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

• Pre-protocol questions: Do you know how to write in the format for the wiki?
  • See https://www.mediawiki.org/wiki/Help:Formatting here for help with formatting.
  • To create a bulleted list, use an asterisk before each section. To create a numbered list, use a # before each section. For subheadings that explain steps in further detail, place a #: before that section.
  • To indent text, place a : in front of text for single indent. For double indent, place : : in front of text. For multiple indent, place : : : : : in front of text.

Protocol

1. Include a goals section at the beginning of the protocol that explains what the protocol will accomplish.
   • Mention here if there are other protocol options, such as choosing between growth rate curves.
2. Include a “pre-protocol questions” section following the goals section that addresses any questions that readers may have before starting the protocol.
   • This is also a good place to remind readers what materials they need, especially if those materials would take a long time to make.
3. Number all the steps of the protocol
   • This helps when you are trying to refer to a specific step in the protocol and need to give an exact number
   • Keep these numbered steps concise. If you feel that you need to include more detail for people who have not done the experiment before, include sub-steps like we have here.
4. Include where to find less-common materials, such as the nanodrop or specialized equipment. Never mention very common materials such as pipet tips.

1. Include a “clean-up” section that lists all the steps necessary to properly clean up the lab after the experiment is finished
2. Have other students read your protocol before posting it to the lab wiki
   • Preferably, you would have first- and second-year students review the protocol, not just third- or fourth-years

Protocol Review

• Include the initials of students who read and checked the protocol to keep track of which protocols have been reviewed.

Watch out!!!

This protocol is über-rough; edit it with Eric before attempting!!!! As written, it is wrong!!!! Move materials descriptions to General Laboratory section of wiki

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