---++ Working with <i>Snodgrassella alvi</i> _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 [[https://wkkwong.com][Waldan Kwong]] in the laboratory of [[http://web.biosci.utexas.edu/moran/][Dr. Nancy Moran]] and are described in depth in [[http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.044875-0][here]]. ---+++ _In vitro_ culture conditions ---++++ Plate Growth _Snodgrassella alvi_ strain wkB2 (type strain) is generally cultured at 35°C and 5% CO<sub>2</sub>. 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. <Br> Perform conjugation as described here: [[ProtocolsConjugation][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. %TABLE{headeralign="left,center,center,center,center,left" dataalign="left,center,center,center,center,left,center,left"}% | *Reagent* | *Abb* | *Stock Conc.* | *Working Conc.* | *Dilution* | *Solvent* | *MSDS* | *Notes* | | Ampicillin | Amp | 100 mg/mL | 20 µg/mL | 5,000× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=A9518&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fa9518%3Flang%3Den][ 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 | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=C1389&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fc1389%3Flang%3Den][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 | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=C0378&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fc0378%3Flang%3Den][link]] | | | Gentamicin | Gen | 20 mg/mL | 25 µg/mL | 800× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927175][link]] | For non-canonical experiments: <br />Use 20 µg/mL in culture from 20mg/mL 1000× stock | | Kanamycin | Kan | 50 mg/mL | 20 µg/mL | 2,500× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927199][link]] | Plates start to lose sensitivity after 2 weeks; [[https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/k1377pis.pdf][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 | [[http://www.sciencelab.com/msds.php?msdsId=9926169][link]] | | | Rifampicin | Rif | 50 mg/mL | 30 µg/mL | 1667× | MeOH + drops of 10M NaOH | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=R3501&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fr3501%3Flang%3Den][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× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=S4014&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fs4014%3Flang%3Den][link]] | | Streptomycin | Str | 100 mg/mL | 20 µg/mL | 5,000× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927414][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. | *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.
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Topic revision: r7 - 2022-09-26 - ZuberiAshraf