Jul 01, 2026
An optimized cleaning protocol for sustainable reuse of Drosophila plastic vials
- Kazuha Abe1,
- Motoharu Kono1,
- Kasumi Kihara1,
- Kengo Mitsunari2,
- Mitsuru Aoyagi1,
- Morihiro Okada1
- 1Prefectural University of Hiroshima;
- 2Ootsuka Kikai Co., Ltd.
Protocol Citation: Kazuha Abe, Motoharu Kono, Kasumi Kihara, Kengo Mitsunari, Mitsuru Aoyagi, Morihiro Okada 2026. An optimized cleaning protocol for sustainable reuse of Drosophila plastic vials. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz4p3xlx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 25, 2026
Last Modified: July 01, 2026
Protocol Integer ID: 315726
Keywords: Drosophila, Recycle, plastic vial
Funders Acknowledgements:
KAKENHI
Grant ID: 24K02245
Abstract
Drosophila melanogaster is one of the most widely used model organisms, owing to its sophisticated genetic
toolkit and suitability for large-scale genetic screening. Its broad use across fundamental biology and biomedical research has created substantial demand for polystyrene (PS) vials used for fly rearing and experiments, the vast
majority of which are discarded after a single use. The disposal of these vials imposes growing environmental and economic burdens, highlighting the need for sustainable reuse strategies in Drosophila research. Although vial recycling systems have been introduced in several Drosophila facilities, broader adoption has been limited by the absence of a practical, standardized protocol. Existing methods are often labor-intensive, time-consuming, and generate unpleasant odors, reducing their feasibility for routine use in many laboratories. To address this challenge, we systematically evaluated and optimized each step of the vial recycling workflow. Here, we present a simplified, reproducible, and broadly applicable protocol for the reuse of Drosophila PS vials, offering a practical framework for reducing plastic waste and facilitating the broader adoption of sustainable vial recycling across Drosophila
laboratories.
Materials
- Polystyrene vials (diameter: 22 mm × height: 96 mm; cat. no. KFB-5S; Chiyoda Science, Japan)
- Sponge plugs (diameter: 25 mm × height: 35 mm; HIGH TECH, Japan)
- Benzine (normal hexane) (Sansei Kakou Co., Ltd., Japan)
- Bamboo round disposable chopsticks (Amazon)
- Plywood board (thickness: 5 mm; Amazon)
- Drill Driver (cat. no. DS12DA; HiKOKI, Japan)
- Stainless steel extension springs (0.9 mm wire diameter × 8 mm outer diameter × 70 mm free length; cat. no. SR-414, Waki Sangyo, Japan)
- Stainless steel eye bolts (outer diameter: 19 mm × inner diameter: 11 mm × thread length: 11 mm; Amazon)
- Epoxy resin kit (Amazon)
- Sieve (diameter: 300 mm × height: 45 mm; Amazon)
- Stainless steel tray (length: 500 mm × width: 350 mm × depth: 150 mm; Amazon)
- Nitrile chemical-resistant gloves (cat. no. SS24-50; SHOWA, Japan)
- Stainless steel rack (hole diameter: φ21 mm, 5 × 10 array; cat. no. SS24-50, SANWA KAKEN KOGYO CO., LTD., Japan)
- Fly food (standard diet: 1.0% agar, 7.3% glucose, 3.6% corn flour, 5.0% dry yeast, 0.6% wheat germ, 0.5% propionic acid, and 0.5% butyl p-hydroxybenzoate; modified diet for developmental timing and lifespan measurements: 1.5% agar, 5.0% glucose, 1.0% dry yeast, 0.5% propionic acid, and 0.5% butyl p-hydroxybenzoate)
Before start
Prior to starting, construct the following two custom-made devices: (1) a chopstick-based cleaning device for the simultaneous removal of organic contents from multiple vials, and (2) a plywood lid for the simultaneous inversion of multiple vials during benzine treatment. Detailed descriptions of both devices, together with schematic illustrations and photographs, are provided in the "Removal of organic contents" section (Figures 3 and 4).
Freezing
2d
Place used vials in stainless steel rack (capacity: up to 50 vials)(Figure 1a).
Freeze at -20°C or below for a minimum of 48 hours to ensure complete eradication of flies and mites across all developmental stages (Figure 1b) .
Figure 1. (a) Used vials arranged in a stainless steel rack prior to freezing.
(b) Vials in a stainless steel rack being frozen at −20°C.
2d
Thawing and plug removal
50m
Remove vials from the freezer and allow them to thaw at room temperature (RT) to loosen residual contents and facilitate their removal. For rapid processing, immerse the vials in a 60°C water bath to accelerate thawing.
40m
Once thawing is complete, remove the plugs manually. Wash the removed plugs with a household detergent, either by hand or in a washing machine, and dry in a temperature-controlled oven prior to reuse. Note that the processing time reported here represents the duration required to process 100 vials.
Figure 2. Used vials arranged in a stainless steel rack after plug removal.
10m
Removal of organic contents
25m
Remove vials from the freezer and allow them to thaw at room tempePerform all subsequent cleaning procedures inside the safety cabinet while wearing nitrile chemical-resistant gloves.rature (RT) to loosen organic contents and facilitate their removal. For rapid processing, immerse the vials in a 60°C water bath to accelerate thawing.
Place the vials, together with their stainless steel rack, into a stainless steel tray. Add benzine to each vial until completely filled.
5m
Remove organic contents from each vial, including residual diet, feces, larvae, and eggs, using the custom-made cleaning device in place of a spatula. The device consists of disposable chopsticks mounted at regular intervals on a plywood board, enabling organic contents to be removed simultaneously from multiple vials by insertion and back-and-forth movement. Approximately 10 back-and-forth movements are sufficient to remove most organic contents. Note that benzine treatment alone is insufficient to completely remove organic contents. The device consists of twenty disposable chopsticks trimmed to 12 cm in length, mounted at regular intervals on a plywood board, enabling simultaneous processing of multiple vials. Schematic illustrations and photographs of the device are provided in Figure 3.
Figure 3. Schematic illustration (a) and representative photograph (b) of the custom-made cleaning device, consisting of disposable chopsticks mounted at regular intervals on a plywood board. Schematic illustration (c) and representative photograph (d) of vials arranged in a stainless steel rack with the device inserted.
5m
To discharge organic contents simultaneously from multiple vials, mount the custom-made plywood lid onto the stainless steel rack and secure it with stainless steel extension springs to prevent vial displacement during inversion. Plywood is recommended for its benzine resistance, low cost, and ease of fabrication. Note that plywood readily absorbs benzine; therefore, coat its surface with resin to minimize benzine consumption. Simpler alternatives, including aluminum foil and plastic film, were evaluated; however, both exhibited insufficient adhesion and durability and were therefore deemed unsuitable.
Equip the plywood lid with circular apertures (23 mm in diameter), slightly smaller than the outer vial diameter (25 mm), arranged at regular intervals, such that upon inversion, organic contents fall through the apertures while the vials remain securely retained within the rack.Schematic illustrations and photographs of the device are provided in Figure 4.
Figure 4. Schematic illustration (a) and representative photograph (b) of the custom-made plywood lid. Schematic illustration (c) and representative photograph (d) of vials arranged in a stainless steel rack fitted with the custom-made plywood lid.
5m
Invert the stainless steel rack to discharge organic contents from each vial onto a sieve, separating them from the benzine. Benzine can be reused for at least 10 cleaning cycles. For recovery of higher-purity benzine, evaporation is recommended.
Figure 5. The stainless steel rack fitted with the custom-made plywood lid being inverted to discharge organic contents from each vial onto a sieve.
5m
Scrub the inner wall of each vial thoroughly with a brush and household detergent to remove remaining adherent material, and rinse repeatedly with tap water.
5m
Autoclave
3h
Autoclave the retained organic contents and dispose of them as combustible waste.
3h
Final Rinse
10m
Rinse each vial thoroughly with distilled water to ensure complete removal of any remaining impurities prior to reuse.
10m
Acknowledgements
Yuya Ohhara, University of Shizuoka