A Technique to Prepare a Monoclonal Bacterial Culture

Abstract

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

This video demonstrates the preparation of a monoclonal bacterial culture for mineral production through serial dilution and colony isolation on an agar plate. By shaking the plate with glass beads, individual cells are spatially separated to form genetically uniform colonies, which are then transferred to liquid media for growth. The culture with the highest turbidity is selected, indicating the densest monoclonal population and the greatest potential for mineral output.

Protocol

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 powder of LB-Lennox broth in a 500 mL laboratory bottle with 400 mL of water.
2. Stir the contents with a polytetrafluoroethylene (PTFE) 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 in the biosafety cabinet (100 mL each). Cap the three bottles tightly. 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 receives ~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 homogeneous 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 homogeneous 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 homogeneous 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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
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
Incubator	Customer made	custom made	heat to 40 °C or cool to 18 °C with time cotrol
Biosafety cabinet	Zong Yen co., LTD	ZYBH-420	All bacteria related process are done here
Autoclave	Tomin Medical Equipmenco, Ltd., Taipei City, Taiwan, ROC	TM-329	heat to 120 °C for 10 min