Dual Layer Assays

Dual Layer Assays

Goal

• 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.
• TTC (Triphenyl tetrazolium chloride) is used to color the cells red. There may be a microcentrifuge tube in the refridgerator; if not, thaw one from the "Molecular Reagents" box in the -20 degree freezer

Protocol

1. Number all the steps of the protocol

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