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.
Special Reagents Needed
Protocol
- 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.
- 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.
- 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.
- Include where to find less-common materials, such as the nanodrop or specialized equipment. Never mention very common materials such as pipet tips.
- Include a “clean-up” section that lists all the steps necessary to properly clean up the lab after the experiment is finished
- 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