Working with Snodgrassella alvi

Snodgrassella alvi is a member of the honey bee (Apis mellifera) gut core microbiota that can be cultivated in vitro. Its isolation and characterization were recently carried out by Waldan Kwong in the laboratory of Dr. Nancy Moran and are described in depth in here.

In vitro culture conditions

Plate Growth

Snodgrassella alvi strain wkB2 (type strain) is generally cultured at 35°C and 5% CO₂. It grows robustly on agar plates after 48 hours on the following media:

• Heart Infusion Agar
• Heart Infusion Agar supplement with Sheep's Blood
• Columbia Agar supplement with Sheep's Blood (CBA)
• R2 Agar

Liquid Media

Snodgrassella alvi can be cultivated in the following liquid media (same general culture conditions as above, without shaking):

• Insectagro (proprietary media)
• Salt-free LB
• R2 Liquid Media

Conjugation

Snodgrassella alvi can be conjugated using E. coli donor strains and mobilizable rsf1010-based plasmids. Refer to the Bee Microbiome Toolkit paper for full description of working with this species.
Perform conjugation as described here: Conjugation, with the following specifics: use 10:1 ratio of S. alvi to donor E. coli pooled on CBA plates. Incubate overnight, then resuspend and plate on selective CBA plates.

Antibiotic concentrations and stock solutions

This table lists “standard” concentrations of antibiotics commonly used in microbiology laboratories. These concentrations apply to S. alvi (wkB2). Antibiotic selection in other organisms may use different concentrations, so check primary literature for more info.

Reagent	Abb	Stock Conc.	Working Conc.	Dilution	Solvent	MSDS	Notes
Ampicillin	Amp	100 mg/mL	20 µg/mL	5,000×	ddH2O	link	Ampicillin degrades quickly in both plates and stock solutions. Culture plates with Amp can be stored at 4°C for about 2 weeks. Stock solutions can be stored at 4°C for 2 weeks but can last as long as 4-6 months when stored at -20°C. In general, use carbenicillin for ampicillin selection, because it is more chemically stable.
Carbenicillin	Crb	100 mg/mL	50 µg/mL	2,000×	50% EtOH	link	Carbenicillin can be used in place of Ampicillin at the same working concentration. Carbenicillin is more stable than Ampicillin, and is generally preferable.
Chloramphenicol	Cam	20 mg/mL	20 µg/mL	1,000×	EtOH	link
Gentamicin	Gen	20 mg/mL	25 µg/mL	800×	ddH2O	link	For non-canonical experiments: Use 20 µg/mL in culture from 20mg/mL 1000× stock
Kanamycin	Kan	50 mg/mL	20 µg/mL	2,500×	ddH2O	link	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.
Nalidixic Acid	Nal	30 mg/mL	30 µg/mL	1,000×	1M NaOH	link
Rifampicin	Rif	50 mg/mL	30 µg/mL	1667×	MeOH + drops of 10M NaOH	link	Toxic to humans, use PPE! Rifampicin is TOXIC to humans, light-sensitive, and degrades in solution rather quickly. It needs to be stored wrapped in foil and replaced every 2 months.
Spectinomycin	Spec	60 mg/mL	30 µg/mL	2,000×	ddH2O	link
Streptomycin	Str	100 mg/mL	20 µg/mL	5,000×	ddH2O	link
Tetracycline	Tet						S. alvi is naturally resistant to Tet. Use 10 µg/mL. Can be used to isolate S. alvi from other bacteria or contaminants during bee colonization assays without other antibiotics.
Trimethoprim	TMP						S. alvi is naturally resistant to TMP. Use 20 µg/mL.

References:

• Kwong, Waldan K., and Nancy A. Moran. "Cultivation and characterization of the gut symbionts of honey bees and bumble bees: description of Snodgrassella alvi gen. nov., sp. nov., a member of the family Neisseriaceae of the Betaproteobacteria, and Gilliamella apicola gen. nov., sp. nov., a member of Orbaceae fam. nov., Orbales ord. nov., a sister taxon to the order ‘Enterobacteriales’ of the Gammaproteobacteria." International journal of systematic and evolutionary microbiology 63.Pt_6 (2013): 2008-2018.

• Leonard, Sean P., et al. "Genetic engineering of bee gut microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids." ACS synthetic biology 7.5 (2018): 1279-1290.