Dual Layer Assays

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Dual Layer Assays


  • Goal: To plate two strains of bacteria (most commonly 'S. pneumoniae') in a way to test bacteriocin production. There are two strains — the producer strain (which may produce a bacteriocin, and therefore is at higher concentrations) and the responder strain (which is being killed by the producer strain).

Pre-Protocol Questions

  • Which strains are you working with, and do you have both strains grown overnight on plates? Fresh plates (i.e. plated the day before) are necessary.
  • Do you have 6-well plates with 2ml Tryptic Soy Broth agar pipetted into each well?

Special Reagents Needed

  • Check if the lab has filtered catalase at 30,000U / ml of PBS, located in the refrigerator.
  • TTC (Triphenyl tetrazolium chloride) is used to color the cells red. There may be a microcentrifuge tube in the refrigerator; if not, thaw one from the "Molecular Reagents" box in the -20 degree freezer.
  • Check if we have Tryptic Soy Broth soft (0.75%) agar, located in the microbiology media cabinet, on the bottom shelf. This is also called top agar — it's the same thing.


  1. All parts of this protocol must be done in the BSL-2 hood.
  1. Move a dry block heater and small vortex into the BSL-2 hood.
  1. Move the prepared 6-well plates (prepared with 2ml TSB hard agar in each well) into the BSL-2 hood.
  1. Add 3ml Tryptic Soy Broth (TSB) to a sterile 13mm test tube; add 100ul catalase and many (more than one) colony from the overnight plate into this tube, using an inoculating loop. Give a quick vortex to distribute equally. This needs to be done for both the producer and the responder strain.
  1. Place strains in the 37 degree incubator with 5% CO2 until the OD 600nm is at 0.3, but not above 0.4 (approximately 3-5 hours). Discard the sample if the OD is significantly above 0.4, as this could indicate that the cells have entered stationary, and immediately afterward, death phase.
  1. During this incubation, prepare soft agar tubes. Place multiple sterile 13mm tubes into the dry block heater and set the temperature to 40 degrees. Allow time for these tubes to reach 40 degrees before adding anything to them (15-30 minutes should be sufficient).
  1. Melt TSB soft agar in the microwave until the entire bottle is melted. Pipette 4ml of soft agar into each tube in the dry block heater. It is OK if the soft agar is very hot — there will be time for it to cool down to 40 degrees.
  1. Once the cells have grown to over 0.3, add TSB to these tubes to adjust their OD 600nm back down to 0.3. This can be done repeatedly to co-ordinate multiple cultures — and we can even dilute down past OD 0.3. The key that each culture can not reach significantly above OD 600 of 0.4.
  1. Add xul of the producer strain as a droplet onto one of the wells

  1. Include a “clean-up” section that lists all the steps necessary to properly clean up the lab after the experiment is finished

Protocol Review

  • No one has reviewed this protocol yet.

Protocal dual layer assay experiment:

Materials: 9 cm diameter Petri dishes Complete Transformation Medium (CTM) prepared from 3% Tryptic Soy Broth (Oxoid Ltd, England) and 0.1 % yeast extract (Melford laboratories Ltd, Ipswich, UK) at pH 7.8. Agar plates prepared from 3% Tryptic Soy Broth, 1.6% Agar and 0.1% solution of 5% antifoam. Soft agar prepared from 3% Tryptic Soy Broth, 0.8% Agar and 0.1% solution of 5% antifoam. When adding Catalase and/or live cells, always use soft agar at 45 degrees, not more than that. Catalase (33 000 u/ml -> might be wrong, exact instruction in the pack)

Aliquots: 200 microliters of cells at OD 0.3 and 50 microliters glycerol 80%

Day 1: Aliquots are inoculated in 3 ml CTM and then grown at 37 degrees Celsius and 5% CO2 until they reach OD 0.3. Dilute 10 times. Plate the potential killers using a pin replicator (approx 1 microliter of the potential killer strain.) Incubate Petri dishes for 1 hour (to make sure that potential killer mix on top of agar is dry). Add an overlay of 5 ml soft agar + 100 microliters of Catalase.

Day 2: Inoculate aliquots in 3 ml CTM and then grow at 37 degrees Celsius and 5% CO2 until they reach OD 0.3. Prepare the overlay: • 3 ml of soft agar at 45 degrees C • 30 microliters of cells at OD 0.3 (you can change the amount, depending on how dense you want the lawn of target strains, but always document it) • 100 microliters of Catalase Add the overlay on the Petri dishes. Incubate.

Day 3 Stain the plates with 15 ml of 2% solution of Methilane blue, two times + two washes. Leave to dry.

Day 4,5,6… Assess the interactions