Feb 16, 2022
Neurospora culture and basic imaging
This protocol is a draft, published without a DOI.
- gamclaug 1
- 1University of North Carolina at Chapel Hill
- GladfelterLab
Protocol Citation: gamclaug 2022. Neurospora culture and basic imaging. protocols.io https://protocols.io/view/neurospora-culture-and-basic-imaging-b47cqziw
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: February 16, 2022
Last Modified: February 16, 2022
Protocol Integer ID: 58308
Abstract
Details about culturing Neurospora, imaging, making freezer stocks, and media recipe
Guidelines
Neurospora spores can travel through the air, so work with the spores/cells in a fume hood to avoid contamination. Only open slants and plates in the fume hood.
http://www.fgsc.net/Neurospora/neurospora.htmlis a good neurospora resource
Materials
Vogel’s Minimal Media recipe:
Slant tubes/plates are made by combining Vogel’s media with 1.5% agar.
Intro
Intro
Neurospora spores can travel through the air, so work with the spores/cells in a fume hood to avoid contamination. Only open slants and plates in the fume hood.
http://www.fgsc.net/Neurospora/neurospora.htmlis a good neurospora resource
Cell Culture
Cell Culture
Neurospora spores are generally mailed between labs on small pieces of filter paper. To start a new slant tube from them, push the filter paper into the agar at the bottom of the slant tube. Put on the lid, and leave the tube at room temp. There will be plenty of mycelia in 1-2 days.
To start a new plate, simply insert a sterile applicator stick into the slant tube and then touch that to the center of a plate. The plate can then be left to grow at room temp. This slant tube can be used many times for this for about 2 weeks.
After 2 weeks, transfer the cells from the original slant tube to a new slant tube (with the same method as above- except stick the applicator stick into the new slant instead of the plate).
Imaging
Imaging
Grow the cells on the plate at room temp until they cover about 2/3 of it. This usually takes about 12h.
Cut out 2cm x 1cm pieces of agar around the edge of the colony (if your intention is to image the apical regions):
Put 20uL of Vogel’s Minimal Media in a 15u-Slide 4 well Ibidi chamber (80426), and then put the agar pieces into the wells with cells facing down:
For imaging high SNR things like nuclei using agar plates is fine. Agarose plates might be better for dimmer signals.
It can also work to put the agar pieces onto a coverslip, but this is not as stable as the ibidi chambers. It works okay to do this on Ann, but does not work very well on Bronty when taking z-stacks.
Making freezer stocks
Making freezer stocks
Start with a slant tube that has mycelia growing in it. Pour 2mL of 25% glycerol into the tube, put the cap on, and vortex. Pipette this into cryto tubes and store at -80.
To start a new slant from the freezer stock, use a sterile applicator stick to scrape out a small amount of the freezer stock and put in the middle of the plate. Do not let it thaw, just scrape off the top.