terrabacteria Firmicutes Bacilli Bacillales Yes 4 Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Citrobacter No 5 Bacteria Firmicutes Bacilli Bacillales Paenibacillaceae Brevibacillus Yes 6 Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Serratia No 7 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Delftia No 8 Bacteria Proteobacteria Betaproteobacteria Rhodocyclales Rhodocyclaceae Azospira Yes 9 Eubacteria Firmicutes Bacilli Bacillales Bacillaceae Lysinibacillus No 10 Bacteria Proteobacteria Gammaproteobacteria Xanthomonadales Xanthomonadaceae Stenotrophomonas No 11 Bacteria Proteobacteria Betaproteobacteria Burkholderiales Comamonadaceae Comamonas No 12 Bacteria Proteobacteria Beta Proteobacteria Burkholderiales Alcaligenaceae Achromobacter Yes 13 Bacteria Proteobacteria Gammaproteobacteria Pseudomonadales Pseudomonadaceae Pseudomonas No 14 Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Enterobacter Yes 15 Bacteria Proteobacteria Epsilonproteobacteria Campylobacterales Campylobacteraceae Sulfurospirillum No 16 Bacteria Firmicutes Bacilli Bacillales Bacillaceae Fictibacillus No 17 Bacteria Proteobacteria Gamma Proteobacteria Enterobacteriales Enterobacteriaceae Pantoea No 18 Bacteria Actinobacteria Actinomycetales Micrococcales Micrococcaceae Micrococcus No 19 Bacteria Proteobacteria Gammaproteobacteria Enterobacteriales Enterobacteriaceae Atlantiacter No The 19 genera listed in this table are the results obtained by cultivation
aThe genera that appeared in the 16S rRNA sequencing results were marked as "Yes", otherwise "No". Only six genera were appeared in sequencing results
Table 2. Presence of cultured microorganisms in the 16S rRNA sequencing data (genus level)