Name: fast colony forming unit counting in 96-well plate format applied to the drosophila microbiome
Uploaded: 2023-01-13T13:50:00
Duration: 12 min 55 s
Description: Watch this Scientific Journal Video about Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome at JoVE.com

Dr. Kerwyn Casey Huang, Dr. Andr&#233;s Aranda-D&#237;az, Ted Cooper, and members of the Ludington lab gave valuable input on the development of this protocol. Funding was provided by NSF IOS grant 2032985, NIH grant DP5OD017851, a Carnegie Institution of Canada grant, and the Carnegie Institute for Science&#39;s Endowment.

1. Fly colonization
NOTE: This method is suitable for quantitative analysis of flies with a culturable gut microbiome by growth plating of CFUs. A detailed protocol for rearing gnotobiotic flies has been previously published12.
<ol>
	<li>Establish stable colonization by a commensal bacterial species.
	<ol>
		<li>Prepare a fresh bacterial culture, pellet by centrifugation at 400 x g for 3 min at room temperature, and resuspend the cell pellet in PBS to an OD600 of 1.0. For this protocol, Lactiplantibacillus plantarum (previously known as Lactobacillus plantarum) was grown overnight to an OD600 of 2.0.</li>
		<li>Pipette 100 &#181;L onto the food in a fly vial (see Table of Materials). Spread evenly and allow the liquid to absorb for 15-30 min.</li>
		<li>Transfer 20 germ-free flies into this vial using the standard vial to vial flipping technique34. Each fly will eat a roughly equal quantity of the bacterial dose35. Alternatively, germ-free eggs can be added.</li>
		<li>Transfer the flies to fresh sterile food daily for 3 days. Do all this in a biosafety cabinet and use sterile technique (Figure 1A-1).</li>
	</ol>
	</li>
	<li>Prior to measuring CFU load, transfer the flies to a vial containing sterile agar-water for 4 h to clear transient bacteria from the gut. The agar-water vials provide a source of moisture for the fly but no nutrition for the fly or bacteria on the surface. They are prepared in the same sterile fly vials as with standard food but contain only deionized water and 1.5% agar.</li>
</ol>
2. Homogenizing flies individually in a 96-well PCR plate
<ol>
	<li>Prepare bead beater plates in advance.
	<ol>
		<li>Pour 0.5 &#181;m glass beads (see Table of Materials) onto the bead measuring tray (3D printed with the help of Supplemental Coding File 1 S1-bead-measurer.stl). Spread the beads on the tray so that all the wells are full and level, and then brush off excess beads into a conical tube to recover them.</li>
		<li>Place a semi-skirted PCR plate (see Table of Materials) upside down on the measuring tray and align it with the wells by fitting it into the indentation on the measuring tray. Then, quickly flip it over to transfer the beads.</li>
		<li>Remove excess beads from the PCR plate surface and cover it with foil. Inspect the PCR plate to ensure that all the wells contain beads. Use a weigh scoop if required to add beads to a single well. If static electricity is causing beads to stick on the plate surfaces, wipe or spray the back of the measuring tray and PCR plate with 70% ethanol.</li>
		<li>Cover with aluminum foil. Many plates can be prepared in this way and then autoclaved and stored for later use. When ready for use, add 100 &#181;L of PBS to each well using the 96-channel pipettor (see Table of Materials).</li>
	</ol>
	</li>
	<li>Surface-sterilize the microbe-colonized flies (see step 1) with 70% ethanol before homogenization.
	<ol>
		<li>Anesthetize the flies with 100% CO2 for 5 s. Transfer anesthetized flies from the vial into a 1.5 mL microcentrifuge tube using a small funnel (Figure 1A-2). In this study, 25 flies were typically added per tube.</li>
		<li>Immediately spray ~1 mL of 70% ethanol into the tube, close the tube and mix by inversion for 10 s. Then, aspirate the ethanol with a P1000 pipette, being careful not to aspirate any flies. Repeat once again with ethanol, then twice with sterile PBS (Figure 1A-3).</li>
	</ol>
	</li>
	<li>After the final PBS wash, close the tube and, with the cap side down, tap it hard a few times on the bench so the flies go into the cap.</li>
	<li>Open the tube and dispense flies into the wells using forceps (Figure 1A-4). Place one fly in each well (Figure 1A-5). Keep the plate on ice while loading to keep the flies anesthetized.</li>
	<li>Seal the plate using Thermal Bond heat sealing foil (see Table of Materials).
	<ol>
		<li>First, remove any stray beads that are close to the wells as these can cause leakage in the foil seal. Make sure the dull side of the foil is oriented down on the plate and the shiny side up toward the heat sealer. Press the heat sealer (see Table of Materials) down firmly for 5 s (Figure 1A-6). Burnish with the hand applicator (see Table of Materials) to secure the foil. 
		NOTE: Poor sealing is a cause of sample loss.</li>
	</ol>
	</li>
	<li>Secure the plate in the plate shaker. Homogenize for 5 min (Figure 1A-7). Spin down the bead plate for 30 s in a mini-plate spinner (see Table of Materials) at 350 x g to remove liquid from the sealing foil.</li>
	<li>Remove the foil while holding the plate so as not to splash droplets of fly homogenate out of the wells.</li>
</ol>
3. Serial dilution of fly homogenate and spotting onto CFU plates
<ol>
	<li>Place four rectangular MRS agar growth plates (see Table of Materials) in the biosafety cabinet and remove their lids. In this protocol, MRS agar was used for the growth of L. plantarum.&#160;Leave these plates to dry for at least 10 min (20 min if they are freshly poured or cold from storage). 
	NOTE: If the plates are wet, the droplets from the 96-well plate will run together and ruin the counts.</li>
	<li>Prepare three dilution plates by adding 100 &#181;L of PBS to each well of a sterile 96-well plate using a 96-channel pipettor. Load a rack of P20 tips onto the 96-channel pipettor.</li>
	<li>Make a 1:10 dilution by aspirating 11.1 &#181;L of homogenate from the sample plate prepared in section 2 (Figure 1A-8). Be sure to draw from the middle of the wells rather than the bottom of the wells because the fly homogenate and glass beads will clog the pipettes. The beads sink, and the fly particulates float, leaving the middle layer mostly clear.</li>
	<li>Dispense the 11.1 &#181;L into the first dilution plate, which already contains 100 &#181;L of sterile PBS per well. Keep the dilution plate on the plate shaker for 10 s at 600 rpm. Mix again by pipetting up and down for five cycles. Transfer 11.1 &#181;L from the first dilution plate to the second dilution plate, and repeat the mixing steps for the next two dilution plates.</li>
	<li>Perform dilution series plating as described below.
	<ol>
		<li>Retrieve the growth plates from the biosafety cabinet.</li>
		<li>Starting with the most dilute plate, spot 2 &#181;L from each well onto the agar plates using the 96-well pipettor (Figure 1A-9).
		<ol>
			<li>Lower the pipettor head slowly onto the plate, taking care not to stab into the agar. Examine the plate carefully and ensure all the spots have been dispensed; if not, manually add 2 &#181;L to the appropriate position. Check that the liquid spots quickly soak into the agar and do not run together.</li>
			<li>If the spots run together, dry a new set of fresh agar plates for a longer period and redo the spotting. If there are multiple plate types (e.g., different media or with antibiotics), spot those too. Dispense any remaining solution back into the dilution plate and proceed to the next higher concentration. 
			NOTE: When spotting dilutions, mix the next dilution five times to ensure that the contents of the pipette tips are cleared of the previous dilution. The dilution plates can be stored &#62;8 h at 4 &#176;C without affecting the colony counts35.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Repeat the plating process as described in step 3.5 for the remaining dilution plates, progressing from most dilute to most concentrated until the plate with the original homogenate.</li>
	<li>When all the liquid has been absorbed into the agar, invert the plates and place them in the incubator (Figure 1A-10). For Lactiplantibacillus and Acetobacter from Drosophila, the optimal temperature is 30 &#176;C on MRS media. Incubate until the colonies have reached optimal size: colonies are large enough to clearly see them but not so large that they merge or interfere with one another&#39;s growth (see the representative results for quantification of the optimal size). 
	NOTE: Optimal growth conditions are strain-dependent and must be determined empirically. For Lactiplantibacillus from Drosophila, the optimal time of incubation is 26 h to 30 h on MRS agar. For Acetobacter from Drosophila, the optimal time of incubation is 30 h to 48 h on MRS depending on the strain.</li>
	<li>After incubation, store the plates at 4 &#176;C if required until ready for counting.</li>
</ol>
4. Quantification of CFUs
<ol>
	<li>Quantify CFUs by photographing the plates and then counting the colonies using automated software, as detailed below. If the plates were stored at 4 &#176;C, first allow them to reach room temperature so there is no condensation on the plates, which produces glare.</li>
	<li>Organize the plates in a logical sequence and keep them in that order while photographing &#8212; it makes it easier to name the files. Orient all the plates so that A1 is in the upper left corner for all the plates. It is recommended to label the A1 corner with a unique ID to ensure no mix up of the photos. Stack each dilution series in order.</li>
	<li>Remove the plate lid and place the plate on the stage with A1 oriented in the correct corner. Designs are included for the plate photo box (Supplemental File 1, Supplemental File 2), which is optimized for photographing these tray plates and includes lighting and filter options to image fluorescent colonies.</li>
	<li>Image the plates. Use the camera (see Table of Materials) with manual settings to achieve a consistent exposure level between plates. A long focal length setting is recommended to minimize perspective distortion. Capture the photo using a remote shutter to minimize blur from camera shake.</li>
	<li>Transfer the images to a computer and rename them, including the experiment name, the type of media, the dilution factor, and any other pertinent details. Some operating systems (see Table of Materials) have a useful batch renaming feature in Finder by right-clicking on a selection of files.</li>
</ol>
5. Automated colony counting using the Count-On-It suite (Supplemental File 3)
<ol>
	<li>Crop the images using the provided Croptacular plugin (Supplemental Coding File 2) for ImageJ. This plugin helps to divide the image into an orderly array of subregions (e.g., 8 x 12 for a 96-well plate). The subregions will be counted individually. 
	NOTE: A separate plugin for counting circular plates called Circus is described in the Supplemental Coding File 3 Circus_.ijm.
	<ol>
		<li>Organize the images to be processed for quantification into a folder. Choose filenames that distinguish the plates, as these filenames become column titles for each set of counts . Within this folder, make subfolders named &#34;cropped&#34; and &#34;receipts&#34; .</li>
		<li>Launch Croptacular,&#160;and click OK to start cropping. 
		NOTE:The default settings are displayed and are usually sufficient. Depending on the resolution of the image, the lighting, the spot size, etc., it can be helpful to adjust the base parameters.</li>
		<li>If the image is already straight, simply press Space. Otherwise, straighten the image by drawing a line along an edge that should become horizontal. Redraw the line as many times as needed if the image still does not appear straightened.</li>
		<li>Next, draw a boundary box of the area for which counting is to be done. Adjust the size and proportion until all the spots are within their cells. Drag the cursor outside the boundary box to refresh the grid. When the grid looks good, press Space.</li>
		<li>Ensure that the next image automatically rotates to the same angle as the first one; the plugin assumes all the photos are aligned the same. If this is accurate, press Space to continue. Otherwise, straighten the image as before. The grid also recalls the same position as the previous image; adjust if necessary, and then press Space.</li>
	</ol>
	</li>
	<li>Enumerate the colonies on the plates using the provided Count-On-It: Gridiron plugin for ImageJ (Supplemental Coding File 4). Detailed instructions for installing and using the plugin are included in Supplemental File 3.
	<ol>
		<li>Launch Count-On-It &#62; Gridiron. Use the same grid settings as with Croptacular. Users can batch an entire folder, analyze a single image, or start from the current image.</li>
		<li>Set the threshold based on an upper and a lower pixel intensity value. Make the threshold as stringent as possible while still selecting all the colonies. Ideally, there will be some space between the selected colonies, but the software is capable of segmenting the blobs to a degree. Click OK on the &#34;Action Required&#34; dialogue box when the threshold is satisfactory .</li>
		<li>To inspect the results, zoom in and look more closely at the colony counts. Clicking cancel will abort the plugin. Click OK to continue.</li>
		<li>On the first image, ensure that the option is given to proceed or return to the setup menu, for example, to change the minimum colony size. To proceed with the batch process, click OK. Then, select a folder to keep the receipts and results table. Use the &#34;receipts&#34; folder or create a new folder.</li>
		<li>After the first image, the next images will default to the same threshold as the previous settings. Click OK to use these settings or adjust the settings. If the photos are consistent and the setting is accurate, Click OK on the &#34;Action Required&#34; dialogue box. 
		NOTE: Once all the images in the batch are completed, the results table saves automatically in the same folder as the receipts.</li>
		<li>Review the count receipts that are produced by the software and manually correct any counting errors. The ImageJ plug-in is statistically accurate, but errors do occur. Proof the receipts to examine outliers and identify miscounts. The software saves the CFU data as a .csv file in the same folder as the receipts.</li>
	</ol>
	</li>
	<li>Analyze the data using the user-preferred software.</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+single+plate-serial+dilution+spotting+(SP-SDS)+with+sample+anchoring+as+an+assured+method+for+bacterial+and+yeast+cfu+enumeration+and+single+colony+isolation+from+diverse+samples.">Optimization of single plate-serial dilution spotting (SP-SDS) with sample anchoring as an assured method for bacterial and yeast cfu enumeration and single colony isolation from diverse samples.</a> Biotechnology Reports. 8, 45-55 (2015).</li><li>Nu&#241;ez, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+cost+and+open+source+multi-fluorescence+imaging+system+for+teaching+and+research+in+biology+and+bioengineering.">Low cost and open source multi-fluorescence imaging system for teaching and research in biology and bioengineering.</a> PLoS One. 12 (11), 0187163 (2017).</li><li>Putman, M., Burton, R., Nahm, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+method+to+automatically+count+bacterial+colony+forming+unit.">Simplified method to automatically count bacterial colony forming unit.</a> Journal of Immunological Methods. 302 (1-2), 99-102 (2005).</li><li>Ashburner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Drosophila. A Laboratory Handbook. , (1989).</li><li>Dodge, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gut+commensal+niche+regulates+stable+association+of+a+multispecies+microbiota.">A gut commensal niche regulates stable association of a multispecies microbiota.</a> bioRxiv. , (2021).</li><li>Obadia, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probabilistic+invasion+underlies+natural+gut+microbiome+stability.">Probabilistic invasion underlies natural gut microbiome stability.</a> Current Biology. 27 (13), 1999-2006 (2017).</li><li>Vega, N. M., Gore, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+assembly+produces+heterogeneous+communities+in+the+Caenorhabditis+elegans+intestine.">Stochastic assembly produces heterogeneous communities in the Caenorhabditis elegans intestine.</a> PLoS Biology. 15 (3), 2000633 (2017).</li><li>Jaime, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+high-throughput+WAFFL+system+for+treating+and+monitoring+individual+Drosophila+melanogaster+adults.">The high-throughput WAFFL system for treating and monitoring individual Drosophila melanogaster adults.</a> bioRxiv. , (2018).</li><li>Ren, C., Webster, P., Finkel, S. E., Tower, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+internal+and+external+bacterial+load+during+Drosophila+aging+without+life-span+trade-off.">Increased internal and external bacterial load during Drosophila aging without life-span trade-off.</a> Cell Metabolism. 6 (2), 144-152 (2007).</li></ol>

The authors have nothing to disclose.

The detailed techniques presented here enable a &#62;100-fold increase in the number of samples that can be evaluated in a CFU counting experiment. This technique advances existing methods for microbiome experiments in Drosophila12,35,36 by using the 96-well plate format to assay individual flies. Furthermore, it applies a more efficient spot plating method19,31 and an automated workflow with a photography and colony counting platform. The significance of this method for Drosophila is to standardize experiments to the 96-well plate format, which enables the simultaneous handling of a large number of biological replicates with automation to achieve high-throughput quantification of CFUs.
The 96-well platform shows that increased frequency of transfer and the &#34;clearing&#34; of transient bacteria causes a significant reduction in both the mean abundance and the variation between samples (Figure 1B,C), demonstrating the stringency of this improved protocol. One limitation of co-housing flies is the horizontal transfer of bacteria between flies. A proposed solution is to keep flies individually in a 96-well plate format, such as the Whole Animal Feeding Flat38.
Although a significant reduction in bacterial load was not observed when flies were washed in ethanol, these flies had only been kept in the presence of external bacteria for 3 days. Housing for longer time periods could allow a greater bacterial load to accumulate39. Therefore, washing in ethanol is still recommended.
Transferring flies into the 96-well plate is the critical first step for setting up the workflow (Figure 1A). Once the flies are washed, they are distributed into the wells one at a time. A plate chart is useful at this stage to note which conditions are present in each well and add any notes such as &#34;fly was lost&#34;. Homogenization is another critical step with some important caveats. Bacteria survive the homogenization process when in the presence of a fly (Figure 1D,E), and presumably, this principle is also true when the bacteria are inside the fly&#39;s gut. However, bacteria are also killed when they are homogenized in bead beater plates alone, demonstrating that the homogenization can kill the bacterial cells in certain circumstances, a limitation which may be important if one is homogenizing dissected guts, for example. Notably, the loss of CFUs when homogenizing is dependent on the number of CFUs in the sample, and the loss is minimal when ~105 CFUs per well are used. Further preservation of CFUs can be achieved by stopping homogenization halfway through and cooling the plate on ice.
Homogenization was performed for 5 min in this protocol based on control experiments where a known number of CFUs were mixed with a germ-free fly, and this worked empirically for fly bacteria. Less beating caused larger fly parts to be present, which interferes with pipetting, while significantly longer homogenization times of ~10 min made CFU counts more variable. The smaller volume and angled shape of conical plate wells were observed to reduce the efficiency of bead beating as compared to cylindrical 2 mL tubes. Many variations on this general approach are possible, including which bacterial strains, which bead beating container, which beads, and which fly genotype are used. Individual user cases need to use positive controls to establish their approach.
The spot plating method was employed in a specific way: 1:2 dilutions of L. plantarum WF in PBS were spotted onto MRS plates and incubated at 30 &#176;C for 2 days (shorter incubation times may be implemented to generate smaller colony sizes). The method requires some up-front investment in equipment, primarily the bead beater and the 96-channel pipettor (Figure 2A), which is necessary for both the dilution series and spot plating. However, less expensive options are available, including a 96-well plate replicator with slotted pins. The dilution series is a critical step that affects the accuracy of the CFU counting results. In terms of failure modes, it is possible to clog the pipette tips with fly parts or glass beads and for the pipette tips to not seal properly onto the pipettor or to fail for some other reason. All these problems result in undercounting of the affected wells and should be monitored. Adequate mixing at each step of the dilution series is also crucial. Each dilution should be mixed thoroughly either by putting the plate on a plate shaker or by pipetting up and down 15-20 times, which also serves to rinse the tips. By spotting from the most dilute to least dilute plate, the tips can be reused for the whole dilution series. With 1:2 dilutions, counting is accurate over a range of 2-25 colonies, spanning an order of magnitude (Figure 2C). Therefore, 1:10 dilutions save time and materials. Another variable that can be taken advantage of is incubation time, which can be adjusted to produce smaller colonies and, thus, increase the range of countable spots by preventing the merging of adjacent colonies.
A quality photo of the plate is essential as it becomes the raw source data from which the CFUs are analyzed and can be archived indefinitely. The FluoroBox is designed to produce photos of the plates with uniform light intensity, which minimizes glare on the agar surface. Additionally, the design is capable of selectively photographing fluorescent colonies using single-color LED lights and colored photo filters (Figure 3A). The construction of a setup like the FluoroBox, with controlled lighting and camera settings, can greatly increase the reproducibility of CFU images, which is important for automated analysis. Colony morphologies, fluorescence intensity, and the effects of time or density on colony growth are just a few of the properties that can be analyzed using the photographs. The photo box can be constructed without the color filters and single-color lights if no fluorescent bacteria are used, reducing the cost and complexity. Different excitation lights and emission filters could be substituted for the ones recommended here if a different fluorophore is used by a lab. A camera that is connected to a tablet over WiFi using an app is useful both for the automated shutter feature to prevent shaking and for ease of data transfer. Images can be transferred to the tablet and then to a laptop using wireless file transfer software. Recommended cameras with these capabilities are indicated in the Table of Materials.
Count-On-It is a plug-in written in ImageJ. The automated CFU counting software segments the plate image into a uniform 96-well grid, counts the colonies in each grid cell, and batches the results into a simple spreadsheet. As there is always variation in the position of the spot grid on the plate and in the photo, the user must manually conform the grid to the photo using the Croptacular plug-in. This also helps exclude areas near the plate edge, which have glare. Setting the threshold is key to getting the most accurate CFU count from the image. If the threshold is set too high, the colonies will merge; if the threshold is set too low, the colonies will be excluded. Once the threshold is set, the macro applies gaussian blur to soften the edges and reduce aliasing, the watershed filter divides overlapping colonies, and the blobs are counted using analyze particles. 
Sometimes, the density of colonies is too high in a particular spot. Count-On-It provides a way to deal with this. To estimate the number of colonies in a grid cell with partially merged colonies, first the average area of a circular blob from the entire plate is taken as Cavg. Then, the area of blob A1 is divided by the average area of a circular blob A1/Cavg. This number is rounded to the nearest integer, and that is the estimate for how many colonies are in a blob. This function is one reason why the threshold can influence the counting results: the relative average colony area versus a merged blob area will be different depending on how the threshold affected merged blobs.
The methods presented have several limitations. These include the need for equipment to dispense liquid media accurately from 96-well plates. This equipment, either a 96-channel pipettor or a slotted replicator pin tool, can cost thousands of dollars to obtain. Cheaper alternatives exist but are less accurate. Automated counting via Count-On-It also presents some limitations. For instance, if two colony types in a mixed population were delimited based on size alone, blob counting would not be able to assign colonies to the correct type. In this case, spots with blobs would need to be eliminated from counts. Further differentiation of colonies based on morphology would be a valuable extension of the method that is not currently implemented. The use of selective media including strain-specific nutrients and antibiotics simplifies the need for complex image analysis.
Maintaining fruit fly&#160;experiments in 96-well plates multiplies the number of samples and conditions that can be tested in a single experiment and can facilitate high-throughput screens in Drosophila-bacteria association phenotypes. We envision that this method can be extended by using selective media to differentiate many bacterial strains in complex mixtures. The method is not limited to the study of the fly microbiome. Quantification of CFUs is common in many applications of microbiology, from coliform counts in drinking water to the identification of pathogens. The CFU plating system presented here enables high-throughput screens, as well as automated acquisition, processing, storage, and delivery of results.

The relationship between intestinal microbiota and their animal host is increasingly at the forefront of biological studies1, which show that the strain composition of the microbiome is important for host physiology2,3,4. The pace of discovery has been limited by confounding factors such as high natural inter-individual variation and high diversity of colonizing bacteria5,6,7. The fruit fly, Drosophila melanogaster, has emerged as a promising model due to its naturally low-diversity microbiome, ease of handling, and robust host genetics8,9,10,11. Flies can be made germ-free and re-associated with a defined microbiota12, and it has been shown that the flora influence fly physiological traits13,14. The innate flora consists of a limited set of bacteria that are all culturable, and close relatives of these are also endogenous to the mammalian gut, including Lactobacilli, Proteobacteria, and Enterococci15.
Studying the influence of the microbiome on host traits requires quantification of the microbiome in terms of both which species are present and their absolute abundances16. The predominant ways of analyzing the fly microbiome are colony forming unit (CFU) counts17, 16S rRNA gene amplicon sequencing9, and qPCR of the 16S rRNA gene18. CFU counts can be obtained without expensive reagents, and they confirm the viability of the bacterial cells19. The 16S techniques including qPCR have advantages in that taxonomic identities of microbes can be ascertained without regard to their growth requirements or colony morphologies20.
In the case where experiments use flies with a defined microbiome of known bacteria (gnotobiotic flies), CFU counts have particular advantages over sequencing21. Sequencing is expensive, requiring DNA extraction, PCR-based library preparation, and high-throughput sequencing to obtain relative abundances22. Due to the high cost, high-throughput sequencing methods typically need to be performed in bulk to reduce the per-sample price23. Further methods such as qPCR are required to obtain absolute abundances16. In contrast, CFU counts are fast and cheap and give the absolute numbers of viable cells. Drosophila8 and other small microbiome models24, including the worm, Caenorhabditis elegans25,26, and the larval zebrafish, Danio rerio, grown gnotobiotically27,&#160;have a limited range of bacteria, which have known growth characteristics28. In these cases, particularly with gnotobiotic animals, CFU counting can differentiate all the species of bacteria within the multispecies gut communities21,27,29. Higher-throughput CFU counting methods would further improve the cost-effectiveness and speed of measuring the composition of the microbiome and could be applied to many other microbiology experiments.
Counting CFUs by serial dilution plating of a bacterial suspension on agar-based growth media is a standard method across the field of microbiology. The colonies that grow on the plates are then counted manually. The dilutions allow the researcher to select a density of colonies that is countable (e.g., ~100 CFUs per plate), meaning the colonies are not growing into one another and can be counted in a reasonable amount of time. Most microbiologists use essentially the same CFU counting method developed 140 years ago in the lab of Robert Koch, and for many applications, this method is still adequate. However, a problem arises when seeking to quantify a large number of samples. A single sample may require plating 1 to 10 serial dilutions of the sample to obtain countable CFUs, so experiments involving more than a few dozen samples become taxing. Various methods have been developed to increase the efficiency of CFU enumeration. Automated spiral plating eliminates the need for serial dilutions30, making only one plate necessary for CFU counting but increasing the time to plate the sample. Single plate-serial dilution spotting (SP-SDS) allows &#65279;CFU estimations from fewer plates per sample31. These methods are an improvement on the traditional spread-plating method but still require handling and plating of bacterial samples singly and are, thus, not ideal for high throughput. Handling samples in 96-well plates and spot plating those 96 samples on rectangular agar plates greatly improves the throughput of samples19.
Microbiomes are typically composed of multiple strains and species. While species can often be distinguished by colony morphology or growth media, fluorescence can be employed to further distinguish different types of bacteria and their growth traits32. For instance, different genotypes of the same species can be labeled with different genetically-encoded fluorescent proteins. Plate imaging methods that incorporate fluorescence allow researchers to take advantage of these genetic techniques in CFU-based assays32. Incorporating fluorescence into high-throughput CFU counting methods would further enhance their utility.
Counting CFUs manually becomes cumbersome when there is a large number of samples. Automated counting of CFUs can be conducted by photographing the plate and processing the image using specialized software33. Sieuwerts et al. combined the improved plating efficiency of spot-plating with automated colony counting using conventional digital photography and ImageJ software19.
A high-throughput method to screen the phenotypes of specific microbe and host associations would aid studies of microbiome community assembly and the impact on host health and fitness. For studies of the Drosophila microbiome, a high-throughput microbiology platform would incorporate handling of fly samples in 96-well plate format, fly lysis without bacterial lysis, the efficiency of spot-plating, the ability to use fluorescence and distinguish multiple fluorophores, a controlled light environment for reproducible imaging of CFU plates, and a reliable automated colony counting software. This article describes a method optimized for the assay of CFUs in gnotobiotic flies, which is simple, fast, and automated. This protocol outlines a novel workflow combining the best of previously published methods and optimized for exploring the gut microbiome in Drosophila.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Bead beating and spot plating</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fly vials</td>
			<td>Genesee</td>
			<td>32-121</td>
			<td>autoclavable</td>
		</tr>
		<tr>
			<td>Fly vial stoppers</td>
			<td>Genesee</td>
			<td>59-201</td>
			<td>autoclavable</td>
		</tr>
		<tr>
			<td>Hand Applicator</td>
			<td>3M</td>
			<td>3M PA1</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Sealer</td>
			<td>Eppendorf</td>
			<td>5390</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Beadbeater 96</td>
			<td>BioSpec</td>
			<td>1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-BeadBeater Glass Mill Beads, 0.5 mm</td>
			<td>BioSpec&#160;</td>
			<td>11079105</td>
			<td></td>
		</tr>
		<tr>
			<td>MPS 1000 Mini PCR Plate Spinner</td>
			<td>Labnet</td>
			<td>LI-CF-P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Rainin BenchSmart 96 semi-automated pipettor</td>
			<td>Mettler Toledo</td>
			<td>30296705</td>
			<td>Less expensive options available, including slotted 96 well pin tool from VP Scientific</td>
		</tr>
		<tr>
			<td>TempPlate semi-skirted 96-well PCR plate, straight skirt, natural</td>
			<td>USA scientific</td>
			<td>1402-9220</td>
			<td>Must be polypropylene for heat sealer</td>
		</tr>
		<tr>
			<td>Thermal Bond Heat Seal Foil</td>
			<td>4titude</td>
			<td>4ti-0591</td>
			<td>Keep sterile</td>
		</tr>
		<tr>
			<td>Tray Plate,128 x 85 mm, Polystyrene, Sterile</td>
			<td>SPL Life Sciences</td>
			<td>31001</td>
			<td>For making rectangular agar plates</td>
		</tr>
		<tr>
			<td>Photobox construction</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#188;&#8221;-20 X &#189;&#8221; Bolts (X2)</td>
			<td>Amazon</td>
			<td>ASIN: B07BP1WR3H</td>
			<td>To attach the camera bracket. Brand not important. Any 1/4&#34;-20 1/2&#34; bolt works.</td>
		</tr>
		<tr>
			<td>&#188;&#8221;-20 x &#189;&#8221; Connector Nut&#160;</td>
			<td>Amazon</td>
			<td>UPC: 799862376780</td>
			<td>AKA cap nut or connector bolt. This is for attaching the rubber bands on the plate holder. Brand not important.</td>
		</tr>
		<tr>
			<td>&#188;&#8221;-20 x &#190;&#8221; bolts (X3)</td>
			<td>Amazon</td>
			<td>ASIN: B003QZSZY4</td>
			<td>For the plate holder. Brand not important.</td>
		</tr>
		<tr>
			<td>1/8&#8221; x &#189;&#8221; washer</td>
			<td>Amazon</td>
			<td>UPC: 611982484599</td>
			<td>Washer for the cap nuts on outside of box. Brand not important. Spray paint black before attaching to blend with the acrylic.</td>
		</tr>
		<tr>
			<td>18 Gauge Wire - Two Conductor Power Wire - 18 AWG Power Wire &#8211; 10ft</td>
			<td>Superbrightleds.com</td>
			<td>WP18-2</td>
			<td></td>
		</tr>
		<tr>
			<td>22-10 AWG Red Wire Nut - WN-R2210 &#8211; Quantity 4</td>
			<td>Superbrightleds.com</td>
			<td>WN-R2210</td>
			<td></td>
		</tr>
		<tr>
			<td>22-18 AWG 3/16in Female Push On Connector - 22-18 AWG &#8211; Quantity 3</td>
			<td>Superbrightleds.com</td>
			<td>SCFP-2218</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#34; Solderless Clamp-On Jumper Connector - 8mm Single Color LED Strip Lights - Quantity 3</td>
			<td>Superbrightleds.com</td>
			<td>SBL-MA2P-8-2</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#34; Solderless Clamp-On Pigtail Adaptor - 8mm Single Color LED Strip Lights &#8211; Quantity 3</td>
			<td>Superbrightleds.com</td>
			<td>SBL-MA2P-8-1&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#8221; drawer handle</td>
			<td>Amazon</td>
			<td>ASIN: B07Z331P99</td>
			<td>Any drawer handle should work.</td>
		</tr>
		<tr>
			<td>6&#8221; Drawer slides</td>
			<td>Btibpse</td>
			<td>UPC: 712243424979</td>
			<td>Trim the soft close rubber stoppers on the drawer sliders.</td>
		</tr>
		<tr>
			<td>8-32 x &#189;&#8221; Cap Nuts (x4)</td>
			<td>Amazon</td>
			<td>ASIN: B00HYLZB98</td>
			<td>Attaches drawer slide bolts on outside of box. Brand not important. Nylon won&#39;t damage the acrylic. Spray paint black before attaching to blend with the acrylic.</td>
		</tr>
		<tr>
			<td>8-32 x &#189;&#8221; Nylon Bolts (x4)</td>
			<td>Amazon</td>
			<td>ASIN: B07KX9T7NF</td>
			<td>To attach drawer slides. Brand not important. Any bolt or machine screw meeting the specifications works. Nylon won&#39;t damage the acrylic and allows you to cut the bolt flush with the nut. Spray paint black before attaching to blend with the acrylic.</td>
		</tr>
		<tr>
			<td>Acrylic Glue</td>
			<td>SCIGRIP</td>
			<td>Ean: 7844908489337</td>
			<td>SCIGRIP Weld-On #4 Adhesive, Pint and Weld-On Applicator Bottle with Needle</td>
		</tr>
		<tr>
			<td>Black Cable Ties - 10 Pack - 4 Inch Long&#160;</td>
			<td>Superbrightleds.com</td>
			<td>CT-B04-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera L-Bracket</td>
			<td>WLPREOE, Vikerer, Unbranded</td>
			<td>ASIN: B09X46YKQZ</td>
			<td>The bracket should be &#34;reversed&#34; from its intended configuration so that the camera is on the &#34;outside&#34; of the L. Some brackets come in two pieces and allow for this alternate configuration, some don&#39;t, you&#39;ll need one that can be flipped. Also should have 1/4&#34; holes for attachment. WLPREOE, Vikerer, Unbranded. Amazon Serial Identification Number given as an example.</td>
		</tr>
		<tr>
			<td>Canon T series camera for tethering option OR</td>
			<td>Canon, Panasonic, Sony, Nikon, etc..</td>
			<td>1894C002</td>
			<td>The Canon Ti series cameras are a good option and can tether to a computer. Use with the 18-55mm standard kit lens. Used options are recommended from the Canon T5 to T7 (current model).</td>
		</tr>
		<tr>
			<td>Custom Length Single Color LED Strip Light - Eco Series Tape Light - 24V - IP20 - 250 lm/ft - Blue - 2 meters</td>
			<td>Superbrightleds.com</td>
			<td>STN-BBLU-B6A-08C1M-24V</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Length Single Color LED Strip Light - Eco Series Tape Light - 24V - IP20 - 250 lm/ft - Green &#8211; 2 meters</td>
			<td>Superbrightleds.com</td>
			<td>STN-BGRE-B6A-08C1M-24V</td>
			<td></td>
		</tr>
		<tr>
			<td>Custom Length Single Color LED Strip Light - Eco Series Tape Light - 24V - IP20 - 250 lm/ft - Natural White 4000K &#8211; 2 meters&#160;</td>
			<td>Superbrightleds.com</td>
			<td>STN-A40K80-B6A-08C1M-24V</td>
			<td></td>
		</tr>
		<tr>
			<td>Drill with &#188;&#8221;, 1/8&#8221; drill bits</td>
			<td>Black &#38; Decker</td>
			<td>&#8206;BDCDD12PK</td>
			<td>Brand not important.&#160;</td>
		</tr>
		<tr>
			<td>Flat Black Spray Paint,&#160; 2X Ultra-Matte</td>
			<td>Rustoleum</td>
			<td>331182</td>
			<td>Paint the interior of everything FLAT black. Brand not important.</td>
		</tr>
		<tr>
			<td>Laser Cut Acrylic Walls</td>
			<td>Big Blue Saw</td>
			<td>www.bigbluesaw.com</td>
			<td>Use the attached PDF, delete all cutouts in the top piece except desired hole for camera&#160;</td>
		</tr>
		<tr>
			<td>Mean Well LED Switching Power Supply - LPV Series 20-100W Single Output LED Power Supply - 24V DC - 20 Watt&#160;</td>
			<td>Superbrightleds.com</td>
			<td>LPV-20-24</td>
			<td></td>
		</tr>
		<tr>
			<td>Panasonic ZS100 for wifi connection to a phone, tablet, or computer</td>
			<td>Canon, Panasonic, Sony, Nikon, etc.</td>
			<td>DMC-ZS100K</td>
			<td>Panasonic cameras can be wirelessly connected to a computer for data transfer and remote shutter options. Used options are good.</td>
		</tr>
		<tr>
			<td>Quick Release Plate</td>
			<td>Neewer Aluminium 50mm Quick Release Plate QR Clamp 3/8-inch with 1/4-inch</td>
			<td>ASIN: B07417F21D</td>
			<td>Add this to the bracket so the camera can be easily removed for changing color filters. Amazon Serial Identification Number given as an example.</td>
		</tr>
		<tr>
			<td>Rubber Bands, Assorted sizes</td>
			<td>BAZIC Products</td>
			<td>Alliance Rubber 26649</td>
			<td>Rubber bands go on the plate holder. Brand not important.</td>
		</tr>
		<tr>
			<td>Screw/Adhesive Cable Tie Mounting Bases - 3/4 inch base &#8211; Quantity 4</td>
			<td>Superbrightleds.com</td>
			<td>CTMB-20</td>
			<td></td>
		</tr>
		<tr>
			<td>SPST Round Rocker Switch - No LED &#8211; Quantity 3</td>
			<td>Superbrightleds.com</td>
			<td>RRS-SP</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiffen 29 Filter (Red) 72 mm&#160;</td>
			<td>Tiffen</td>
			<td>72R29</td>
			<td></td>
		</tr>
		<tr>
			<td>Tiffen 58 Filter (Green) 72 mm</td>
			<td>Tiffen</td>
			<td>7258</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ64</td>
			<td>https://imagej.net/downloads</td>
			<td>N/A</td>
			<td>Free. Just cite: Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., &#8230; Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676&#8211;682. doi:10.1038/nmeth.2019</td>
		</tr>
		<tr>
			<td>MacOSX</td>
			<td>Apple</td>
			<td>N/A</td>
			<td>Has a useful batch rename feature in Finder to rename a group of photos to facilitate organizing and analyzing in Count-on-it.</td>
		</tr>
		<tr>
			<td>Unix</td>
			<td>BSD</td>
			<td>N/A</td>
			<td>64 bit</td>
		</tr>
		<tr>
			<td>Windows</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td>64 bit</td>
		</tr>
	</tbody>
</table>

fast colony forming unit counting in 96-well plate format applied to the drosophila microbiome

The intestines of animals are colonized by commensal microbes, which impact host development, health, and behavior. Precise quantification of colonization is essential for studying the complex interactions between host and microbe both to validate the microbial composition and study its effects. Drosophila melanogaster,&#160;which has a low native microbial diversity and is economical to rear with defined microbial composition, has emerged as a model organism for studying the gut microbiome. Analyzing the microbiome of an individual organism requires identification of which microbial species are present and quantification of their absolute abundance. This article presents a method for the analysis of a large number of individual fly microbiomes. The flies are prepared in 96-well plates, enabling the handling of a large number of samples at once. Microbial abundance is quantified by plating up to 96 whole fly homogenates on a single agar plate in an array of spots and then counting the colony forming units (CFUs) that grow in each spot. This plating system is paired with an automated CFU quantification platform, which incorporates photography of the plates, differentiation of fluorescent colonies, and automated counting of the colonies using an ImageJ plugin. Advantages are that (i) this method is sensitive enough to detect differences between treatments, (ii) the spot plating method is as accurate as traditional plating methods, and (iii) the automated counting process is accurate and faster than manual counting. The workflow presented here enables high-throughput quantification of CFUs in a large number of replicates and can be applied to other microbiology study systems including in vitro and other small animal models.

Watch this Scientific Journal Video about Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome at JoVE.com

Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome

This method quantifies microbial abundance using a 96-well plate format to plate colony forming units (CFUs) and is applied to the Drosophila microbiome in whole fly homogenate samples. CFUs are counted with an automated image analysis software provided here.

Fast Colony Forming Unit Counting in 96-Well Plate Format Applied to the Drosophila Microbiome

The intestines of animals are colonized by commensal microbes, which impact host development, health, and behavior. Precise quantification of colonization ...

Research

JoVE Journal

Biology

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