Feb 02, 2016
Base agar plates
- Mathias Middelboe1,
- Amy M. Chan1,
- and Sif K. Bertelsen1
- 1Manual of Aquatic Viral Ecology
- VERVE Net
- Suttle Laboratory of Marine Molecular Microbiology and Virology
Protocol Citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. Base agar plates. protocols.io https://dx.doi.org/10.17504/protocols.io.dp75rm
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
Created: September 01, 2015
Last Modified: February 27, 2018
Protocol Integer ID: 1503
Keywords: isolation of cyanophage, cyanophage, plaque assay, base agar plates for use, base agar plate, agar plate, plaque
Abstract
For use in "Isolation of cyanophages by plaque assays"
Guidelines
For example, purified agar or agarose (1% w/v) is added to your media of choice and autoclaved. This will provide a support base for the top agar/agarose overlay as well as nutrients for the host cells. For best results, use plates within 1 week of pouring.
Tip: plates can be fast-tracked: dry plates at 37°C; leave lids slightly ajar; monitor closely to prevent over drying.
Considerations: Depending on the composition of the media used, the addition of solidification agents (in particular the combination of high salinity seawater-based media and common agar such as Bacto Agar) can often result in the formation of precipitates when autoclaved together. These flocks can sometimes interfere with interpretation of the plaque assay. Moreover, impurities in common agar can negatively affect the growth of the host cells. Here are some suggestions on how to reduce the formation of these precipitates. Some testing may be required to determine the best combination to use for your particular situation.
a. Do not use common agar; rule of thumb the whiter the agar, the cleaner it is.
b. Use commercially available purified agar or agarose; or clean common agar using a washing procedure such as the one outlined in Waterbury and Willey (1989).
c. Reduce the salinity of seawater media with purified water; e.g., to 20-25 psu.
d. Add purified agar or agarose to autoclaved media aseptically and then melt the agar/agarose in the microwave (bring to a short boil 2-3 times to completely dissolve the agar/agarose).
e. For cells that will grow in artificial media, prepare media and gelling agent at 2x concentration and autoclave separately. When cooled to ca. 60°C, gently mix the gelling agent into the media and dispense immediately.
f. In the case of artificial media, add agar/agarose to filter-sterilized media and melt the gelling agent in the microwave
Troubleshooting
Safety warnings
If the surface of the bottom agar is too moist, the top agar/agarose will not stick to the bottom plate and will slide off when the plate is inverted.
Prepare base plates
Add 5 g purified agar or agarose to 500 mL culture media in a 1-L Erlenmeyer or media bottle.
Gently stir to disperse the agar/agarose.
Autoclave for 20 to 25 min to sterilize.
00:25:00
When cooled to about 60°C, dispense 15 to 20 mL per plate.
Note
the suggested volume is suitable for 15 x 100 mm diameter petri plates; adjust accordingly for smaller or larger plates
To reduce condensation forming on the insides of the lids, leave lids slightly ajar to allow escape of steam or stack the plates immediately after pouring.
Invert plates once the agar has solidified to prevent condensation from dripping onto the surface of the agar.
Plates can be used about 12 h after pouring if the agar surface is not wet; a longer time is needed if conditions are humid.
12:00:00
Note
If the surface of the bottom agar is too moist, the top agar/agarose will not stick to the bottom plate and will slide off when the plate is inverted.