Oct 05, 2025
Colony Picking
- igemnthuth 1
- 1iGEM_NTHU_Taiwan
- iGEM NTHU
Protocol Citation: igemnthuth 2025. Colony Picking. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvw4mo9lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 04, 2025
Last Modified: October 05, 2025
Protocol Integer ID: 228962
Keywords: colony picking isolate, subsequent liquid culture, colonies on culture media, experimental material, reproducibility of experimental material, colony, frozen storage, single strain, strain, frozen stock, material
Abstract
Isolate a single strain from frozen stock or colonies on culture media, then purify and establish subsequent liquid culture or frozen storage to ensure uniformity and reproducibility of experimental materials.
Materials
- Frozen glycerol stock culture or colonies from LB agar plates
- LB broth (liquid medium)
- LB agar plate (or species-specific medium, e.g., Pseudomonas isolation agar)
- Sterile PBS (optional, if dilution is required)
- Sterile glycerol (for preparing glycerol stocks, final concentration 20%)
- Sterile inoculation loop
- 50 mL Falcon tube or other liquid culture vessel
- Shaking incubator (30 or 37 °C, 140–220 rpm, depending on optimal growth conditions for the strain)
- Spectrophotometer or microplate reader (for measuring OD₆₀₀)
- Incubator (30 or 37 °C)
Troubleshooting
Initial Inoculation
Take a single colony from a glycerol stock or agar plate.
Using a sterile inoculating loop, pick a small amount of the colony and inoculate it into 10 mL LB broth (in a 50 mL Falcon tube).
Gently swirl the inoculating loop to ensure the bacteria are suspended.
Loosen the cap of the culture tube (to maintain aerobic conditions) and incubate in a shaking incubator at 30 or 37 °C, 140 rpm, for 12–14 hours.
Check Growth Status
Observe whether the culture has turned turbid.
If necessary, measure OD₆₀₀ = 0.6–0.8 to confirm it is in log phase.
Streaking for Isolation
Prepare a fresh LB agar plate (equilibrated to room temperature).
Dip a sterile inoculating loop into the overnight culture and streak three parallel lines on the agar.
Using a new sterile loop dipped in sterile LB broth, streak three more lines at an angle, making sure the first line crosses the previous streaks.
Rotate the plate 90°, use a new sterile loop, and streak three “dry lines.”
Rotate the plate another 90° and repeat streaking three more dry lines, with the final streak extending toward the center of the plate.
Invert the plate and incubate at 30 or 37 °C for 24 hours.
Inspection and Picking of Single Colonies
On the following day, check for single colonies approximately 1–2 mm in diameter.
If growth is insufficient: extend incubation for an additional 12–24 hours.
If growth is excessive: dilute the culture before streaking (e.g., 1:4 dilution).
Using a sterile inoculating loop, carefully pick a single colony and inoculate into 10 mL LB broth.
Incubate in a shaking incubator at 30 or 37 °C, 140 rpm, for 12–14 hours.