(r8) ProtocolsCulturingSnodgrassellaAlvi < Lab

---++ 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. |
| 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.
Barrick Lab > ProtocolList > ProtocolsCulturingSnodgrassellaAlvi
Topic revision: r8 - 2022-09-28 - 18:59:02 - Main.ZuberiAshraf