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

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

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

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.

-- Main.SeanLeonard - 23 Feb 2017

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Contributors to this topic Edit topic SeanLeonard, ZuberiAshraf
Topic revision: r2 - 2025-03-20 - 22:41:16 - Main.ZuberiAshraf
 
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