This video demonstrates the quantification of colony-forming units (CFUs) of gut-associated bacteria in Drosophila flies using a serial dilution and plating approach. Homogenized fly samples containing gut bacteria are serially diluted, plated on agar, and incubated to allow colony growth. Images of the plates are then analyzed using software to align, segment, and count colonies in each spot, enabling accurate CFU quantification.
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Method Article
Quantification of Colony-Forming Units of Gut-Associated Bacteria in Drosophila Flies
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October 30th, 2025
In This Article
Abstract
Protocol
1. Homogenizing flies individually in a 96-well PCR plate
- Prepare bead-beater plates in advance.
- Pour 0.5 µm glass beads (see Table of Materials) onto the bead measuring tray. 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.
- Place a semi-skirted polymerase chain reaction (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 tray. Then, quickly flip it over to transfer the beads.
- Remove excess beads from the PC....
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Results
Figure 1: The colonization assay measures CFUs in hundreds of individual flies using a 96-well plate format. <.......
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Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Bead beating and spot plating | |||
| Fly vials | Genesee | 32-121 | autoclavable |
| Fly vial stoppers | Genesee | 59-201 | autoclavable |
| Hand Applicator | 3M | 3M PA1 | |
| Heat Sealer | Eppendorf | 5390 | |
| Mini-Beadbeater 96 | BioSpec | 1001 | |
| Mini-BeadBeater Glass Mill Beads, 0.5 mm | BioSpec | 11079105 | |
| MPS 1000 Mini PCR Plate Spinner | Labnet | LI-CF-P1000 | |
| Rainin BenchSmart 96 semi-automated pipettor | Mettler Toledo | 30296705 | Less expensive options available, including slotted 96 well pin tool from VP Scientific |
| TempPlate semi-skirted 96-well PCR plate, straight skirt, natural | USA scientific | 1402-9220 | Must be polypropylene for heat sealer |
| Thermal Bond Heat Seal Foil | 4titude | 4ti-0591 | Keep sterile |
| Tray Plate,128 x 85 mm, Polystyrene, Sterile | SPL Life Sciences | 31001 | For making rectangular agar plates |
| Photobox construction | |||
| ¼”-20 X ½” Bolts (X2) | Amazon | ASIN: B07BP1WR3H | To attach the camera bracket. Brand not important. Any 1/4"-20 1/2" bolt works. |
| ¼”-20 x ½” Connector Nut | Amazon | UPC: 799862376780 | AKA cap nut or connector bolt. This is for attaching the rubber bands on the plate holder. Brand not important. |
| ¼”-20 x ¾” bolts (X3) | Amazon | ASIN: B003QZSZY4 | For the plate holder. Brand not important. |
| 1/8” x ½” washer | Amazon | UPC: 611982484599 | Washer for the cap nuts on outside of box. Brand not important. Spray paint black before attaching to blend with the acrylic. |
| 18 Gauge Wire - Two Conductor Power Wire - 18 AWG Power Wire – 10ft | Superbrightleds.com | WP18-2 | |
| 22-10 AWG Red Wire Nut - WN-R2210 – Quantity 4 | Superbrightleds.com | WN-R2210 | |
| 22-18 AWG 3/16in Female Push On Connector - 22-18 AWG – Quantity 3 | Superbrightleds.com | SCFP-2218 | |
| 4" Solderless Clamp-On Jumper Connector - 8mm Single Color LED Strip Lights - Quantity 3 | Superbrightleds.com | SBL-MA2P-8-2 | |
| 4" Solderless Clamp-On Pigtail Adaptor - 8mm Single Color LED Strip Lights – Quantity 3 | Superbrightleds.com | SBL-MA2P-8-1 | |
| 6” drawer handle | Amazon | ASIN: B07Z331P99 | Any drawer handle should work. |
| 6” Drawer slides | Btibpse | UPC: 712243424979 | Trim the soft close rubber stoppers on the drawer sliders. |
| 8-32 x ½” Cap Nuts (x4) | Amazon | ASIN: B00HYLZB98 | Attaches drawer slide bolts on outside of box. Brand not important. Nylon won't damage the acrylic. Spray paint black before attaching to blend with the acrylic. |
| 8-32 x ½” Nylon Bolts (x4) | Amazon | ASIN: B07KX9T7NF | To attach drawer slides. Brand not important. Any bolt or machine screw meeting the specifications works. Nylon won'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. |
| Acrylic Glue | SCIGRIP | Ean: 7844908489337 | SCIGRIP Weld-On #4 Adhesive, Pint and Weld-On Applicator Bottle with Needle |
| Black Cable Ties - 10 Pack - 4 Inch Long | Superbrightleds.com | CT-B04-10 | |
| Camera L-Bracket | WLPREOE, Vikerer, Unbranded | ASIN: B09X46YKQZ | The bracket should be "reversed" from its intended configuration so that the camera is on the "outside" of the L. Some brackets come in two pieces and allow for this alternate configuration, some don't, you'll need one that can be flipped. Also should have 1/4" holes for attachment. WLPREOE, Vikerer, Unbranded. Amazon Serial Identification Number given as an example. |
| Canon T series camera for tethering option OR | Canon, Panasonic, Sony, Nikon, etc.. | 1894C002 | 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). |
| Custom Length Single Color LED Strip Light - Eco Series Tape Light - 24V - IP20 - 250 lm/ft - Blue - 2 meters | Superbrightleds.com | STN-BBLU-B6A-08C1M-24V | |
| Custom Length Single Color LED Strip Light - Eco Series Tape Light - 24V - IP20 - 250 lm/ft - Green – 2 meters | Superbrightleds.com | STN-BGRE-B6A-08C1M-24V | |
| Custom Length Single Color LED Strip Light - Eco Series Tape Light - 24V - IP20 - 250 lm/ft - Natural White 4000K – 2 meters | Superbrightleds.com | STN-A40K80-B6A-08C1M-24V | |
| Drill with ¼”, 1/8” drill bits | Black & Decker | BDCDD12PK | Brand not important. |
| Flat Black Spray Paint, 2X Ultra-Matte | Rustoleum | 331182 | Paint the interior of everything FLAT black. Brand not important. |
| Laser Cut Acrylic Walls | Big Blue Saw | www.bigbluesaw.com | Use the attached PDF, delete all cutouts in the top piece except desired hole for camera |
| Mean Well LED Switching Power Supply - LPV Series 20-100W Single Output LED Power Supply - 24V DC - 20 Watt | Superbrightleds.com | LPV-20-24 | |
| Panasonic ZS100 for wifi connection to a phone, tablet, or computer | Canon, Panasonic, Sony, Nikon, etc. | DMC-ZS100K | Panasonic cameras can be wirelessly connected to a computer for data transfer and remote shutter options. Used options are good. |
| Quick Release Plate | Neewer Aluminium 50mm Quick Release Plate QR Clamp 3/8-inch with 1/4-inch | ASIN: B07417F21D | Add this to the bracket so the camera can be easily removed for changing color filters. Amazon Serial Identification Number given as an example. |
| Rubber Bands, Assorted sizes | BAZIC Products | Alliance Rubber 26649 | Rubber bands go on the plate holder. Brand not important. |
| Screw/Adhesive Cable Tie Mounting Bases - 3/4 inch base – Quantity 4 | Superbrightleds.com | CTMB-20 | |
| SPST Round Rocker Switch - No LED – Quantity 3 | Superbrightleds.com | RRS-SP | |
| Tiffen 29 Filter (Red) 72 mm | Tiffen | 72R29 | |
| Tiffen 58 Filter (Green) 72 mm | Tiffen | 7258 | |
| Software | |||
| ImageJ64 | https://imagej.net/downloads | N/A | Free. Just cite: Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., … Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods, 9(7), 676–682. doi:10.1038/nmeth.2019 |
| MacOSX | Apple | N/A | Has a useful batch rename feature in Finder to rename a group of photos to facilitate organizing and analyzing in Count-on-it. |
| Unix | BSD | N/A | 64 bit |
| Windows | Microsoft | N/A | 64 bit |
