Feb 05, 2022
Soft agar for yeast imaging
This protocol is a draft, published without a DOI.
- leonhard.bandilla 1
- 1(independent)
- LBandilla
Protocol Citation: leonhard.bandilla 2022. Soft agar for yeast imaging . protocols.io https://protocols.io/view/soft-agar-for-yeast-imaging-b4q8qvzw
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: In development
We are still developing and optimizing this protocol
Created: February 05, 2022
Last Modified: February 05, 2022
Protocol Integer ID: 57856
Keywords: soft agar for yeast, soft agar, yeast without immobilization, yeast, inverted microscope, soft gel, imaging, agar, described medium, medium constists of ypd, final medium, 1l of final medium, medium constist
Disclaimer
This is an unverified and experimental protocol. Use this protocol at your own risk, use this for informational purposes only.
Abstract
The here described medium can be used to image yeast without immobilization or compression while allowing for growth in all directions. The medium constists of YPD with agar added at a very low concentration to form a soft gel that allows for free growth in all directions. The media can be poured into any multiwell plate or dish to be imaged with an inverted microscope. This protocol is written for 1l of final medium but smaller quanities can be used.
Guidelines
When transfering the warm media to the final container, allow for enough height to image and for innoculation.
The final product is fragile and should not be tilted, shaken or inverted.
Troubleshooting
Safety warnings
Wear standard PPE for the biosafety level of the laboratory where this protocol is performed.
The contents of the flask can remain dangerously hot for a long time after autoclaving. Always follow the instructions of your autoclave, tightening bottles can be dangerous due to the pressure differences involved.
When autoclaving or heating, only fill bottles to half their volume to avoid foaming, splashing and spills.
Read the SDS of all materials involved before following the protocol.
To an autoclavable bottle add per 1l:
- 10 g Yeast extract
- 20 g Peptone
- 1 g Agar
5m
Add 950 mL of water of appropriate quality per 1l of final media
1m
Invert the bottle to dissolve everything apart from the agar
2m
Autoclave or heat up sufficiently to dissolve the agar and insure sterility
20m
Per 1l add 50 mL of sterile 40% glucose and mix, avoid introducing air bubbles
1m
Let the solution cool down so that it can be safely handled and pour or pipette into the final container in which the imaging will take place. Avoid air bubbles as they can interfere with imaging. Make sure to avoid contamination by working near a bunsen burner or in a laminar flow hood
10m
Wait for the agar solidify over night before innoculating the media. This media takes significantly longer to set, allow for multiple hours before any change in consistancy is noticible, be aware that the final product is very soft and can easily be mistaken for liquid media
12h