Tag Archives: death

Extent of death post-holiday – Cut Glass and Petri Dishes

Following more detailed review of cell flasks, there are some hardy ‘survivors’ of my lab-free holiday.

Cut Glass CellLight microscope image of cut glass dish with media containing dead cell debris and evidence of a small number of surviving cells. 

After removing the old media, it was easier to see the remaining cells:

Cell Survivors - Cut Glass

Light microscope image of cut glass dish in PBS showing evidence of a small number of surviving cells. 

This image shows even more evidence of cell survival:

Cell Survivors - Cut GlassLight microscope image of cut glass dish in PBS showing further evidence of a small number of surviving cells. 

 Cell Survivors - Cut GlassLight microscope image of cut glass dish in PBS with likely cells circled. There are a few additional potential cells visible, but I have only circled the most obvious. 

To get an even better sense of survivors, I will fix (in 4% PFA) and H&E stain 2/3 of the cut glass dishes. The flatter cut glass dish and Petri dish (with more potential for cell survival will be maintained in the incubator to see how they fare over the next week).

Petri Dish Light microscope image of Petri dish with fresh complete media with live cells. 

I will also fix and stain the glass vessels. It is less likely that these will yield anything interesting, but it will help me troubleshoot how to do the protocol with the tiny openings – it is very difficult to effectively remove the media – even with a 20ul pipette and tip 🙁

 

 

Post-holiday update

I have returned from the festive season break and started back in the lab.

Let’s start with the good news! There is no visible infection in any of the vessels including cut glass dishes and vials. My flasks are doing OK and there are cells actively growing (despite evidence of cell death indicated by cell debris).

Now for the bad news…

There has been mass death. Despite the slow growth rates of my cells, 3 weeks is just too long to leave cells starving and without ongoing maintenance. That said, there is evidence (in cell debris) that a good number of cells did grow in the vessels during my absence. This shows that the overall plan should work.

The plan for today is:

  1. Make up new media and FBS aliquots.
  2. Feed cells (i.e. replace media with fresh solution)
  3. Remove dead cells from all cut glass dishes.
  4. Collect dead cells via centrifugation and fix in PFA.
  5. Fix cells in some of the older flasks, fix in PFA and stain with H&E.

If I have time, I will also:

  1. Bleach and wash glassware and prepare for autoclaving.
  2. Autoclave glassware.