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 ...

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Research

JoVE Journal

Biology

6.4K Views.  Carnegie Institution for Science. 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.

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

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor mutations in components of age-regulatory pathways. If these mutations perturb the function of the age-regulatory pathway and therefore alter the lifespan of the entire organism, they provide important mechanistic insights1-3.
Another strategy to investigate the regulation of lifespan is to use small molecules to perturb age-regulatory pathways. To date, a number of molecules are known to extend lifespan in various model organisms and are used as tools to study the biology of aging4-16. The number of molecules identified thus far is small compared to the genetic "toolset" that is available to study the biology of aging.
Caenorhabditis elegans is one of the principle models used to study aging because of its excellent genetics and short lifespan of three weeks. More recently, C.elegans has emerged as a model organism for phenotype based drug screens5,7,16-20 because of its small size and its ability to grow in microtiter plates.
Here we present an assay to measure C.elegans lifespan in 96 well microtiter plates. The assay was developed and successfully used to screen large libraries for molecules that extend C.elegans lifespan7. The reliability of the assay was evaluated in multiple tests: first, by measuring the lifespan of wild type animals grown at different temperatures; second, by measuring the lifespan of mutants with altered lifespans; third, by measuring changes in lifespan in response to different concentrations of the antidepressant Mirtazepine. Mirtazepine has previously been shown to extend lifespan in C.elegans7. The results of these tests show that the assay is able to replicate previous findings from other assays and is quantitative. The microtiter format also makes this lifespan assay compatible with automated liquid handling systems and allows integration into automated platforms.

Measuring Caenorhabditis elegans Life Span in 96 Well Microtiter Plates

In this protocol we present a method to measure Caenorhabditis elegans lifespan in 96 well microtiter plates.

Lifespan is a biological process regulated by several genetic pathways. One strategy to investigate the biology of aging is to study animals that harbor ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Rapid and Efficient Generation of Neurons from Human Pluripotent Stem Cells in a Multititre Plate Format

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are multistep procedures involving the isolation and expansion of neural precursor cells, prior to terminal differentiation. In comparison to these time-consuming approaches, we have recently found that combined inhibition of three signaling pathways, TGF&beta;/SMAD2, BMP/SMAD1, and FGF/ERK, promotes rapid induction of neuroectoderm from hPSCs, followed by immediate differentiation into functional neurons. Here, we have adapted our procedure to a novel multititre plate format, to further enhance its reproducibility and to make it compatible with mid-throughput applications. It comprises four days of neuroectoderm formation in floating spheres (embryoid bodies), followed by a further four days of differentiation into neurons under adherent conditions. Most cells obtained with this protocol appear to be bipolar sensory neurons. Moreover, the procedure is highly efficient, does not require particular expert skills, and is based on a simple chemically defined medium with cost-efficient small molecules. Due to these features, the procedure may serve as a useful platform for further functional investigation as well as for cell-based screening approaches requiring human sensory neurons or neurons of any type.

Protocols for neuronal differentiation of pluripotent human stem cells (hPSCs) are often time-consuming and require substantial cell culture skills. Here, we have adapted a small molecule-based differentiation procedure to a multititre plate format, allowing simple, rapid, and efficient generation of human neurons in a controlled manner.

Existing protocols for the generation of neurons from human pluripotent stem cells (hPSCs) are often tedious in that they are  multistep procedures ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...