Targeted Bacterial Colony Isolation Using Light-Patterned Hydrogel Degradation

Abstract

Source: Fattahi, N., et al. Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation. J. Vis. Exp. (2021).

This video demonstrates the extraction of bacterial colonies from a photodegradable hydrogel using UV light. A specific colony is selected, and a patterned light exposure is designed to initiate localized hydrogel degradation. After adjusting light intensity and duration, the UV light is applied, releasing the colony. The colony is then aspirated through tubing and transferred into a tube for downstream analysis.

Protocol

1. Bacterial strains and culture protocols

1. Streak colonies of bacteria on agar plates supplemented with appropriate growth media. In this report, B. subtilis (strain 1A1135, Bacillus Genetic Stock Center) is cultured on ATGN (0.079 M KH₂PO₄ (Potassium dihydrogen phosphate), 0.015 M (NH₄)₂SO₄ (Ammonium sulfate), 0.6 mM MgSO₄·7H₂O (Magnesium sulfate heptahydrate), 0.06 mM CaCl₂·2H₂O (Calcium chloride dihydrate), 0.0071 mM MnSO₄·H₂O (Manganese(II) sulfate monohydrate), 0.125 M FeSO₄·7H₂O (Ferrous ....

Results

Figure 1: Hydrogel formation on thiolated glass coverslips. (A) Spacers with a thickness of 12.7.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(3-Mercaptopropyl)triethoxysilane	Sigma-Aldrich	175617-25G	> 95%
Alconox detergent powder	Alconox	1104
Ammonium sulfate	Fisher Chemical	A702-500	Certified ACS Granular
Autoclave SK300C	Yamato Scientific	18016
Bacillus subtilis 1A1135	Bacillus Genetic Stock Center	1A1135
Brightfield upright microscope	Olympus Corporation	BX51
Calcium chloride, anhydrous	Fisher Chemical	C614-500	For Desiccators Pellets, 4-20 Mesh
Centrifuge 5702	Eppendorf	5702
Citric acid monohydrate	Sigma-Aldrich	C1909-500G	ACS reagent, > 99.0%
D-Glucose (Dextrose)	VWR Amresco Life Science	0188-1KG
DOWSIL 184 silicone elastomer base	Dow Silicones Corporation		Storage temperature: -30-60 °C
DOWSIL 184 silicone elastomer curing agent	Dow Silicones Corporation		Storage temperature: < 32 C
EPOCH2 microplate spectrophotometer	BioTek Instruments	EPOCH2
Escherichia coli ATCC 25922	Thermo Fisher Scientific	R19020
Ethanol, anhydrous	Fisher Chemical	459844
Fisherbrand microscope cover glass	Fisher Scientific	12540A
Fisherfinest premium microscope slides plain	Fisher Scientific	12-544-4
Hydrogen peroxide solution	Sigma-Aldrich	216763-500ML	Contains inhibitor, 30 wt% in H2O
Incu-Shaker Mini	Benchmark	E5-0014-01
Isopropanol	Sigma-Aldrich	190764
Magnesium sulfate, 7-hydrate	Macron Fine Chemicals	6066-04	Avantor Performance Materials, Inc.
Methanol	Sigma-Aldrich	179337-4L	ACS reagent, > 99.8%
NanoDrop One spectrophotometer	Thermo Scientific	ND-ONEC-W
Nitrogen, compressed	Matheson	UN1066
Oxygen plasma cleaner	Harrick Plasma	PDC-001-HP
Pentaerythritol tetra(mercaptoethyl) polyoxyethylene (4 arm-PEG)	NOF America Corporation	PTE-100SH	Sunbright-PTE-100SH
Phosphate buffered saline (PBS), 10X	VWR Amresco Life Science	K813-500ML	Store between 15 °C–30 °C
Polydimethyl siloxane (PDMS) Slygard 184	Dow Corning	4019862
Polygon400	Mightex	DSI-D-000
Premium microscope slides	Fisher Scientific	12-544-4	25 x 75 x 1 mm
Sodium chloride	Sigma Life Science	S5886-500G	Bioreagent, suitable for cell culture
Sodium hydroxide	Sigma-Aldrich	S8045-500G	BioXtra, > 98%, pellets (anhydrous)
Sodium phosphate monobasic dihydrate	Sigma-Aldrich	71505-250G	BioUltra, for molecular biology, > 99.0% (T)
Stainless steel thickness gage	Precision Brand Products	77739
Sulfuric acid	Sigma-Aldrich	320501-2.5L
Toluene, anhydrous, 99.8%	Sigma-Aldrich	244511-1L	Anhydrous, > 99.8%
Trichloro (1H,1H,2H,2H perfluorooctyl) silane (TPS), 97%	Sigma-Aldrich	448931-10G
Tryptic soy broth	Sigma-Aldrich	22092-500G	For microbiology
Ultrasonic sonicator	Fischer Scientific	FS-110H