Difference: ProtocolsCulturingFirm_5 (1 vs. 2)

Revision 22025-03-20 - ZuberiAshraf

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Working with Lactobacillus _"Firm-5"

The Lactobacillus "Firm 5" clade are gram positive bacterial symbionts of the honey bee ( Apis mellifera) gut core microbiota that can be cultivated in vitro. Their isolation and characterization were recently carried out by Waldan Kwong in the laboratory of Dr. Nancy Moran and are described in depth here. Genomes for two Firm-5 strains, wkB8 and wkB10 are described here
In vitro culture conditions

Plate Growth

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Firm 5 strains wkB8 and wkB10 are generally cultured at 35°C and 5% CO₂. They grow robustly on agar plates after 48 hours on the following media:

Columbia Agar supplement with Sheep's Blood

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Firm 5 strains wkB8 and wkB10 are generally cultured at 35°C and 5% CO₂. They grow robustly on agar plates after 48-72 hours on the following media:

Columbia Agar supplement with 5% Sheep's Blood

MRS agar

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Colonies will appear tiny on CBA agar but will have a slight clearing of blood (alpha hemolysis), along the streaks/path where they are growing. The hemolysis/clearing will increase over time if left in the incubator.

Liquid Media

Firm 5 strains wkB8 and wkB10 can be cultivated in the following liquid media (same general culture conditions as above, without shaking):

MRS broth

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Antibiotic Concentrations

This table lists some of the antibiotics tested on Lactobacillus Firm-5 strains. These concentrations apply to Lactobacillus (wkB8 and wkB10). Antibiotic selection in other organisms may use different concentrations, so check the primary literature for more info.

Reagent	Abb	Stock Conc.	Working Conc.	Dilution	Solvent	MSDS	Notes
Kanamycin	Kan	50 mg/mL	50 µg/mL	2,500×	ddH₂O	link	Lactobacillus Firm-5 wkB8 and wkB10 are both naturally resistant to Kan. Use 50 µg/mL. Plates start to lose sensitivity after 2 weeks; recommended Kanamycin plates only last a few weeks at 4°C; best to make fresh Kan plates every 2 - 4 weeks.
Erythromycin	Ery	250 mg/mL	250 µg/mL	1,000×	EtOH	link	May crystallize when added to liquid culture. Still works. Selection strength may vary in non-model bacteria.
Tetracycline	Tet	10 mg/mL	10 µg/mL	1,000×	70% EtOH	link	Light sensitive, stability < 2 weeks.

-- SeanLeonard - 23 Feb 2017

Revision 12017-02-23 - SeanLeonard

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