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 do....

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