This is a snapshot of the protocol as practiced on May 10, 2022. The latest version of the protocol is here.
- at least 25 mL of LB media
- six 14 mL culture tubes
- six sterile microcentrifuge tubes AKA Eppendorf tubes (1.7 mL capacity each)
- overnight liquid cultures of Agar Art E. coli, at least 1 mL each:
- Pink
- GFP
- Teal
- Blue
- Purple
- DH5α as control
- 1000X chloramphenicol stock solution (in BosLab −20°C freezer), at least 25 µL
- Three micropipettors of the following types, plus compatible tips:
- at least 20 mL 1M NaCl sterile solution
- 1M means 1 molar concentration, i.e. 1 mole NaCl per liter of solution
- at least 50 mL 1 M trehalose sterile solution
- Dry culture carrier comprising either:
- sterile petri dishes and pieces of filter paper cut to fit in them, or
- new unused zip lock bags and pieces of filter paper cut to fit in them
- 37°C plate incubator to use for drying cells (consider drying at 30°C if cells don't revive)
- 37°C shaker incubator for liquid cultures
- tube vortexer
- PCR workstation, or laminar flow workbench, or clean work surface with bunsen burner to create a sterile airspace in which to work
- Work in a sterile airspace such as the BosLab PCR workstation (a.k.a. “PCR hood”).
- Labeled 6 culture tubes with your initials, the date, and as follows:
- LB+chloro Pink
- LB+chloro GFP
- LB+chloro Teal
- LB+chloro Blue
- LB+chloro Purple
- LB only, DH5α control
- With a P1000 micropipette, transfer 900 µL LB media to each tube, TWICE, for a total of 1800µL LB into each tube.
- Using a fresh P1000 tip, transfer 1000 µL of 1M (1 molar) NaCl to each tube THREE TIMES, for a total of 3000 µL into each tube.
- With a P20 micropipette, transfer 5 µL of 1000X chloramphenicol stock solution to each of the “LB+chloro” tubes, being sure to skip the “LB only, DH5α control” tube.
- Vortex each tube for 10 seconds so contents are well mixed.
- Using a P200 micropipette to transfer 195 µL GFP Agar Art E. coli liquid culture to the LB+chloro GFP tube.
- Same with the other colors into the corresponding LB+chloro color tube.
- Incubate tubes in the orbital shaker at 37°C and 260 RPM for 36 hours. See Ref. 5 below for justification for the 36 hours. Alas, we were sharing the BosLab incubator with a long-running 30°C project, so we incubated at that temperature. Also, because of schedule availability, we ended up incubating for 4 days.
- While incubating, label six sterile microfuge tubes with your initials, the date, and:
- LB+chloro Pink
- LB+chloro GFP
- LB+chloro Teal
- LB+chloro Blue
- LB+chloro Purple
- Label 6 sterile Petri dishes the same way.
- Sterile transfer a piece of autoclaved paper into each Petri dish.
- Pipette 1500 µL from each incubated culture tube to the correspondingly labeled microfuge tube.
- We used a P1000 and pipetted 750 µL twice.
- Centrifuge the microfuge tubes to pellet cells. Discard supernatant into liquid biohazard waste.
- Resuspend cells in 150 µL of 1 M trehalose by vortexing for 15-30 seconds, or until the pellet is gone.
- Pipette 50 µL per piece of paper. This covers an area of approximately 1 cm x 1 cm.
- Next time, laser-print some boxes or circles on the paper so we know where to pipette the culture.
- Our sterile paper pieces were more like 4 cm x 6 cm, so we pipetted the full 150 µL of a tube onto new piece of sterile paper.
- Put paper in sterile petri dishes with lids closed but NOT parafilmed or sealed. Use two small pieces of tape to keep the lids from opening if the dishes are dropped.
- Or put paper in new unused zip lock bags (leave bags open). This might work also but we haven't tried it yet.
- Place petri dishes or bags in plate incubator to dry. May take 1-3 days depending on ambient humidity.
- Due to scheduling, we incubated for a full 7-day week.
- Remove petri dishes and/or bags and store in a cool dry place.
- Autoclave a pair of scissors and two pairs of tweezers.
- Do all the steps below in a sterile atmosphere, e.g. BosLab PCR hood, or clean work surface within a meter of a Bunsen flame.
- Label plates with your initials, date, “LB+chloro”, and either “Pink Agar Art” or “GFP Agar Art”.
- Pour LB agar plates with the appropriate antibiotic. For the BosLab agar art E. coli we're using, this is chloramphenicol.
- Under the light of the PCR hood, note the location of the dried culture on each piece of paper.
- Using the sterile tweezers and scissors, cut small squares of dried cultured paper and place onto the LB agar plates.
- Wet each piece of paper with 100 µL distilled H2O.
- Cover each plate with its lid. Wrap plate(s) in parafilm or tape closed.
- Now the plates can be safely taken out of the sterile atmosphere.
- If not using parafilm wrapping, place plates in closed ziplock bags to prevent drying during incubation.
- Incubate plates at 37°C for 2-3 days, or until growth is noticeable around the edges of the inoculating papers.
- Cells now revived and ready for further use!
- Let's say we want to rehydrate and revive N cultures. For today, N=2.
- Autoclave a pair of scissors and two pairs of tweezers.
- Perform all steps below in a sterile atmosphere.
- Label N 14 mL culture tubes with your initials, date, “LB+chloro”, and the name of the bacterial strain you're reviving. For today, there are two: “Pink Agar Art E.coli” and “GFP Agar Art E.coli”.
- Sterile transfer (N+1) × 2 mL of LB to a sterile container. This could be another 14 mL culture tube if the quantity fits, else use sterile glassware such as an autoclaved 100 mL bottle.
- For today, N=2, so use a sterile 10 mL serological pipette to transfer 6 mL LB to a sterile culture tube that we'll label “LB+chloro”.
- Antibiotic stock solutions at BosLab are made at 1000X concentration and stored in the “Antibiotics” box inside the −20°C chest freezer. Sterile transfer (N+1) × 2 µL of 1000X chloramphenicol stock solution to the LB. Mix the LB and chloramphenicol thoroughly.
- For today, use a P20 micropipette to transfer 6 µL of chloro stock to the LB. Use the vortex to help mix the LB and chloro.
- Using the sterile tweezers and scissors, cut small squares of dried cultured paper and insert into the correspondingly-labeled culture tube. Close and tap each tube as necessary to ensure the paper goes to the bottom of the tube.
- Sterile transfer 2 mL of the LB+chloro into each culture tube to wet the dried paper within.
- Cap each tube but be sure the cap is in the “loose” position so air can get in.
- Incubate tubes in the shaking incubator at 250 rpm and 37°C for 1-2 days or until desired growth stage is reached.