Barrick Lab :: ProtocolsCulturingFirm

---++ Working with Lactobacillus _"Firm-5"_
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The <em>Lactobacillus "Firm 5" </em>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 <a target="_top" href="https://wkkwong.com/" style="text-decoration: none; color: #666666; border-color: #58438b; border-style: none none dotted; border-width: 1px">Waldan Kwong</a> in the laboratory of <a target="_top" href="http://web.biosci.utexas.edu/moran/" style="text-decoration: none; color: #666666; border-color: #58438b; border-style: none none dotted; border-width: 1px">Dr. Nancy Moran</a> and are described in depth <a target="_top" href="http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.044875-0" style="text-decoration: none; color: #666666; border-color: #58438b; border-style: none none dotted; border-width: 1px">here</a>. Genomes for two Firm-5 strains, wkB8 and wkB10 are described [[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4241670/][here]]

_In vitro_ culture conditions
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---++++ <a name="Plate_Growth"></a>Plate Growth
 _Firm 5_ strains wkB8 and wkB10 are generally cultured at 35°C and 5% CO<sub style="font-family: 'Palatino Linotype', 'Book Antiqua', Palatino, serif">2</sub>. 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.

---++++ <a name="Liquid_Media"></a>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

---++++ <a name="Antibiotic Concentrations"></a>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.  
%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* |
| Kanamycin | Kan | 50 mg/mL | 50 µg/mL | 2,500× |  ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927199][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; [[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. |
| Erythromycin | Ery | 250 mg/mL | 250 µg/mL | 1,000× |  EtOH | [[https://www.fishersci.com/store/msds?partNumber=AAJ6227909&productDescription=ERYTHROMYCIN+CEL+CULTURE+G+10G&vendorId=VN00024248&countryCode=US&language=en][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 | [[https://www.fishersci.com/store/msds?partNumber=AC233100025&productDescription=TETRACYCLINE+HYDROCHLORI+2.5GR&vendorId=VN00032119&countryCode=US&language=en][link]] | Light sensitive, stability < 2 weeks. |

-- Main.SeanLeonard - 23 Feb 2017
Barrick Lab > ProtocolList > ProtocolsCulturingFirm_5
Contributors to this topic
ZuberiAshraf, SeanLeonard
Topic revision: r2 - 2025-03-20 - 22:41:16 - Main.ZuberiAshraf