This protocol describes a method for isolating new bacteria from fecal samples. The goal is to diversify the lab culture collection to enable us It uses multiple types of rich/selective media to try to enrich different populations. This isolates can then be identified by 16S rRNA sequencing and stored as frozen glycerol stocks in the freezer for future experiments. Note: all materials should be placed in the anaerobic chamber 1-2 days ahead of time to ensure they have degassed their oxygen content.
- Fecal sample (~100mg aliquot that has been flash frozen at -80˚C).
- PBS + 0.1% L-cysteine hydrochloride (autoclaved)
- mGAM broth
- One plate per sample of each media in Table 1
- 20 µL tips
- 20 µL pipette
- Sterile falcon tubes (VWR 60819-524)
- Crimp Top Vials (Sigma 854964)
- Crimp Seals (Sigma 854144)
- Crimping Device (Ex. Sigma Z114243-1EA)
- 1.5 mL Cryovial (External threads, ex Simport T310-2A)
- Appropriate growth media
- Sterile 50% glycerol (mix 50:50 with water and autoclave)
- Anaerobic chamber running 5% Hydrogen, 10-20% carbon dioxide, balance Nitrogen
| Media | Expected Target |
|---|---|
| BHI CHVR | not specific |
| mGAM | not specific |
| FAA | not specific |
| MRS | lactic acid bacteria, Enterococcus, Bifidobacterium |
| TCCFA | C. difficile (yellow), C. inocuum (yellow), Enterococcus spp. (will be pink)* |
| TSA Blood | not specific |
| TOS Proprionate | Bifidobacterum spp. |
| BBE | Bacteroides spp. |
| BE | Enterococcus and Streptococcus |
| CIN | Proteobacteria, Yersinia spp., Aeromonas spp. |
| *See recipes here |
- Add 1mL of reduced PBS to 100mg feces aliquot
- Vortex well to mix
- Using disposable loop or pipette tip, do four quadrant streak on each of the plates in Table 1
- Incubate plates for 4-7 days at 37˚C in anaerobic chamber
- Inspect plates for growth noting the number of different colonies which differ in their morphology (shape, colour, edges, texture) and transfer 2 plates for each unique colony into the anaerobic chamber. Label each plate according to the sample of origin, the media on which it grew, and a unique number. For example: BZH16 mGAM2.
- Using a Pipette tip, pick a single well-isolated colony from the original plate and do a quadrant streak on a new plate (of the same type) to purify.
- Incubate new plate at 37˚C for 72h.
- Inspect the new plates to ensure that the colonies are homogenous in morphology.
- Using a pipette tip, pick a single colony from new plates and four quadrant streak onto a new plate (of the same type) to further purify and ensure that we will have a single isolate in the future.
- Incubate new plate at 37˚C for 24-72h depending on speed of growth of organism.
- Aliquot 3 mL of mGAM into 1 falcon tube per isolate (plus 1 more for a sterile control) and move to anaerobic chamber
- Transfer a single colony from the third round of streak purification into 3mL mGAM.
- Note the colony morphology and approximate growth rate of the culture in your lab notebook.
- Incubate for 2-3 days.
- Transfer 200 µL of liquid culture to a 1.5mL microcentrifuge tube to proceed to sanger 16S rRNA sequencing. Note: this can be frozen at -80˚C and processed later.
- Transfer 1mL of liquid culture to each of a crimp top vial and a screw top vial
- Add 0.5 mL 50% glycerol to each tube and mix by pipetting
- Seal both vials
- Using a marker label the cryovials (ex BZH16 mGAM2)
- Remove vials and transfer to a rack in the -80˚C freezer
- After Sanger sequencing, enter the strain into the main lab collection sheet (see #strains on slack). Include all relevant information and a copy of the 16S sequence.
- Print a label with the Species, Strain, Box Position, and Date of Preparation.
- Breifly remove stock from -80˚C, wipe surface with ethanol-damp paper towel and affix the label.
- Transfer the cryovial to the lab strain collection working stocks, and the crimp top of the archival stocks.