Synthesis of Metal Oxide Microcapsules Using Metal-Reducing Bacteria

Samenvatting

Source: Lin, P., et al. Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion. J. Vis. Exp. (2018).

This video demonstrates the procedure for synthesizing rigid metal oxide microcapsules by culturing metal-reducing bacteria with metal ions, followed by sequential purification through sonication, centrifugation, and ethanol washing.

Protocol

CAUTION: Use latex gloves, protective eyeglasses, and a laboratory coat for performing the experiment. Whenever using the biosafety cabinet, turn on the cabinet fan and keep the cabinet door half-closed.

1. Preparation of Glass Beads

1. Place 100 glass beads of 3 mm diameter in a 100-mL laboratory bottle, and then cap it tightly.
2. Autoclave the contents at 120 °C for 10 min.
3. Leave the bottle to cool down to room temperature, then place it in the biosafety cabinet.

2. Preparation of Lysogeny Broth (LB)

1. Dissolve 8 g of powder of LB-Lennox broth in a 500-mL laboratory bottle with 400 mL of water.
2. Stir the contents with a PTFE ( Polytetrafluoroethylene) magnetic stirring bar for 20 min, and then cap it tightly.
3. Autoclave the contents at 120 °C for 10 min.
4. Leave the solution to cool down to room temperature and place it in the biosafety cabinet.
5. Using a pipette, aliquot the broth into eight 15-mL centrifuge tubes (12.5 mL each) in the biosafety cabinet.
6. Aliquot the remaining broth into three 100-mL laboratory bottles (100 mL each) in the biosafety cabinet. Cap the three bottles tightly and keep them in the biosafety cabinet.

3. Culture of Shewanella algae

1. Use the deep-frozen cryopreserved strain.
2. In the biosafety cabinet, pick out 1 mL of the frozen material from the frozen tube with a stainless-steel spatula, and place it into a centrifuge tube prepared in step 3.5.
3. Incubate the cultures for 24 h in a 37 °C incubator.

4. Preparation of LB-Lennox (Broth with Agar) Petri Dishes

1. Dissolve two tablets of LB-Lennox (broth with agar) into a 100-mL laboratory bottle with 100 mL of water.
2. Stir the contents with a PTFE magnetic stirring bar for 20 min and then cap it tightly.
3. Autoclave the contents at 120 °C for 10 min.
4. In the biosafety cabinet, aliquot by hand 100 mL of solution into 4 Petri dishes, ensuring each receive ~25 mL. Leave the solution to cool down to room temperature.

5. Preparation of Monoclonal Bacteria

1. In the biosafety cabinet, label the three bottles prepared in step 2.6, #1, #2, and #3, respectively.
2. Pipette 0.1 mL of the resultant bacterial suspension in step 3.3 into bottle #1. Cap the bottle and swing it by hand for 1 min to get a homogenous solution.
3. Pipette 0.1 mL of the resultant bacterial liquid in step 5.2 into bottle #2. Cap the bottle and swing it by hand for 1 min to get a homogenous solution.
4. Pipette 0.1 mL of the resultant bacterial liquid in step 5.3 into bottle #3. Cap the bottle and shake it by hand for 1 min to get a homogenous solution.
5. Pipette the liquid in bottle #3 into the 4 Petri dishes prepared in step 4.4, using a volume of 0.02 mL each.
6. Put the glass beads prepared in step 1.3 into the 4 Petri dishes used, 4 beads in each dish.
7. Close the lids of the Petri dishes and shake them by hand for 1 min.
8. Turn the Petri dishes upside down and incubate in a 37 °C incubator for 24 h.

6. Multiplication of Monoclonal Bacteria

1. Fetch 7 tubes prepared in step 2.5.
2. Pick out the resultant monoclonal bacteria from the 4 Petri dishes prepared in step 5.8 with a stainless steel-spatula, and put them into 7 tubes separately.
3. Leave the 7 tubes in a 37 °C incubator for 24 h.
4. Pick out the one with the largest light scattering using the visual colorimetric method.

7. Preparation of LB-Lennox Broth with Glucose and Salt

1. Put 10 g of LB-Lennox broth, 10 g of NaCl, and 10 g of glucose into a 500-mL laboratory bottle. Add water until the volume reaches 450 mL.
2. Stir the contents with a PTFE magnetic stirring bar for 20 min.
3. Autoclave the contents at 120 °C for 10 min.

8. Preparation of Sodium Tungstate

1. Put 16.5 g of Sodium Tungstate Na₂WO₄．2H₂O into a 100-mL laboratory bottle with a stainless-steel spatula. Add water until the volume reaches 50 mL.
2. Stir the contents with a PTFE magnetic stirring bar for 20 min.
3. Autoclave the contents at 120 °C for 10 min.
4. In the biosafety cabinet, get filtrate via a vacuum fiberglass filter with pores of 1 µm.

9. Preparation of LB with Glucose, Salt, and Sodium Tungstate

1. In the biosafety cabinet, pour the filtrate gained in step 8.4 by hand into the solution with glucose and salt prepared in step 7.3.
2. In the biosafety cabinet, aliquot with a pipette the 500 mL resultant solution in step 9.1 into 10 x 50 mL centrifuge tubes.

10. Culture of Bacteria

1. In the biosafety cabinet, fetch the liquid prepared in step 6.4 and aliquot it with a pipette into the 10 test tubes prepared in step 9.2, with each tube receiving 0.05 mL.
2. Incubate the 10 tubes in a 37 °C incubator for 120 h.

11. Harvest of BME minerals

1. Ultrasonicate each of the 10 tubes in step 9.2 at 20 KHz with 150 W for 1 h.
2. Centrifuge the tubes at 2,025 x g for 1 h.
3. Remove the clear liquid in the tubes with a pipette, add water, and then repeat steps 11.1 and 11.2 one more time.
4. Remove the clear liquid in the tubes with a pipette, add alcohol, and then ultrasonicate them at 20 KHz with 150 W for 1 h.
5. Centrifuge the tubes at 2,025 x g for 1 h.
6. Repeat steps 11.4 and 11.5 one more time
7. Harvest BME minerals by removing the clear liquid in the tubes with a pipette; afterwards, immediately cap the tubes without running any drying process.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
LB(Lennox)broth with agar tablets	Sigma-Aldrich	L7075	1 tablet for 50 mL broth with agar
LB (Lennox) broth	Sigma-Aldrich	L3022-1KG	LB (Lennox) powder 1 kg
Dextrose anhydrous	Nihon Shiyaku Reagent	PL 78695	glucose
Sodium Tungstate	Nihon Shiyaku Reagent	PL 76050	Na2WO4 · 2H2O
Sodium Chloride	Nihon Shiyaku Reagent	PL 68131	NaCl
Ethanol 99.5%	Acros organics	AC615090040	CH3CH2OH
Water	Made in our university		de-ionlized water
Autoclave	Tomin Medical Equipmenco, Ltd., Taipei City, Taiwan, ROC	TM-329	heat to 120 °C for 10 min
Centrifuge	Digit System Laboratory System, New Taipei City, Taiwan, ROC	DSC302SD	centrifuge at 2025 x g
-80 °C Refrigerator	Panasonic	MDF-U3386S	Use to deep-freeze cryopreserve strain
Ultrasonic Homogenizer Sonicator Processor Cell Disruptor	Lenox	UPS-150	frequency 20 KHz power 150 W
Incubator	Customer made	custom made	heat to 40 °C or cool to 18 °C with time cotrol
Reciprocal shaking baths	Kingtech Scientific Co., Ltd	WBS-L
Digital Stirring Hot Plate	Corning	#6797-620D	use with PTFE magnetic stirring bar
Biosafety cabinet	Zong Yen co., LTD	ZYBH-420	All bacteria related process are done here
50 mL centrifudge tube	Falcon	14-432-22
15 mL centrifudge tube	Falcon	14-959-53A
Laboratory bottle 100 mL	Duran	21 801 24 5
Laboratory bottle 500 mL	Duran	21 801 44 5
Stainless steel spatula	Chemglass	CG-1981-10
PTFE Disposable Stir Bars	Fisher	S68066
Plastic Petri Dishes	Fisher	S33580A
Shewanella algae	Courtesy of author #3	Courtesy of author #3