Digital Plating: A Universal and Versatile Microbiological Technique

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Here, we introduce a digital plating (DP) platform that integrates digital assays with traditional plate culturing. Using a high-density microwell array chip covered with an agar medium sheet, the DP platform not only enables accurate bacterial quantification but also facilitates the isolation of single bacteria from complex communities for further characterization. The high flexibility afforded by the replaceable agar medium cover allows the DP platform to support complex microbial culturing, thereby broadening its potential applications. We demonstrated its versatility in accurate bacterial quantification, efficient isolation, identification, and clonal culture of specific bacteria from complex communities, rapid antibiotic susceptibility testing, and detailed investigation of microbial interactions. The DP system’s simplicity, cost-effectiveness, and versatility demonstrate its potential to substitute traditional plating techniques and enable rapid and scalable bacterial assays that were previously unattainable. Biological sciences/Biological techniques Biological sciences/Microbiology digital plating isolation identification recovery antibiotic susceptibility testing microbial interaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Isolation, identification, quantification, and clonal cultivation of bacteria are crucial from fundamental microbiology to infectious disease diagnosis to industrial microbiology 1,2 . Culture-based methods, though reliable and well-established, are labor-intensive, time-consuming, and inefficient. To achieve rapid bacteria detection, various molecular methods have been proposed, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) 3-6 . Despite high sensitivity and specificity, these methods often require extensive sample pretreatment, costly reagents, labor-intensive procedures, and skilled operators, which limit their practical use. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a routine tool for rapid bacterial identification in clinical microbiology 7-10 . Despite its success, MALDI-TOF MS struggles with differentiating closely related bacterial species, offers limited quantification capabilities, and involves complex technical requirements and high costs 11 . Moreover, it often necessitates pre-detection sampling processes to enhance bacterial concentration or purity. Recently, droplet microfluidic technology, capable of encapsulating individual bacteria in microscale droplets for the micro-confined growth, has gained recognition as a powerful tool in microbiology. This technology enables various applications, including early detection of bacteria 15,16 , isolation of rare and uncultured microbes 17-20 , precise quantitation of bacteria 21,22 , characterization of the heterogeneity in bacterial populations 23,24 , selection of improved strains 25,26 , and exploration of microbial interactions 27-29 . Although this technology has greatly expanded the scope and context of microbiology, it faces inherent challenges such as droplet fusion during cultivation, limited substance exchange within droplets, and the requirement for expensive, complex equipment. To address these challenges, we developed a digital microbial assay, termed "digital plating" (DP), which is based on a solid medium-covered PicoArray device. In the DP platform, a bacterial suspension is partitioned into numerous picoliter microwells via a pre-degassing-induced vacuum 30,31 , followed by coverage with a solid agar medium sheet for incubation and analysis. Unlike droplet-based assays, this well-based format eliminates the risk of droplet fusion during cultivation, ensuring the isolation of individual bacteria. Moreover, the immobilized array format of the well-based assay is also beneficial for time-lapse analysis since the isolated droplets can be spatially indexed. More importantly, the replaceability of the covering solid medium sheet allows precise control and flexible regulation of microbial growth conditions, which gives access to several new possibilities – from precise selection of individuals with desired properties to rapid antibiotic susceptibility testing (AST) to cultivation of uncultivable microbes. Attributed to its unique advantages, the DP is expected to serve as a generic platform for microbiologists to explore the world of microorganisms. Results Concept and workflow of the DP platform An ideal microbial assay platform should be user-friendly (requiring no expensive instruments and well-trained personnel), rapid (producing results within a time frame suitable for point-of-care testing), precise (enabling sensitive detection of bacteria in a quantitative manner), reliable (with a low probability of false positive or false negative results), flexible (easily accommodating a wide range of microbiological applications), and cost-effective (readily accessible to any global setting). To this end, we propose the "digital plating" (DP) technique. The principal strategy of our DP technique is to integrate a powerful digital assay format with the conventional plating principle for rapid, precise, reliable, and cost-effective microbial detection and analysis. Fig. 1a illustrates the concept of our DP technique. This technique involves two principal components: a PicoArray device for stochastic compartmentalization of single bacteria to enable digital assays and a covering agar sheet that serves as a solid culture medium to support bacterial growth. The DP workflow comprises four major steps: sample discretization, assembly of the agar/chip, device incubation, and microscopic examination (Fig. 1b and Movie S1 in the Supplementary Information). To facilitate sample discretization, a pre-degassing-driven self-pumping mechanism is used to partition the bacteria suspension into a large number of picowells and stochastically encapsulate single bacteria into compartments 30,31 ; after compartmentalization, an agar-based solid medium sheet is conformally attached to the device to seal all picowells, which provides a physical barrier to trap motile cells within the picowells; next, the device is incubated and the growth of bacteria in microwells is examined microscopically to determine phenotypic characters. Due to its digital format, this technique offers several main advantages: (1) eliminating the interspecies competition and biases due to growth rate differences, which facilitates recovery of rare or slow-growing microorganisms from complex ecosystems; (2) allowing the study of the heterogeneity of bacteria that are masked in ensemble measurements; (3) absolutely quantifying the concentration of bacteria, which enables highly sensitive detection of bacteria over a wide range of concentrations in a quantitative manner; (4) facilitating timely detection of pathogenic bacteria owing to microconfinement-induced rapid accumulation of detectable metabolites. Meanwhile, the agar sheet provides culture conditions analogous to those in the conventional plating techniques, thus allowing smooth and seamless transfer of well-established characterization and analysis methods from conventional plating techniques to this platform and easy interpretation of the obtained results. More importantly, the replaceability of the covering agar sheet offers more flexibility for digital microbial assays. For instance, if an agar sheet containing a selective medium is applied on the PicoArray device, the DP technique can isolate a particular strain of microorganisms; if an agar sheet containing a differential medium is applied on the PicoArray device, the DP technique can identify and differentiate closely-related microorganisms; if an agar sheet containing a certain antibiotic is applied on the PicoArray device, the DP technique can analyse the response of bacteria to the antibiotic. Furthermore, our DP platform can sequentially combine the PicoArray with multiple different medium sheets, i.e. , allowing a wide range of culture conditions to be applied on a single device. Thanks to its similarities to conventional plate methods, our DP platform allows in situ clonal culturing of single bacteria isolated from heterogeneous samples without requiring extra sorting and inoculation steps, and the cultivated pure microbes can be recovered directly for further off-chip culture or genotyping. Characterization of the DP platform A key premise for digital bioassays is stochastic encapsulation of target biospecimens into massive numbers of equal-volume small compartments. To achieve precise and reliable digital bioassays, the homogeneity of partitions is a critical factor 32,33 . Here, we first quantified the variance of partition volumes created by the self-discretization based on the combination of pre-degassing pumping and capillary force-assisted dewetting 34 . To investigate the distribution of the partitioned volumes across the device, the fluorescence microscopy images of the partitioned Rhodamine B droplets from different positions on the device were captured using a CCD camera. It is assumed that Rhodamine B solution is homogeneous, so the total fluorescence intensity of each Rhodamine B droplet is directly proportional to its volume, and then the uniformity of the total fluorescence intensities of partitioned droplets can reflect their volume uniformity. The captured images of droplets from different areas on the device were analyzed using an image analysis software (ImageJ) to determine the total fluorescence intensity of each droplet. From six random areas of the device, 3,200 droplets were selected for measurement and calculation. The calculated total fluorescence intensities for all these droplets were then plotted into a volume distribution histogram, showing that the coefficient of variation (CV) of the total fluorescence intensities measured from 3,200 randomly selected droplets is about 4.78% (Extended Data Fig. 1a). This demonstrates that the PicoArray device, combined with the self-discretization mechanism, achieves highly uniform discretization for digital microbial assays. Furthermore, for our DP platform, effective diffusion of molecules from the covering agar sheet to the picowells is crucial for nutrient delivery and microenvironment regulation. To investigate whether the nutrient or drug molecules could diffuse from the covering agar sheet into the picowells, we conducted a simulation experiment by attaching a red dye-stained agar sheet to the PicoArray device and subsequently monitoring the color change in the picowells of the device. After attaching for 15 min and then peeling off the agar sheet, the color of the solution in all picowells of the device changed to red (Extended Data Fig. 1b and Fig. S1 in the Supplementary Information), confirming that the dye molecules within the agar sheet could effectively and homogeneously diffuse into the picowells of the device. Such diffusion-induced molecule transport allows us to use the agar sheet for supplying nutrients to microbes confined in picowells and chemically regulating the microbial growth microenvironment in picowells, which offers unparalleled flexibility for various microbiological studies, such as monitoring microbial response upon perturbation, screening novel antibiotics, and culturing the uncultured microorganisms. Furthermore, this diffusion also allows reverse molecule transport, i.e. , from picowells to the covering agar sheet, thus enabling facile detection of metabolites and signaling molecules secreted by bacteria confined in picowells. That is, this platform allows us to perform biochemical assays to identify microcolonies formed in picowells if the covering agar sheet contains differential media. Our DP method is based on the fact that when single bacteria partitioned into picowells are incubated, each of the microconfined single viable bacteria will proliferate forming a visible and independent colony. This allows for precise and absolute quantification of bacteria in samples. To achieve accurate enumeration, it is essential to culture individual bacterial cells until their colonies are visible, while also ensuring that each colony remains independent, avoiding fusion with neighboring colonies. To determine the optimal time point for microcolony counting using the PicoArray device, we examined the effect of cultivation time on the visibility and independence of microcolonies formed in picowells. A time-lapse sequence of E. coli cultured in the PicoArray device covered with a Luria-Bertani (LB) agar sheet is shown in Extended Data Fig. 1c. In these images, dark spots represent microwells where bacteria grew and formed microcolonies, while blank spots indicate microwells that either initially lacked cells or where the cells failed to form colonies, likely due to low growth rates. Most microcolonies were nearly invisible before 5 h. After incubation for 6 h, most microcolonies were discernible and confined to individual picowells without fusion between neighboring colonies. However, after 7 h of incubation, there was a noticeable overgrowth of the formed microcolonies, which spread into adjacent picowells. Thus, to ensure a precise enumeration, counting of E. coli microcolonies should be performed between 6 and 7 h of incubation. It is worth noting that the optimal counting time varies across different microorganisms due to significant differences in their growth rates. To demonstrate the capability of our DP platform to rapidly and accurately quantify bacteria, we loaded serially diluted samples over a range of starting cell densities from a previously quantified Salmonella stock with Mueller-Hinton broth into the device, followed by discretization and incubation. After 6 h of incubation, all devices were examined by microscopy to count the positive microwells containing microcolonies in each chip. Extended Data Fig. 2a shows representative micro-images of PicoArray devices loaded with different concentrations of Salmonella after incubation. As expected, reducing the bacterial input concentration resulted in fewer picowells occupied by microcolonies. The proportion of positive microwells in each device was then counted, and the cell concentration in colony-forming units per milliliter (CFU/mL) was calculated using Poisson statistical analysis 32,33 . This count was plotted against the CFU/mL measured in parallel by conventional colony counting on agar plates. As shown in Extended DataFig. 2b, the Salmonella concentrations measured using the PicoArray-based digital CFU method correspond very well to those obtained via the conventional plate counting method (R 2 = 0.9992) in the entire 5 log range of the measured sample concentrations, demonstrating the capability of the PicoArray-based digital CFU method to precisely quantify microbes. Importantly, the entire process for the digital CFU method took only 6 h, significantly shorter than that with the conventional plating method, which is beneficial to rapid diagnosis and prompt treatment of infectious diseases. Isolation of single bacteria from a heterogeneous sample In nature, microorganisms typically grow as mixed populations rather than as single species. Therefore, isolating individual microorganisms from complex microbial communities is an essential prerequisite for downstream phenotypic and genomic characterization, as well as for scaled-up culture. Due to its ability to compartmentalize single cells into massively large numbers of subunits for clonal growth, our DP platform is ideal for the isolation of individual bacteria from heterogeneous samples. To demonstrate the DP platform's capacity for single-cell separation and cultivation, we prepared a simple two-strain microbial community consisting of S. aureus and green fluorescent protein (GFP)-expressing E. coli (BL21). First, both strains were cultivated overnight, diluted 10,000 times in LB broth, respectively, and then mixed in a ratio of 1:1 (v/v) to obtain a microbial mixture. After discretization in the device and incubation at 37 ºC for 8 h, three distinct populations of microwells were observed via microscopy (Fig. 2a). One population of microwells was empty microwells, which was predicted to represent compartments containing no bacteria. The remaining microwells contained microcolonies with two distinct morphologies: densely packed microcolonies and thin microcolonies with fewer cells. Given that E. coli showed slower growth under the used conditions, the population of microwells containing thinner microcolonies was predicted to represent compartments containing E. coli strain. The population of microwells containing denser microcolonies was assigned to correspond to compartments containing the best-growing bacteria, here, S. aureus . This hypothesis was confirmed through fluorescence microscopy. Under 523 nm excitation light, only the thinner microcolonies exhibited green fluorescence, indicating the presence of GFP-expressing E. coli , while the denser microcolonies showed no fluorescence, confirming them as S. aureus (Fig. 2a). Based on the total number of picowells and the count of microwells occupied by fluorescent and dense microcolonies, we calculated the numbers of S. aureus and E. coli using a method similar to digital PCR 35 . As shown in Fig. 2b, the calculated numbers of S. aureus and E. coli corresponded closely with the expected number of S. aureus and E. coli (estimated via agar plating). These results demonstrate that the DP allows for rapid and highly effective separation of different species coexisting in a complex sample. This suggests that our DP platform can simultaneously count and isolate different bacterial cells in mixed microbial communities by using vision analysis or light scattering based methods. Enrichment and identification of specific bacterial species from mixed-species microbial communities In addition to enabling spatial separation of different bacteria in a single sample, our DP platform can also be combined with selective and/or differential media to enrich and identify specific bacteria in complex samples. To demonstrate the selective-medium-assisted enrichment capability of the DP platform, we prepared a microbial mixture consisting of S. aureus and GFP-expressing E. coli and loaded it into a PicoArray device. When the device was covered with an agar sheet containing LB medium and incubated at 37 ºC for 8 h, both bacterial strains grew into microcolonies (see the left column in Fig. 3a-i). The two species were easily distinguished by comparing bright field and fluorescent microimages: one population exhibited green fluorescence, while the other did not. In contrast, when the device was covered with an agar sheet made with NaCl-based selective medium (containing 7.5% NaCl) and incubated at 37 ºC for 8 h, only non-fluorescent microcolonies appeared (see the right column inFig. 3a-i), implying that none of the isolates of E. coli but only the isolates of S. aureus grew in the NaCl-based medium-covered PicoArray device. This is because Staphylococci species can tolerate high salt concentrations and grow on this selective medium agar, whereas other bacteria may not. Additionally, the number of non-fluorescent microcolonies in the PicoArray device covered with LB medium was nearly equal to the total number of microcolonies formed in the device covered with NaCl-based medium (Fig. 3a-ii), further confirming the selectivity of the NaCl-based medium. These results demonstrate that the DP platform, when coupled with selective media, can effectively enrich target microorganisms from complex microbial communities with high specificity. In addition, we also performed an experiment to demonstrate the differential-medium-assisted identification ability of our DP platform. Here, MacConkey agar (MCA), one of the earliest and most common differential media, was chosen to demonstrate the applicability of “DP + differential medium” to distinguishing specific bacterial species from other bacterial types. The experiment was performed by discretizing a S. aureus suspension,an E. coli suspension, and a mixture of the two in three PicoArray devices, respectively, covering each device with an MCA sheet, and then incubating them at 37 ºC for 8 h. As shown in Fig. 3b, the microcolonies formed in three devices displayed different appearances in color: the E. coli -loaded device exclusively exhibited red microcolonies, the S. aureus -loaded device displayed only colorless microcolonies, and the device loaded with the mixture of both species presented both red and colorless microcolonies. This differentiation is attributed to the composition of the MCA medium, which contains lactose and neutral red (pH indicator). E. coli can ferment lactose to produce lactic acid, thereby decreasing the pH of the agar and turning the indicator (neutral red) pink. In contrast, S. aureus cannot utilize lactose, resulting in colorless colonies due to the absence of pH alteration and indicator color change. Rapid phenotypic AST Another important application scenario of the DP platform is AST, a fundamental mission of the clinical microbiology laboratory, which is paramount for effective infection management. In particular, a rapid and precise AST assay can assist clinicians to choose appropriate antimicrobial therapies for various pathogen infections and avoid unnecessary overprescription. Thanks to its ability to miniaturize bacterial cultures to picoliter levels which can dramatically alter early colonization and self-regulated quorum signaling 36 , the DP platform enables faster measurement of the ability of individual bacteria to form microcolonies at different antibiotic concentrations. This is achieved through microconfinement-induced rapid increases in cell density or the accumulation of detectable metabolites, thus enabling rapid AST assays. To demonstrate the applicability of the DP platform to AST assays, we used E. coli as a model bacterium and ampicillin sodium as a model antibiotic to perform AST experiments. Multiple PicoArray devices were run in parallel, each loaded with the same concentration of E. coli suspensions and covered with an agar sheet containing a different concentration of ampicillin sodium. In addition, a control device was run in parallel, which was loaded with the same bacterial concentration as the others but covered with an agar sheet without any antibiotic. This control allows us to estimate the number of cells initially loaded into each DP chip. After 6 h incubation at 37℃, the chips were imaged via microscopy. It was observed that the bacterial growth was inhibited in the presence of antibiotics. As shown in Fig. 4a, the number of microwells containing microcolonies decreased with increasing concentrations of ampicillin sodium, and no cells survived at concentrations above 200 μg/mL. The percentages of surviving cells at 6 h were plotted against the concentrations of ampicillin sodium, exhibiting a monophasic dose-response curve (Fig. 4b). The minimum inhibitory concentration (MIC) value, obtained by fitting the bacterial growth data to a Gompertz model 37 , was 196.9 μg/mL, which closely matches the MIC determined by the broth microdilution method (193.4 μg/mL) (Fig. S2 in the Supplementary Information). Compared to conventional AST methods, our DP platform not only consumes fewer reagents (~20 μL) but also allows faster antibiotic susceptibility assays (< 6 h). More importantly, it can characterize physiological responses of individual cells to different antibiotic concentrations, which enables the quantitative phenotyping of heterogeneous resistance, or heteroresistance, with single-cell resolution 23 . These advantages make it an excellent alternative to standard phenotypic AST with potential applications in clinical diagnostics and point-of-care testing, enabling rapid clinical decision-making, improvement of infectious disease management, and facilitation of antimicrobial stewardship. In addition to digital AST, the DP platform also provides a unique ability to track the temporal evolution of the bacterial colonies within each picowell. This is due to its confinement of isolated droplets into static, spatially-defined arrays, enabling the indexing and time-lapse microscopy monitoring of droplet contents over time. This feature allows for the exploration of the progeny of individual cells as they respond to antibiotic stress. For example, E. coli cells exposed to ampicillin sodium underwent adaptive morphogenesis, as shown in Fig. 4c. In the absence of antibiotics, nearly all bacteria within the picowells were in their planktonic state, actively swimming within the droplets (Movie S2) and exhibiting the characteristic size and morphology of E. coli in culture (see the top row in Fig. 4c). Conversely, at sub-MIC concentrations (100 μg/mL) of ampicillin sodium (see the bottom row in Fig. 4c), the cells exhibited morphological changes, elongating into filamentous forms, and became immobile (Movie S3). This filamentation is a self-preservation response triggered by exposure to ampicillin sodium, during which cell division halts, but cellular metabolism continues, leading to increased cell volume. These elongated cells may either resume division after several hours or remain in their filamentous state. This observation is consistent with results obtained on agar plates 38 . The DP platform's ability to follow morphological changes over time in a large number of individual cells allows the comparison of different bacterial strains' responses to antibiotics with varying mechanisms of action. This capability is invaluable for elucidating the biological mechanisms that enable cells to survive antibiotic stress at sub-MIC concentrations. Investigating microbial interactions Understanding microbe-microbe interactions is critical to predict microbiome function and to construct communities for desired outcomes. However, investigating these interactions poses a significant challenge due to the lack of suitable probing tools. The DP platform, with its ability to compartmentalize single cells and its assemblability, provides an ideal tool for studying microbial interactions. As a proof of concept, we used the DP platform to explore the interaction between Bacillus (an unreported strain isolated from the mucus layer of a slug by our group) and Salmonella . As depicted in Fig. 5a, 10 μL of Bacillus suspension was pipetted onto the center of a solidified agar medium sheet and evenly spread across the surface using a glass rod spreader. Then, the inoculated sheet was incubated at 37°C for 3 h. Next, 10 7 CFU/mL of Salmonella suspension was prepared and loaded into a PicoArray device. After discretization, the device was overlaid with the agar medium sheet inoculated with Bacillus . Following incubationat 37°C for 5 h, the Bacillus cells grew to dense microcolonies and there are clear zones around the microcolonies, indicating inhibition of Salmonella growth (Fig. 5b). This suggests an antagonistic relationship between Bacillus and Salmonella , where Bacillus exerts an inhibitory effect on Salmonella . The “digital” feature of the DP platform allows for precise, quantitative assessment of Bacillus antagonism against Salmonella . This was easily accomplished by dividing the number of microwells in the inhibition zone induced by a Bacillus microcolony by the number of microwells covered by the Bacillus microcolony (more details see Section S1 in the Supplementary Information). Furthermore, by measuring and comparing the antagonism levels of different Bacillus microcolonies (Fig. S3), we can also characterize the heterogeneity in cell-cell interactions, which is crucial for screening highly effective potent antibiotic-producing microorganisms from complex environmental samples. Recovery of target microorganisms A recurring desire of microbiologists is to be able to extract individual colonies of interest for further culture or analysis. This operation is particularly difficult in most microfluidic approaches and remains a blocking point for the adoption of microfluidics by biologists. Thanks to its reversible assembly, our DP platform can be readily used for recovering isolated target microorganisms for further characterization and scaling-up cultivation. To demonstrate the ability of the DP platform to recover target bacterial species, we prepared a two-strain bacterial suspension consisting of S. aureus and E. coli , loaded it into a PicoArray chip, and covered the chip with an agar sheet containing selective medium. The protocol was similar to that used in the experiment to isolate specific bacteria by coupling the DP chip with selective media (see the subsection of “enrichment and identification of specific bacterial species from mixed-species microbial communities”). After incubating the chip at 37℃ for 10 h, microcolonies visible to the naked eye appeared on the chip. Note that, to eliminate the cross-contamination risk during recovery, the bacterial suspension must be diluted enough so that the probability of two single cells located in neighboring microwells or sharing the same microwell is extremely low. We randomly picked three microcolonies from the chip using sterile toothpicks and transferred them to three tubes of liquid medium for scale-up cultivation (Fig. 6a). To identify the recovered microorganisms, genomic characterization was performed by PCR amplification and 16 S rRNA gene sequencing. As expected, 16S rRNA sequencing revealed that all three recovered clones were S. aureus (Fig. 6b), indicating that the recovered cultures are free from contamination and accurately represent the microorganisms enriched by the selective medium. These results suggest that isolation and recovery of target bacterial strains can be conducted effectively with the DP platform. Discussion We present here an attach-and-use digital plating (DP) platform for microbiological analysis. The DP platform, integrating digital assay formats with traditional plate culturing principles, demonstrates significant advantages in terms of efficiency, accuracy, and versatility for microbial detection and analysis. One of its most outstanding features is the ability to encapsulate single cells or small colonies in massively large numbers of compartments for growth, monitoring, and selection. This provides several benefits for microbiological detection and analysis, including: (1) confining bacterial growth in ultra-small volumes, which allows rapid accumulation of metabolic products or secreted molecules, thus significantly reducing the time required for microbial detection. This rapid turnaround is particularly beneficial in clinical settings where timely identification of pathogens can be critical for patient outcomes; (2) isolating individual bacteria from complex communities, which eliminates cultivation bias caused by competition and inhibitory effects, thus facilitating the recovery of rare or slow-growing microorganisms from complex ecosystems; and (3) massively analyzing large numbers of individual bacteria, which enables examination of phenotypic and genetic variability at the single-cell or small population level and high throughput screening of functional microorganisms. Another key feature provided uniquely by the DP platform is the flexibility offered by its assemblability, which allows for the replacement of the covering agar medium sheet to introduce multiple detection or stimuli reagents to microorganisms within picowells, thereby creating well-controlled and variable culture environments. This feature gives access to two new possibilities: (1) tracking the evolution of a population in controllably changing chemical environments, and (2) performing more complex experimental protocols. Furthermore, this feature makes the DP platform compatible with commercial ready-to-use agar plates (more details see Section S2 and Fig. S4 in the Supplementary Information). This compatibility offers several benefits: (1) facilitating seamless integration of the DP chip with existing laboratory protocols which allows researchers to leverage commercially available resources without the need for extensive modifications; (2) eliminating the need for end-users to prepare the solid agar media themselves, which significantly speeds up lab processes; and (3) ensuring high reproducibility and reliability since commercial ready-to-use agar plates are manufactured under controlled conditions. Compared to prevalent droplet microfluidic methods which are limited to cultivating suspended growth microorganisms and are not conducive to observing and examining the growth morphology of microcolonies, the DP platform combines the advantages of both solid culture medium surface-adherent cultivation and liquid culture medium suspension cultivation. This makes it suitable for cultivating a wider range of microorganisms. Moreover, the DP platform allows the expansion of single bacteria to bigger microcolonies since the reversible assembly of agar/chip allows overgrown microcolonies to spread to adjacent picowells, potentially increasing the sensitivity of biological readouts. In addition, downstream harvesting of selected microcolonies is more straightforward from discrete microcompartments than from droplets. The technical innovations of the DP platform will open new possibilities to various fields of microbiology, from defining single-cell phenotypic and genetic heterogeneity to investigating spatiotemporal dynamics of microbial communities, from precise quantitation of microbiota to systematically deciphering microbial interactions, from isolating rare and uncultured microbes to selectively extracting improved microbial strains. We believe that, attributed to its unique advantages, the DP platform will serve as a versatile and powerful tool for microbiologists to explore the world of microorganisms. In conclusion, the DP platform represents a significant advancement in microbiological techniques, offering a rapid, accurate, and versatile alternative to conventional plate culturing. Its ability to streamline microbial isolation, identification, and quantification processes, coupled with its low cost and ease of use, positions the DP platform as a valuable tool for both research and clinical microbiology laboratories. Methods Fabrication of PicoArray devices The PDMS PicoArray device containing an array of 113,137 hexagonal microwells was fabricated using the conventional soft lithography as described previously 30,31 . Briefly, SU-8 3010 and 3050 negative photoresists (MicroChem Corp., Newton, MA) were patterned onto separate silicon wafers to create two molds for the channel layer and microwell layer, respectively. Typical dimensions of the molds are: main channel = 52 mm (length) × 80 μm (width) × 60 μm (height); loading microchannel = 17.9 mm (length) × 30 μm (width) × 20 μm (height); gap of neighboring channels = 48 μm; microwell = 70 μm (diagonal) × 40 μm (height). Subsequently, a thoroughly degassed PDMS prepolymer, consisting of silicone elastomer (Sylgard 184) and curing agent (10:1, w/w), was poured onto the prepared SU-8 molds. After curing at 90 °C for 1 hour, the molded PDMS slabs were carefully peeled off from the molds and an inlet port was created on the PDMS channel layer with a punching tool. Finally, the PDMS channel layer and the PDMS microwell layer were face-to-face aligned and conformally contacted to form a reversible seal for subsequent experiments. Preparation of covering agar solid media sheets Covering agar medium sheets were prepared as follows: 2.5 g LB broth powder (CM158, Beijing Land Bridge Technology, China) and 1.5 g agar powder (Biowest, Spain) were dissolved in 1000 L water and autoclaved. After cooling to 60°C, the appropriate reagents ( e.g. , dye, antibiotics, specific metabolic indicator, etc ) were mixed thoroughly into the agar solution depending on the experimental purposes. Next, the mixture was poured into a sterilized PDMS chamber mold with dimensions of 76 mm × 26 mm × 1 mm and covered with a sterilized plastic sheet. Then, a glass slide and a weight were placed onto the plastic sheet. After solidification at room temperature, the PDMS chamber mold was removed to obtain an agar solid media sheet. Preparation of bacterial suspensions All bacterial samples used in this work were cultured from frozen glycerol stocks. The frozen stock of each species was stored at −80 °C and thawed at room temperature (25 °C) before use. After thawing, the stock was inoculated into a liquid medium and stabilized for over an hour in a shaking incubator at 37 °C. The stabilized bacteria were seeded in an agar plate containing a suitable medium. The agar plates were incubated at 37 °C for 12−24 h until colony formation was visible. Then, a liquid subculture was performed by picking up one colony (or a piece of a colony) with a sterilized inoculating loop, transferring it to a liquid medium, and incubating it at 37 °C overnight in a shaking incubator. The subculture solution was diluted with normal saline to a desired concentration. Antibiotic Ampicillin sodium salt was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The stock solution of ampicillin sodium was prepared by dissolving ampicillin sodium salt in distilled water at 400 μg/mL. The stock solutions were sterilized by filtering through 0.22-μm sterile nylon syringe filters and were stored at –20°C until use. The final concentrations of ampicillin sodium used for AST were between 0 and 400 μg/mL. PCR amplification and sequencing of the 16S rRNA gene Genomic DNA samples were prepared from the subculture solutions inoculated from the recovered microcolonies using the MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0, Takara, Japan). The 16S rRNA gene was PCR-amplified using the universal bacterial primer pair 27F/1492R. The PCR products were sent to a sequencing service provider (Sangon Biotech Co., Ltd., Shanghai, China) for the determination of the nucleotide sequence of the 16S rRNA gene fragment. The sequences were then aligned against the NCBI database using BLAST to identify the closest phylogenetic matches and a phylogenetic tree was constructed using MEGA X software. Images and data analysis Bright-field and Fluorescent images were acquired by an upright fluorescence microscope (CX40, Sunny Optical Technology Co., Ltd., China Germany) equipped with a CMOS camera (OD230R, SOPTOP, China). Image J (https://imagej.net) was used to recognize and count the microcolonies formed in the PicoArray devices. The detailed procedure is as follows: (1) Load an image which is needed to be analyzed. To do this, select: File > open . (2) Convert the image to grayscale. To do this, select: Image > Type > 8-bit . (3) Adjust threshold to highlight the picowells containing microcoloies. To do this, go to Image > Adjust > Threshold . (4) Binary conversion after thresholding is done. To do this, go to Process > Binary > Make Binary . (5) Count the highlighted picowells. To do this, go to Analyze > Analyze Particles . Declarations Data Availability The authors confirm that all data generated or analysed during this study are available within the paper and its supplementary information files. Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 61771078 and 62404194), the Natural Science Foundation of Xinjiang Uygur Autonomous Region (No. 2023D01C179), and Xinjiang Tianchi Yingcai Youth Doctoral Project (No. 51052300578). Author Contributions T.H. designed and carried out the experiments, prepared most of the data and wrote the paper; X.H. carried out and assisted with the experiments; L.W. fabricated the microwell array chips; B.S. consulted on the manuscript and contributed to writing the paper; G.L. proposed the idea, managed the research process and wrote the paper. Competing Interests The authors declare no competing interests. Additional information Supplementary information The online version contains supplementary material available at … References Stolp H, Starr MP. Principles of isolation, cultivation, and conservation of bacteria. In: The Prokaryotes: A Handbook on Habitats, Isolation, and Identification of Bacteria ). Springer (1981). Pommerville JC. Fundamentals of microbiology . Jones & Bartlett Publishers (2013). Lacroix J-M, Jarvi K, Batra SD, Heritz DM, Mittelman MW. PCR-based technique for the detection of bacteria in semen and urine. J. Microbiol. Methods 26, 61–71 (1996). 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Supplementary Files SupplementaryInformation.pdf MovieS1.mp4 MovieS2.mp4 MovieS3.mp4 ExtendedDataFig.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5298212","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":381613945,"identity":"76fef420-44f3-436b-9c86-0ca466b9f16a","order_by":0,"name":"Gang Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyklEQVRIiWNgGAWjYNCCAgk5CIONaC0GEsYka2FIbCBai7z72cMvGAws0vvbcwwYPpQdZuCf3YBfi+GZvDQLoMNyZ5x5Y8A449xhBok7BwhoacgxMwBp2SCRY8DM23YYyE4goKX/DVhLugFIy19itMhL5Bg/ACkDa2EkRouBxBszEGk448yzgoM959J5JG4QsqU/x/gDQ0WdPH978sYHP8qs5fhnELLlAAOb9B8wM4HhAJDkwa8eZEsDA/MHBqiWUTAKRsEoGAVYAQColzvNHI4irQAAAABJRU5ErkJggg==","orcid":"","institution":"Chongqing University","correspondingAuthor":true,"prefix":"","firstName":"Gang","middleName":"","lastName":"Li","suffix":""},{"id":381613946,"identity":"f87338a3-8b48-4fe1-b75e-1225f053c2f8","order_by":1,"name":"Tianbao Hu","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Tianbao","middleName":"","lastName":"Hu","suffix":""},{"id":381613947,"identity":"c84edb37-e5dd-4287-ac6c-389927dcf17e","order_by":2,"name":"Xue Han","email":"","orcid":"","institution":"Chongqing University","correspondingAuthor":false,"prefix":"","firstName":"Xue","middleName":"","lastName":"Han","suffix":""},{"id":381613948,"identity":"f2643911-e6c4-463b-9e20-a48649e48781","order_by":3,"name":"Lei Wu","email":"","orcid":"","institution":"Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Wu","suffix":""},{"id":381613949,"identity":"1af574e2-4079-47a8-8629-7b8866bb5f8f","order_by":4,"name":"Bangyong Sun","email":"","orcid":"","institution":"Xinjiang University","correspondingAuthor":false,"prefix":"","firstName":"Bangyong","middleName":"","lastName":"Sun","suffix":""}],"badges":[],"createdAt":"2024-10-20 11:53:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5298212/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5298212/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":69891260,"identity":"55384b8d-1049-41ce-b74e-ea4fe2570950","added_by":"auto","created_at":"2024-11-26 10:29:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3047618,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the DP (digital plating) technique.\u003c/strong\u003e (a) Schematic of the DP technique for fundamental microbiological practices. (b) Work flow of the DP technique.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/ce20c3990038e30db7ea8cab.png"},{"id":69891258,"identity":"0d41ecd6-a9ee-439e-8579-093d3e37f992","added_by":"auto","created_at":"2024-11-26 10:29:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3860419,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIsolation and cultivation of bacterial cells from a microbial mixture consisting of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eS. aureus\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and GFP-expressing\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e E. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with the DP platform.\u003c/strong\u003e (a) Typical bright-field, fluorescence, and merged microimages of microcolonies grown in a PicoArray device. (b) Enumeration of \u003cem\u003eS. aureus\u003c/em\u003eand GFP-expressing\u003cem\u003e E. coli\u003c/em\u003e in the microbial mixture using the DP platform \u003cem\u003evs.\u003c/em\u003e the agar plating.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/c3b02f9d4474defaea66bf9a.png"},{"id":69891573,"identity":"6f94cdfb-9184-4173-b915-094a93f819de","added_by":"auto","created_at":"2024-11-26 10:37:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3210482,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnrichment and identification of bacteria in complex samples by coupling the DP with selective and differential medium. \u003c/strong\u003e(a) Enrichment of \u003cem\u003eS. aureus\u003c/em\u003e from a microbial mixture consisting of \u003cem\u003eS. aureus\u003c/em\u003e and GFP-expressing \u003cem\u003eE. coli\u003c/em\u003e by coupling the DP platform with a NaCl-based selective medium. (i) Representative bright-field, fluorescence, and merged microimages of microcolonies formed in two PicoArray devices after being covered with LB medium agar sheet and selective medium agar sheet, respectively. (ii) Enumeration of the fluorescent and non-fluorescent microcolonies formed in the PicoArray devices with LB medium agar sheet and selective medium agar sheet, respectively. (b) Representative bright-field microimages of microcolonies formed in three PicoArray devices after being loaded with \u003cem\u003eS. aureus\u003c/em\u003e, \u003cem\u003eE. coli\u003c/em\u003e, and \u003cem\u003eS. aureus\u003c/em\u003e/\u003cem\u003eE. coli\u003c/em\u003e mixture and incubated with MCA sheets.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/c1f2d11d35cb0df97afa49c1.png"},{"id":69891261,"identity":"d38a58ac-40cf-4867-a82c-a895eb22389c","added_by":"auto","created_at":"2024-11-26 10:29:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5111017,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotyping antibiotic resistance using the DP platform. \u003c/strong\u003e(a) Representative bright-field microimages showing \u003cem\u003eE. coli\u003c/em\u003e microcolonies grown in the PicoArray chips under different concentrations of ampicillin sodium. (b) Dose response curve for \u003cem\u003eE. coli\u003c/em\u003e viability after treatment with different concentrations of ampicillin sodium. Calculation of MIC using a Gompertz function fit. Blue vertical dashed line shows the position of the MIC. (c) Representative bright-field microimages showing the morphology changes of \u003cem\u003eE. coli\u003c/em\u003eunder antibiotic stress.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/c8a74b05e5c3cc2a6725cdf9.png"},{"id":69891265,"identity":"c5ae7059-b957-43e0-8579-76cd3a45c4fd","added_by":"auto","created_at":"2024-11-26 10:29:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4285533,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInvestigation of microbial interactions using the DP platform. \u003c/strong\u003e(a) Workflow for investigating microbial interactions based on the DP method. (b) Representative bright-field microimages showing the inhibition zones agaisnt \u003cem\u003eSalmonella\u003c/em\u003e formed around the\u003cem\u003e Bacillus\u003c/em\u003e colonies.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/170aef74f1284123a5a42764.png"},{"id":69891574,"identity":"a5ac5209-2e09-4ede-8475-e3128679b69a","added_by":"auto","created_at":"2024-11-26 10:37:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1245573,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDP platform for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein situ\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e clonal culture and microcolony extraction. \u003c/strong\u003e(a) Workflow of recovering microcolonies from the PicoArray chip for genetic characterization. (b) Neighbour-joining phylogenetic tree based on 16S rRNA gene sequences of the recovered clones and the closest type strains sequences in the NCBI database. The red triangle represents the sequences of the recovered bacteria.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/57dc0632ea46487ce064e659.png"},{"id":69891576,"identity":"acd4a800-e660-4ba8-addd-af6606843b16","added_by":"auto","created_at":"2024-11-26 10:38:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":19767834,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/9bef0eac-1c3b-414b-8d48-5695c7319ebb.pdf"},{"id":69891257,"identity":"45023e44-2d9e-4dd3-abe3-d667aae247cd","added_by":"auto","created_at":"2024-11-26 10:29:48","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":592838,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/416a6c306564f8c7ec86ccfa.pdf"},{"id":69891575,"identity":"d7255330-2483-4943-bb38-a3093c605ea8","added_by":"auto","created_at":"2024-11-26 10:37:48","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3904206,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/20daf84f8a2117dac727324e.mp4"},{"id":69891266,"identity":"14a26e04-ddeb-4002-accf-8eaceaaa7402","added_by":"auto","created_at":"2024-11-26 10:29:48","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12063400,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS2.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/1d8946eaeaaf5f5bdc8c3acf.mp4"},{"id":69891264,"identity":"efd1bc5e-9974-4a3d-9a8c-ecca0763dc71","added_by":"auto","created_at":"2024-11-26 10:29:48","extension":"mp4","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":6811248,"visible":true,"origin":"","legend":"","description":"","filename":"MovieS3.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/d324681b6fea6c933616d3ad.mp4"},{"id":69891263,"identity":"8f779148-104e-4fca-a317-c0aabacf7189","added_by":"auto","created_at":"2024-11-26 10:29:48","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2477438,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedDataFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-5298212/v1/89961114d11f749f997cb9ad.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Digital Plating: A Universal and Versatile Microbiological Technique","fulltext":[{"header":"Introduction","content":"\u003cp\u003eIsolation, identification, quantification, and clonal cultivation of bacteria are crucial from fundamental microbiology to infectious disease diagnosis to industrial microbiology\u003csup\u003e1,2\u003c/sup\u003e. Culture-based methods,\u0026nbsp;though reliable and well-established,\u0026nbsp;are labor-intensive, time-consuming, and inefficient. To achieve rapid bacteria detection, various molecular methods have been proposed, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA)\u003csup\u003e3-6\u003c/sup\u003e. Despite high sensitivity and specificity, these methods often require extensive sample pretreatment, costly reagents, labor-intensive procedures, and skilled operators, which limit their practical use. In recent years, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has emerged as a routine tool for rapid bacterial identification in clinical microbiology\u003csup\u003e7-10\u003c/sup\u003e. Despite its success, MALDI-TOF MS struggles with differentiating closely related bacterial species, offers limited quantification capabilities, and involves complex technical requirements and high costs\u003csup\u003e11\u003c/sup\u003e. Moreover, it often necessitates pre-detection sampling processes to enhance bacterial concentration or purity.\u003c/p\u003e\n\u003cp\u003eRecently, droplet microfluidic technology, capable of encapsulating individual bacteria in microscale droplets for the micro-confined growth, has gained recognition as a powerful tool in microbiology. This technology enables various applications, including early detection of bacteria\u003csup\u003e15,16\u003c/sup\u003e, isolation of rare and uncultured microbes\u003csup\u003e17-20\u003c/sup\u003e, precise quantitation of bacteria\u003csup\u003e21,22\u003c/sup\u003e, characterization of the heterogeneity in bacterial populations\u003csup\u003e23,24\u003c/sup\u003e, selection of improved strains\u003csup\u003e25,26\u003c/sup\u003e, and exploration of microbial interactions\u003csup\u003e27-29\u003c/sup\u003e. Although this technology has greatly expanded the scope and context of microbiology, it faces\u0026nbsp;inherent\u0026nbsp;challenges such as droplet fusion during cultivation, limited substance exchange within droplets, and the requirement for expensive, complex equipment.\u003c/p\u003e\n\u003cp\u003eTo address these challenges, we developed a digital microbial assay, termed \u0026quot;digital plating\u0026quot; (DP), which is based on a solid medium-covered PicoArray device. In the DP platform, a bacterial suspension is partitioned into numerous picoliter microwells \u003cem\u003evia\u003c/em\u003e a pre-degassing-induced vacuum\u003csup\u003e30,31\u003c/sup\u003e, followed by coverage with a solid agar medium sheet for incubation and analysis. Unlike droplet-based assays, this well-based format eliminates the risk of droplet fusion during cultivation, ensuring the isolation of individual bacteria. Moreover, the immobilized array format of the well-based assay is also beneficial for time-lapse analysis since the isolated droplets can be spatially indexed. More importantly, the replaceability of the covering solid medium sheet allows precise control and flexible regulation of microbial growth conditions, which gives access to several new possibilities \u0026ndash; from precise selection of individuals with desired properties to rapid antibiotic susceptibility testing (AST) to cultivation of uncultivable microbes. Attributed to its unique advantages, the DP is expected to serve as a generic platform for microbiologists to explore the world of microorganisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eConcept and workflow of the DP platform\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn ideal microbial assay platform should be user-friendly (requiring no expensive instruments and well-trained personnel), rapid (producing results within a time frame\u0026nbsp;suitable for\u0026nbsp;point-of-care testing), precise (enabling\u0026nbsp;sensitive detection of\u0026nbsp;bacteria in a quantitative manner), reliable (with a low probability of false positive or false negative results), flexible (easily accommodating a wide range of microbiological applications), and cost-effective (readily accessible to any global setting). To this end, we propose the\u0026nbsp;\u0026quot;digital plating\u0026quot; (DP)\u0026nbsp;technique. The principal strategy of our DP technique is to integrate a powerful digital assay format with the conventional plating principle for rapid, precise, reliable, and cost-effective\u0026nbsp;microbial detection and analysis. Fig. 1a illustrates the concept of our DP technique. This technique involves two principal components: a PicoArray device for stochastic compartmentalization of single bacteria to enable digital assays and a covering agar sheet that serves as a solid culture medium to support bacterial growth. The DP workflow\u0026nbsp;comprises\u0026nbsp;four major steps: sample discretization, assembly of the agar/chip, device incubation, and microscopic examination (Fig. 1b and Movie S1 in the Supplementary Information). To facilitate sample discretization, a pre-degassing-driven self-pumping mechanism is used to partition the bacteria suspension into a large number of picowells and stochastically encapsulate single bacteria into compartments\u003csup\u003e30,31\u003c/sup\u003e; after\u0026nbsp;compartmentalization, an agar-based solid medium sheet is conformally attached to the device to seal all picowells, which provides a physical barrier to trap motile cells within the picowells; next, the device is incubated and the growth of bacteria in microwells is examined microscopically to determine phenotypic characters. Due to its digital format, this technique offers several main advantages: (1) eliminating the interspecies competition and biases due to growth rate differences, which facilitates recovery of rare or slow-growing microorganisms from complex ecosystems; (2) allowing the study of the heterogeneity of bacteria that are masked in ensemble measurements; (3) absolutely quantifying the concentration of bacteria, which enables highly sensitive detection of bacteria over a wide range of concentrations in a quantitative manner; (4) facilitating timely detection of pathogenic bacteria owing to microconfinement-induced rapid accumulation of detectable metabolites. Meanwhile, the agar sheet provides culture conditions analogous to those in the conventional plating techniques, thus allowing smooth and seamless transfer of well-established characterization and analysis methods from conventional plating techniques to this platform and easy interpretation of the obtained results. More importantly, the replaceability of the covering agar sheet offers more flexibility for digital microbial assays. For instance, if an agar sheet containing a selective medium is applied on the PicoArray device, the DP technique can isolate a particular strain of microorganisms; if an agar sheet containing a differential medium is applied on the PicoArray device, the DP technique can identify and differentiate closely-related microorganisms; if an agar sheet containing a certain antibiotic is applied on the PicoArray device, the DP technique can\u0026nbsp;analyse the response of bacteria to the antibiotic. Furthermore, our DP platform can sequentially combine the PicoArray with multiple different medium sheets, \u003cem\u003ei.e.\u003c/em\u003e, allowing a wide range of culture conditions to be applied on a single device. Thanks to its similarities to conventional plate methods, our DP platform allows \u003cem\u003ein situ\u003c/em\u003e clonal culturing of single bacteria isolated from heterogeneous samples without requiring extra sorting and inoculation steps, and the cultivated pure microbes can be recovered directly for further off-chip culture or genotyping.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCharacterization of the DP platform\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA key premise for digital bioassays is stochastic encapsulation of target biospecimens into massive numbers of equal-volume small compartments. To achieve precise and reliable digital bioassays, the homogeneity of partitions is a critical factor\u003csup\u003e32,33\u003c/sup\u003e. Here, we first\u0026nbsp;quantified the variance of partition volumes created by the self-discretization based on the combination of pre-degassing pumping and capillary force-assisted dewetting\u003csup\u003e34\u003c/sup\u003e. To investigate the distribution of the partitioned volumes across the device, the fluorescence microscopy images of the partitioned Rhodamine B droplets from different positions on the device were captured using a CCD camera.\u0026nbsp;It is assumed that Rhodamine B solution is homogeneous, so the total fluorescence intensity of each Rhodamine B droplet is directly proportional to its volume, and then\u0026nbsp;the uniformity of the total fluorescence intensities of partitioned droplets can reflect their volume uniformity. The captured images of droplets from different areas on the device were analyzed using an image analysis software (ImageJ) to determine the total fluorescence intensity of each droplet.\u0026nbsp;From six random areas of the device, 3,200 droplets were selected for measurement\u0026nbsp;and calculation. The calculated total fluorescence intensities for all these droplets were then plotted into a volume distribution histogram, showing that the coefficient of variation (CV) of the total fluorescence intensities measured from 3,200 randomly selected droplets is about 4.78% (Extended Data Fig. 1a). This demonstrates that the PicoArray device, combined with the self-discretization mechanism, achieves highly uniform discretization for digital microbial assays.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFurthermore, for our DP platform, effective diffusion of molecules from the covering agar sheet to the picowells is crucial for\u0026nbsp;nutrient delivery and microenvironment regulation.\u0026nbsp;To investigate whether the nutrient or drug molecules could diffuse from the covering agar sheet into the picowells, we conducted\u0026nbsp;a simulation experiment by attaching a red dye-stained agar sheet to the PicoArray device and\u0026nbsp;subsequently monitoring the color change in the picowells of the device. After attaching for 15 min and then peeling off the agar sheet, the color of the solution in all picowells of the device changed to red (Extended Data Fig. 1b and Fig. S1 in the Supplementary Information), confirming that the dye molecules within the agar sheet could effectively and homogeneously diffuse into the picowells of the device. Such diffusion-induced molecule transport allows us to use the agar sheet for supplying nutrients to microbes confined in picowells and chemically regulating the microbial growth microenvironment in picowells, which offers unparalleled flexibility for various microbiological studies, such as monitoring microbial response upon perturbation, screening novel antibiotics, and culturing the uncultured microorganisms. Furthermore, this diffusion also allows reverse molecule transport, \u003cem\u003ei.e.\u003c/em\u003e, from picowells to the covering agar sheet, thus enabling facile detection of metabolites and signaling molecules secreted by bacteria confined in picowells. That is, this platform allows us to perform biochemical assays to identify microcolonies formed in picowells if the covering agar sheet contains differential media.\u003c/p\u003e\n\u003cp\u003eOur DP method is based on the fact that when\u0026nbsp;single bacteria partitioned into picowells are incubated, each of the microconfined single viable bacteria will proliferate forming a visible and independent colony. This allows for precise and absolute quantification of bacteria in samples.\u0026nbsp;To achieve accurate enumeration, it is essential to culture individual bacterial cells until their colonies are visible, while also ensuring that each colony remains independent, avoiding fusion with neighboring colonies.\u0026nbsp;To determine the optimal time point for microcolony counting using the PicoArray device, we examined the effect of cultivation time on the visibility and independence of microcolonies\u0026nbsp;formed in picowells. A time-lapse sequence of\u0026nbsp;\u003cem\u003eE. coli\u003c/em\u003e cultured in the PicoArray device covered with a\u0026nbsp;Luria-Bertani (LB)\u0026nbsp;agar sheet\u0026nbsp;is shown in Extended Data\u0026nbsp;Fig. 1c.\u0026nbsp;In these images, dark spots represent microwells where bacteria grew and formed microcolonies, while blank spots indicate microwells that either initially lacked cells or where the cells failed to form colonies, likely due to low growth rates.\u0026nbsp;Most microcolonies were nearly invisible before 5 h. After incubation for 6 h, most microcolonies were discernible and confined to individual picowells without fusion between neighboring colonies. However, after 7 h of incubation, there was a noticeable overgrowth\u0026nbsp;of the formed microcolonies, which spread into\u0026nbsp;adjacent\u0026nbsp;picowells. Thus, to ensure a precise enumeration, counting of \u003cem\u003eE. coli\u003c/em\u003e microcolonies should be performed between 6 and 7 h of incubation. It is worth noting that the optimal counting time varies across different microorganisms due to significant differences in their growth rates.\u003c/p\u003e\n\u003cp\u003eTo demonstrate the capability of our DP platform to rapidly and accurately quantify bacteria, we loaded\u0026nbsp;serially diluted samples over a range of starting cell densities from\u0026nbsp;a previously quantified \u003cem\u003eSalmonella\u003c/em\u003e stock with Mueller-Hinton broth into the device, followed by discretization and incubation.\u0026nbsp;After 6 h of incubation, all devices were examined by microscopy to count the positive microwells containing microcolonies in each chip. Extended Data Fig. 2a shows representative micro-images of PicoArray devices loaded with different concentrations of\u0026nbsp;\u003cem\u003eSalmonella\u003c/em\u003e after incubation.\u0026nbsp;As expected, reducing the bacterial input concentration resulted in fewer picowells occupied by microcolonies. The proportion of positive microwells in each device was then counted, and the cell concentration in colony-forming units per milliliter (CFU/mL) was calculated using Poisson statistical analysis\u003csup\u003e32,33\u003c/sup\u003e.\u0026nbsp;This count was plotted against the CFU/mL measured in parallel by conventional colony counting on agar plates. As shown in Extended DataFig. 2b, the\u0026nbsp;\u003cem\u003eSalmonella\u003c/em\u003e concentrations measured using the PicoArray-based digital CFU method correspond very well to those obtained \u003cem\u003evia\u003c/em\u003e the conventional plate counting method (R\u003csup\u003e2\u003c/sup\u003e = 0.9992) in the entire 5 log range of the measured sample concentrations, demonstrating the capability of the PicoArray-based digital CFU method to precisely quantify microbes. Importantly, the entire process for the digital CFU method took only 6 h, significantly shorter than that with the conventional plating method, which is beneficial to rapid diagnosis and prompt treatment of infectious diseases.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation of single bacteria from a heterogeneous sample\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn nature, microorganisms typically grow as mixed populations rather than as single species. Therefore, isolating individual microorganisms from complex microbial communities is an essential prerequisite for downstream phenotypic and genomic characterization, as well as for scaled-up culture.\u0026nbsp;Due to\u0026nbsp;its ability to compartmentalize single cells into massively large numbers of subunits for clonal growth,\u0026nbsp;our DP platform is ideal for the isolation of individual bacteria\u0026nbsp;from heterogeneous samples.\u0026nbsp;To demonstrate the DP platform\u0026apos;s capacity for single-cell separation and cultivation, we prepared a simple two-strain microbial community consisting of \u003cem\u003eS. aureus\u003c/em\u003e and green fluorescent protein (GFP)-expressing \u003cem\u003eE. coli\u003c/em\u003e (BL21).\u0026nbsp;First, both strains were cultivated overnight, diluted 10,000 times in LB broth, respectively, and then mixed in a ratio of 1:1 (v/v)\u0026nbsp;to obtain a microbial mixture. After discretization in the device and incubation at 37 \u0026ordm;C for 8 h,\u0026nbsp;three distinct populations of microwells were observed\u0026nbsp;\u003cem\u003evia\u003c/em\u003e microscopy (Fig. 2a). One population of microwells was empty microwells, which was predicted to represent compartments containing no bacteria.\u0026nbsp;The remaining microwells contained microcolonies with two distinct morphologies: densely packed microcolonies and thin microcolonies with fewer cells.\u0026nbsp;Given that \u003cem\u003eE. coli\u003c/em\u003e showed slower growth under the used conditions, the population of microwells containing thinner microcolonies was predicted to represent compartments containing \u003cem\u003eE. coli\u003c/em\u003e strain. The population of microwells containing denser microcolonies was assigned to correspond to compartments containing the best-growing bacteria, here, \u003cem\u003eS. aureus\u003c/em\u003e.\u0026nbsp;This hypothesis was confirmed through fluorescence microscopy. Under 523 nm excitation light, only the thinner microcolonies exhibited green fluorescence, indicating the presence of GFP-expressing \u003cem\u003eE. coli\u003c/em\u003e, while the denser microcolonies showed no fluorescence, confirming them as \u003cem\u003eS. aureus\u003c/em\u003e (Fig. 2a).\u0026nbsp;Based on the total number of picowells and the count of microwells occupied by fluorescent and dense microcolonies, we calculated the numbers of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e using a method similar to digital PCR\u003csup\u003e35\u003c/sup\u003e.\u0026nbsp;As shown in Fig.\u0026nbsp;2b, the calculated numbers of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003ecorresponded closely with the expected number of\u0026nbsp;\u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003e(estimated \u003cem\u003evia\u003c/em\u003e agar plating).\u0026nbsp;These results demonstrate that the DP allows for rapid and highly effective separation of different species coexisting in a complex sample. This suggests that our DP platform can simultaneously count and isolate different bacterial cells in mixed microbial communities by using vision analysis or light scattering based methods.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnrichment and identification of specific bacterial species from mixed-species microbial communities\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to enabling spatial separation\u0026nbsp;of different bacteria in a single sample, our DP platform can also be combined with selective and/or differential media to enrich and identify specific bacteria in complex samples.\u0026nbsp;To demonstrate the selective-medium-assisted enrichment capability of the DP platform, we prepared a microbial mixture consisting of \u003cem\u003eS. aureus\u003c/em\u003e and GFP-expressing \u003cem\u003eE. coli\u003c/em\u003e and loaded it into a PicoArray device.\u0026nbsp;When the device was covered with an agar sheet containing LB medium and incubated\u0026nbsp;at 37 \u0026ordm;C for 8 h, both bacterial strains grew into microcolonies (see the left column in Fig. 3a-i). The two species were easily distinguished by comparing bright field and fluorescent microimages:\u0026nbsp;one population exhibited green fluorescence, while the other did not. In contrast, when the device was covered with an agar sheet made with NaCl-based selective medium (containing 7.5% NaCl) and incubated\u0026nbsp;at 37 \u0026ordm;C for 8 h, only non-fluorescent microcolonies appeared (see the right column inFig. 3a-i), implying that none of the isolates of \u003cem\u003eE. coli\u003c/em\u003e but only the isolates of \u003cem\u003eS. aureus\u003c/em\u003e grew in the NaCl-based medium-covered PicoArray device.\u0026nbsp;This is because \u003cem\u003eStaphylococci\u003c/em\u003e species can tolerate high salt concentrations and grow on this selective medium agar, whereas other bacteria may not. Additionally, the number of non-fluorescent microcolonies in the PicoArray device covered with LB medium was nearly equal to the total number of microcolonies formed in the device covered with NaCl-based medium (Fig. 3a-ii), further confirming the selectivity of the NaCl-based medium. These results demonstrate that the DP platform, when coupled with selective media, can effectively enrich target microorganisms from complex microbial communities with high specificity.\u003c/p\u003e\n\u003cp\u003eIn addition, we also performed an experiment to demonstrate the differential-medium-assisted identification ability of our DP platform. Here, MacConkey agar (MCA), one of the earliest and most common\u0026nbsp;differential media, was chosen to demonstrate the applicability of \u0026ldquo;DP + differential medium\u0026rdquo; to distinguishing specific bacterial species from other bacterial types.\u0026nbsp;The experiment was performed by discretizing a \u003cem\u003eS. aureus\u0026nbsp;\u003c/em\u003esuspension,an\u003cem\u003e\u0026nbsp;E. coli\u0026nbsp;\u003c/em\u003esuspension, and a mixture of the two in three PicoArray devices, respectively, covering each device with an\u0026nbsp;MCA sheet, and then incubating them at 37 \u0026ordm;C for 8 h. As shown in Fig. 3b, the microcolonies formed in three devices displayed different appearances in color:\u0026nbsp;the \u003cem\u003eE. coli\u003c/em\u003e-loaded device exclusively exhibited red microcolonies, the \u003cem\u003eS. aureus\u003c/em\u003e-loaded device displayed only colorless microcolonies, and the device loaded with the mixture of both species presented both red and colorless microcolonies.\u0026nbsp;This differentiation is attributed to the composition of the MCA medium, which contains lactose and neutral red (pH indicator). \u003cem\u003eE. coli\u003c/em\u003e can ferment\u0026nbsp;lactose to produce lactic acid,\u0026nbsp;thereby\u0026nbsp;decreasing the pH of the agar and turning the indicator (neutral red) pink.\u0026nbsp;In contrast, \u003cem\u003eS. aureus\u003c/em\u003e cannot utilize lactose, resulting in colorless colonies due to the absence of pH alteration and indicator color change.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRapid phenotypic AST\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnother important application scenario of the DP platform is AST, a fundamental mission of the clinical microbiology laboratory, which is paramount for effective infection management. In particular, a rapid and precise AST assay can assist clinicians to choose appropriate antimicrobial therapies for various pathogen infections and avoid unnecessary overprescription. Thanks to its ability to miniaturize bacterial cultures to picoliter levels which can dramatically alter early colonization and self-regulated quorum signaling\u003csup\u003e36\u003c/sup\u003e, the DP platform enables faster measurement of the ability of individual bacteria to form microcolonies at different antibiotic concentrations.\u0026nbsp;This is achieved through microconfinement-induced rapid increases in cell density or the accumulation of detectable metabolites, thus enabling rapid AST assays. To demonstrate the applicability of the DP platform to AST assays, we used \u003cem\u003eE. coli\u003c/em\u003e as a model bacterium and\u0026nbsp;ampicillin sodium\u0026nbsp;as a model antibiotic to perform AST experiments. Multiple PicoArray devices were run in parallel, each loaded with the same concentration of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003esuspensions and covered with an agar sheet containing a different concentration of ampicillin sodium.\u0026nbsp;In addition, a control device was run in parallel, which was\u0026nbsp;loaded with the same bacterial concentration as the others but covered with an agar sheet without any antibiotic. This control allows us to estimate the number of cells initially loaded into each DP chip.\u0026nbsp;After 6 h incubation at 37℃, the chips were imaged \u003cem\u003evia\u003c/em\u003e microscopy. It was observed that\u0026nbsp;the bacterial growth was inhibited in the presence of antibiotics.\u0026nbsp;As shown in\u0026nbsp;Fig. 4a,\u0026nbsp;the number of microwells containing microcolonies decreased with increasing concentrations of ampicillin sodium, and no cells survived at concentrations above 200 \u0026mu;g/mL. The percentages of surviving cells at 6 h were plotted against\u0026nbsp;the concentrations of\u0026nbsp;ampicillin sodium,\u0026nbsp;exhibiting a monophasic dose-response curve (Fig. 4b). The minimum inhibitory concentration (MIC) value, obtained by fitting the bacterial growth data to a Gompertz model\u003csup\u003e37\u003c/sup\u003e, was 196.9\u0026nbsp;\u0026mu;g/mL, which closely matches the MIC determined by the broth microdilution method (193.4 \u0026mu;g/mL) (Fig. S2 in the Supplementary Information). Compared to conventional AST methods, our DP platform not only\u0026nbsp;consumes fewer reagents (~20 \u0026mu;L) but also allows faster\u0026nbsp;antibiotic susceptibility\u0026nbsp;assays (\u0026lt; 6 h). More importantly, it can characterize physiological responses of individual cells to different antibiotic concentrations, which enables the quantitative phenotyping of heterogeneous resistance, or heteroresistance, with single-cell resolution\u003csup\u003e23\u003c/sup\u003e. These advantages\u0026nbsp;make it an excellent alternative to standard phenotypic AST with potential applications in clinical diagnostics and point-of-care testing,\u0026nbsp;enabling rapid clinical decision-making, improvement of infectious disease management, and facilitation of antimicrobial stewardship.\u003c/p\u003e\n\u003cp\u003eIn addition to digital AST, the DP platform also provides a unique ability to track the temporal evolution of the bacterial colonies within each picowell. This is due to its confinement of isolated droplets into static, spatially-defined arrays, enabling the indexing and time-lapse microscopy monitoring of droplet contents over time. This feature allows for the exploration of the progeny of individual cells as they respond to antibiotic stress. For example, \u003cem\u003eE. coli\u003c/em\u003e cells exposed to ampicillin sodium underwent adaptive morphogenesis, as shown in Fig. 4c. In the absence of antibiotics, nearly all bacteria within the picowells were in their planktonic state, actively swimming within the droplets (Movie S2) and exhibiting the characteristic size and morphology of \u003cem\u003eE. coli\u003c/em\u003e in culture (see the top row\u0026nbsp;in Fig. 4c).\u0026nbsp;Conversely, at sub-MIC concentrations (100\u0026nbsp;\u0026mu;g/mL) of ampicillin sodium (see the bottom row\u0026nbsp;in Fig. 4c), the cells exhibited morphological changes, elongating into filamentous forms, and became immobile (Movie S3). This filamentation is a self-preservation response triggered by exposure to ampicillin sodium, during which cell division halts, but cellular metabolism continues, leading to increased cell volume. These elongated cells may either resume division after several hours or remain in their filamentous state. This observation is consistent with results obtained on agar plates\u003csup\u003e38\u003c/sup\u003e. The DP platform\u0026apos;s ability to follow morphological changes over time in a large number of individual cells allows the comparison of different bacterial strains\u0026apos; responses to antibiotics with varying mechanisms of action. This capability is invaluable for elucidating the biological mechanisms that enable cells to survive antibiotic stress at sub-MIC concentrations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvestigating microbial interactions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUnderstanding microbe-microbe interactions is critical to predict microbiome function and to construct communities for desired outcomes. However, investigating these interactions poses a significant challenge due to the lack of suitable probing tools.\u0026nbsp;The DP platform, with its ability to compartmentalize single cells and its assemblability, provides an ideal tool for studying microbial interactions.\u0026nbsp;As a proof of concept, we used the DP platform to explore the interaction between \u003cem\u003eBacillus\u003c/em\u003e (an unreported strain isolated from the mucus layer of a slug by our group) and \u003cem\u003eSalmonella\u003c/em\u003e. As depicted in Fig. 5a, 10 \u0026mu;L of \u003cem\u003eBacillus\u003c/em\u003e suspension was pipetted onto the center of a solidified agar medium sheet and evenly spread across the surface using a glass rod spreader. Then, the inoculated sheet was incubated at 37\u0026deg;C for 3 h. Next, 10\u003csup\u003e7\u003c/sup\u003e CFU/mL of \u003cem\u003eSalmonella\u0026nbsp;\u003c/em\u003esuspension was prepared and loaded into a PicoArray device. After discretization, the device was overlaid with the agar medium sheet inoculated with \u003cem\u003eBacillus\u003c/em\u003e. Following incubationat 37\u0026deg;C for 5 h, the \u003cem\u003eBacillus\u003c/em\u003e cells grew to dense microcolonies and there are clear zones around the microcolonies, indicating inhibition of \u003cem\u003eSalmonella\u003c/em\u003e growth (Fig. 5b). This suggests an antagonistic relationship between \u003cem\u003eBacillus\u003c/em\u003e and \u003cem\u003eSalmonella\u003c/em\u003e, where \u003cem\u003eBacillus\u003c/em\u003e exerts an inhibitory effect on \u003cem\u003eSalmonella\u003c/em\u003e.\u0026nbsp;The \u0026ldquo;digital\u0026rdquo; feature of the DP platform allows for precise, quantitative assessment of \u003cem\u003eBacillus\u003c/em\u003e antagonism against \u003cem\u003eSalmonella\u003c/em\u003e. This was easily accomplished by dividing the number of microwells in the inhibition zone induced by a \u003cem\u003eBacillus\u003c/em\u003e microcolony by the number of microwells covered by the \u003cem\u003eBacillus\u003c/em\u003e microcolony (more details see\u0026nbsp;Section S1\u0026nbsp;in the Supplementary Information). Furthermore, by measuring and comparing the antagonism levels of different \u003cem\u003eBacillus\u003c/em\u003e microcolonies (Fig. S3), we can also characterize the heterogeneity in cell-cell interactions, which is crucial for screening highly effective potent antibiotic-producing microorganisms\u0026nbsp;from complex environmental samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecovery of target microorganisms\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA recurring desire of microbiologists is to be able to extract individual colonies of interest for further culture or analysis. This operation is particularly difficult in most microfluidic approaches and remains a blocking point for the adoption of microfluidics by biologists. Thanks to its reversible assembly, our DP platform can be readily used for recovering isolated target microorganisms for further characterization and scaling-up cultivation. To demonstrate the ability of the DP platform to recover target bacterial species, we prepared a two-strain bacterial suspension consisting of \u003cem\u003eS. aureus\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e, loaded it into a PicoArray chip, and covered the chip with an agar sheet containing selective medium.\u0026nbsp;The protocol was similar to that used in the experiment to isolate specific bacteria by coupling the DP chip with selective media (see the subsection of \u0026ldquo;enrichment and identification of specific bacterial species from mixed-species microbial communities\u0026rdquo;).\u0026nbsp;After incubating the chip at 37℃ for 10 h, microcolonies visible to the naked eye appeared on the chip. Note that, to eliminate the cross-contamination risk during recovery, the bacterial suspension must be diluted enough so that the probability of two single cells located in neighboring microwells or sharing the same microwell is extremely low. We randomly picked three microcolonies from the chip using sterile toothpicks and transferred them to three tubes of liquid medium for scale-up cultivation (Fig. 6a). To identify the recovered microorganisms, genomic characterization was performed by PCR amplification and 16\u0026thinsp;S rRNA gene sequencing. As expected,\u0026nbsp;16S rRNA sequencing\u0026nbsp;revealed that all three recovered clones were\u003cem\u003e\u0026nbsp;S. aureus\u003c/em\u003e (Fig. 6b), indicating that the recovered cultures are free from contamination and accurately represent the microorganisms enriched by the selective medium. These results suggest that isolation and recovery of target bacterial strains can be conducted effectively with the DP platform.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe present here an attach-and-use digital plating (DP) platform for microbiological analysis. The DP platform, integrating digital assay formats with traditional plate culturing principles, demonstrates significant advantages in terms of efficiency, accuracy, and versatility for microbial detection and analysis. One of its most outstanding features is the ability to encapsulate single cells or small colonies in massively large numbers of compartments for growth, monitoring, and selection. This provides several benefits for microbiological detection and analysis, including: (1) confining\u0026nbsp;bacterial growth\u0026nbsp;in ultra-small volumes, which allows rapid accumulation of metabolic products or secreted molecules, thus significantly reducing the time required for microbial detection. This rapid turnaround is particularly beneficial in clinical settings where timely identification of pathogens can be critical for patient outcomes;\u0026nbsp;(2) isolating individual bacteria from complex communities, which eliminates cultivation bias caused by competition and inhibitory effects, thus facilitating the recovery of rare or slow-growing microorganisms from complex ecosystems; and (3) massively analyzing large numbers of individual bacteria, which enables examination of phenotypic and genetic variability at the single-cell or small population level and high throughput screening of functional microorganisms. Another key feature provided uniquely by the DP platform is the flexibility offered by its\u0026nbsp;assemblability, which allows for\u0026nbsp;the replacement of\u0026nbsp;the covering agar medium sheet to introduce multiple detection or stimuli reagents to microorganisms within picowells, thereby creating well-controlled and variable culture environments. This feature gives access to two new possibilities: (1) tracking the evolution of a population in controllably changing chemical environments, and (2) performing more complex experimental protocols. Furthermore, this feature makes the DP platform\u0026nbsp;compatible with commercial ready-to-use agar plates (more details see Section S2 and Fig. S4 in the\u0026nbsp;Supplementary Information). This compatibility offers several benefits: (1) facilitating seamless integration of the DP chip with existing laboratory protocols which allows researchers to leverage commercially available resources without the need for extensive modifications; (2) eliminating the need for end-users to prepare the solid agar media themselves, which significantly speeds up lab processes; and (3)\u0026nbsp;ensuring\u0026nbsp;high reproducibility\u0026nbsp;and reliability since\u0026nbsp;commercial ready-to-use agar plates\u0026nbsp;are manufactured under controlled conditions.\u003c/p\u003e\n\u003cp\u003eCompared to prevalent droplet microfluidic methods which are limited to cultivating suspended growth microorganisms and are not conducive to observing and examining the growth morphology of microcolonies, the DP platform combines the advantages of both solid culture medium surface-adherent cultivation and liquid culture medium suspension cultivation.\u0026nbsp;This makes it suitable for\u0026nbsp;cultivating a wider range of microorganisms.\u0026nbsp;Moreover, the DP platform allows the expansion of single bacteria to bigger microcolonies since the reversible assembly of agar/chip allows overgrown microcolonies to spread to adjacent picowells, potentially increasing the sensitivity of biological readouts. In addition, downstream harvesting of selected microcolonies is more straightforward from discrete microcompartments than from droplets.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe technical innovations of the DP platform will open new possibilities to various fields of microbiology, from defining single-cell phenotypic and genetic heterogeneity to investigating spatiotemporal dynamics of microbial communities, from precise quantitation of microbiota to systematically deciphering microbial interactions, from isolating rare and uncultured microbes to selectively extracting improved microbial strains. We believe that, attributed to its unique advantages, the DP platform will serve as a versatile and powerful tool for microbiologists to explore the world of microorganisms.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn conclusion, the DP platform represents a significant advancement in microbiological techniques, offering a rapid, accurate, and versatile alternative to conventional plate culturing. Its ability to streamline microbial isolation, identification, and quantification processes, coupled with its low cost and ease of use, positions the DP platform as a valuable tool for both research and clinical microbiology laboratories.\u0026nbsp;\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eFabrication of PicoArray devices\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PDMS PicoArray device containing an array of 113,137 hexagonal microwells was fabricated using the conventional soft lithography as described previously\u003csup\u003e30,31\u003c/sup\u003e. Briefly, SU-8 3010 and 3050 negative photoresists (MicroChem Corp., Newton, MA) were patterned onto separate silicon wafers to create two molds for the channel layer and microwell layer, respectively. Typical dimensions of the molds are: main channel = 52 mm (length) \u0026times; 80 \u0026mu;m (width) \u0026times; 60 \u0026mu;m (height); loading microchannel = 17.9 mm (length) \u0026times; 30 \u0026mu;m (width) \u0026times; 20 \u0026mu;m (height); gap of neighboring channels = 48 \u0026mu;m; microwell = 70 \u0026mu;m (diagonal) \u0026times; 40 \u0026mu;m (height). Subsequently, a thoroughly degassed PDMS prepolymer, consisting of silicone elastomer (Sylgard 184) and curing agent (10:1, w/w), was poured onto the prepared SU-8 molds. After curing at 90 \u0026deg;C for 1 hour, the molded PDMS slabs were carefully peeled off from the molds and an inlet port was created on the PDMS channel layer with a punching tool. Finally, the PDMS channel layer and the PDMS microwell layer were face-to-face aligned and conformally contacted to form a reversible seal for subsequent experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of covering agar solid media sheets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCovering agar medium sheets were prepared as follows: 2.5 g LB broth powder (CM158, Beijing Land Bridge Technology, China) and 1.5 g agar powder (Biowest, Spain) were dissolved in 1000 L water and autoclaved. After cooling to 60\u0026deg;C, the appropriate reagents (\u003cem\u003ee.g.\u003c/em\u003e, dye, antibiotics,\u0026nbsp;specific metabolic\u0026nbsp;indicator, \u003cem\u003eetc\u003c/em\u003e) were mixed thoroughly into the agar solution depending on the experimental purposes. Next, the mixture was poured into a sterilized PDMS chamber mold with dimensions of 76 mm \u0026times; 26 mm \u0026times; 1 mm and covered with a sterilized plastic sheet. Then, a glass slide and a weight were placed onto the plastic sheet. After solidification at room temperature, the PDMS chamber mold was removed to obtain an agar solid media sheet.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of bacterial suspensions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll bacterial samples used in this work were cultured from frozen glycerol stocks. The frozen stock of each species was stored at \u0026minus;80 \u0026deg;C and thawed at room temperature (25 \u0026deg;C) before use. After thawing, the stock was inoculated into a liquid medium and stabilized for over an hour in a shaking incubator at 37 \u0026deg;C. The stabilized bacteria were seeded in an agar plate containing a suitable medium. The agar plates were incubated at 37 \u0026deg;C for 12\u0026minus;24 h until colony formation was visible. Then, a liquid subculture was performed by picking up one colony (or a piece of a colony) with a sterilized inoculating loop, transferring it to a liquid medium, and incubating it at 37 \u0026deg;C overnight in a shaking incubator. The subculture solution was diluted with normal saline to a desired concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntibiotic\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmpicillin sodium salt was purchased from Shanghai Yuanye Bio-Technology Co., Ltd. The stock solution of ampicillin sodium was prepared by dissolving ampicillin sodium salt in distilled water at 400\u0026nbsp;\u0026mu;g/mL. The stock solutions were sterilized by filtering through 0.22-\u0026mu;m sterile nylon syringe filters and were stored at \u0026ndash;20\u0026deg;C until use. The final concentrations of ampicillin sodium used for AST were between 0 and 400\u0026nbsp;\u0026mu;g/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePCR amplification and sequencing of the 16S rRNA gene\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic DNA samples were prepared from the subculture solutions inoculated from the recovered microcolonies using the MiniBEST Bacteria Genomic DNA Extraction Kit Ver. 3.0, Takara, Japan). The 16S rRNA gene was PCR-amplified using the universal bacterial primer pair 27F/1492R. The PCR products were\u0026nbsp;sent to a sequencing service provider (Sangon Biotech Co., Ltd., Shanghai, China) for the determination of the nucleotide sequence of the 16S rRNA gene fragment. The sequences were then aligned against the NCBI database using BLAST to identify the closest phylogenetic matches and a phylogenetic tree was constructed using MEGA X software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImages and data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBright-field and Fluorescent images were acquired by an upright fluorescence microscope (CX40,\u0026nbsp;Sunny Optical Technology Co., Ltd., China Germany) equipped with a CMOS camera (OD230R, SOPTOP, China). Image J (https://imagej.net) was used to recognize and count the microcolonies formed in the PicoArray devices.\u0026nbsp;The detailed procedure is\u0026nbsp;as\u0026nbsp;follows: (1)\u0026nbsp; \u0026nbsp;\u0026nbsp;Load an image which is needed to be analyzed. To do this, select: \u003cem\u003eFile \u0026gt; open\u003c/em\u003e. (2) Convert the image to grayscale. To do this, select: \u003cem\u003eImage \u0026gt; Type \u0026gt; 8-bit\u003c/em\u003e. (3) Adjust threshold to\u0026nbsp;highlight the picowells containing microcoloies. To do this, go to \u003cem\u003eImage \u0026gt; Adjust \u0026gt; Threshold\u003c/em\u003e. (4)\u0026nbsp;Binary conversion after thresholding is done.\u0026nbsp;To do this, go to \u003cem\u003eProcess \u0026gt; Binary \u0026gt; Make Binary\u003c/em\u003e. (5) Count the highlighted picowells. \u0026nbsp;To do this, go to \u003cem\u003eAnalyze \u0026gt; Analyze Particles\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that all data generated or analysed during this study are available within the paper and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 61771078 and 62404194), the Natural Science Foundation of Xinjiang Uygur Autonomous Region (No. 2023D01C179), and Xinjiang Tianchi Yingcai Youth Doctoral Project (No.\u0026nbsp;51052300578).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eT.H. designed and carried out the experiments, prepared most of the data and wrote the paper; X.H. carried out and assisted with the experiments; L.W. fabricated the microwell array chips; B.S. consulted on the manuscript and contributed to writing the paper; G.L. proposed the idea, managed the research process and wrote the paper.\u003c/p\u003e\n\u003cp\u003eCompeting Interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eAdditional information\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary information\u003c/strong\u003e The online version contains supplementary material available at \u0026hellip;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eStolp H, Starr MP. 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Emergence of antibiotic resistance from multinucleated bacterial filaments. \u003cem\u003eProc. Natl. Acad. Sci. USA\u003c/em\u003e 112, 178\u0026ndash;183 (2015).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"digital plating, isolation, identification, recovery, antibiotic susceptibility testing, microbial interaction ","lastPublishedDoi":"10.21203/rs.3.rs-5298212/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5298212/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Traditional plate culturing remains the “gold standard” in microbiology labs, but it is labor-intensive, time-consuming, and resource-heavy. Here, we introduce a digital plating (DP) platform that integrates digital assays with traditional plate culturing. Using a high-density microwell array chip covered with an agar medium sheet, the DP platform not only enables accurate bacterial quantification but also facilitates the isolation of single bacteria from complex communities for further characterization. The high flexibility afforded by the replaceable agar medium cover allows the DP platform to support complex microbial culturing, thereby broadening its potential applications. We demonstrated its versatility in accurate bacterial quantification, efficient isolation, identification, and clonal culture of specific bacteria from complex communities, rapid antibiotic susceptibility testing, and detailed investigation of microbial interactions. The DP system’s simplicity, cost-effectiveness, and versatility demonstrate its potential to substitute traditional plating techniques and enable rapid and scalable bacterial assays that were previously unattainable.","manuscriptTitle":"Digital Plating: A Universal and Versatile Microbiological Technique","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-11-26 10:29:43","doi":"10.21203/rs.3.rs-5298212/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f735fdf-b721-449a-a2a7-a6dd929f18e8","owner":[],"postedDate":"November 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":40650321,"name":"Biological sciences/Biological techniques"},{"id":40650322,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2024-11-26T10:29:43+00:00","versionOfRecord":[],"versionCreatedAt":"2024-11-26 10:29:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5298212","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5298212","identity":"rs-5298212","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}