Nov 24, 2020
Toxicity Assay
This protocol is a draft, published without a DOI.
- 1In-house protocol
Protocol Citation: Elizabeth Fozo 2020. Toxicity Assay. protocols.io https://protocols.io/view/toxicity-assay-bpwhmpb6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 20, 2020
Last Modified: November 24, 2020
Protocol Integer ID: 44713
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Abstract
Toxicity Assay (based on growth curves protocol)
Rules of thumb
Rules of thumb
Never remove > 1/3 of a culture for a growth curve
Read OD600 (optical density at 600 nm) of 200 microliters of culture added to 800 microliters of fresh media -> 1,000 microliters of fluid in cuvette; = 1:5 dilution
Note
Remember to set the factor equal to 5 when reading from the spectrometer
Easier to measure 800 microliters of fresh media into cuvettes ahead of time; simply store in cuvette box
Easier to measure media into a 50 mL conical for filling fresh cuvettes; less likely to get a contaminated bottle
Easier to use same blank multiple times; suggest every time you refill conical with media for cuvettes, make a new blank with that and use with cuvettes from that mixture.
Place cuvettes into 10% bleach solution when finished.
*This is a must for BSL 2 organisms such as E. faecalis and EHEC.
Making an overnight culture
Making an overnight culture
Measure 5mL appropriate media with appropriate antibiotics into fresh conical (make sure to label conical with date, strain!)
Take 1 colony from a plate with appropriate strain (use pipette to poke that colony)
Inoculate media
Grow overnight (usually start culture ~3-5 p.m. night before culture is needed) In the morning:
In the morning
In the morning
Measure OD600
Use V1C1=V2C2 to make a 0.01 OD600 dilution in media for growth curve
Place culture in flasks, mix, place in the appropriate incubator, start count up timer.
Toxicity Assay:
Toxicity Assay:
The first time point is at 30 min on the count-up timer.
Take time point every 30 min, write down data and graph as you go
Use count up timer to measure time. When it reaches 30 min, 1 hour, 1.5 hours, etc. remove cultures from the incubator, pipette into cuvettes (remember 1/5 dilution, e.g. 200 microliters culture in 800 microliters media)
Remember to mix culture gently before adding to a cuvette (the culture can be clumpy at the bottom, and you want a homogenous solution)
Once the culture reaches OD 0.3 (at about 2-3 hours for E. coli in LB), split cell culture in half.
Note
Try to reach as close to OD 0.3 as possible. May require you to take samples off the 30-minute cycle.
Induce ½ cells with the appropriate inducer (for example, arabinose) at the appropriate final concentration (for ara, 0.2% is saturating).
Continue incubation for all cultures, taking OD600 every 30 minutes after induction.