Telling the difference between a live and dead cell in a sealed tube

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andrewkirk

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Summary
For a fiction story. If a frozen ovum were taken out of its cold storage and left at room temp for a few days, would it die? If so, would we be able to tell whether it was dead just by looking at it through a microscope. Presume the ovum is still in whatever tube (possibly sealed) it was held in the liquid nitrogen bath in.
A friend is writing a story in which somebody steals a frozen, non-fertilised ovum from an egg bank, meaning to do something with it (not sure what). The thief then forgets about it and finds the tube containing the ovum in her pocket a few days later. She looks at it under a microscope. The intended plot requires that she realise upon looking through the microscope that the ovum has died.

I was hoping somebody could tell me if an ovum would die if kept at room temperature in its test tube for a few days. And if so, could one tell that had happened by looking at it through a microscope? I imagine it may depend on the sort of container the ovum is held in and whether it is sealed. I am imagining a small sealed phial containing the ovum, immersed in a bath of liquid hydrogen in a rack along with many other phials. ANy info to correct or add detail to that imagination would be gratefully received!

I am physics guy, rather than biology, but I promised I'd do what I could to help find with the scientific verisimilitude of the story.

thank you
 

jim mcnamara

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AFAIK: Tests for thawed cell viability require samples that contain multiple cells. You sacrifice some cells for testing.

This is not my field, but freezing thawing bovine or equine sperm is a big part of animal husbandry.

What you check is the integrity of the cell membrane, i.e., did the cells burst during freezing - or not.
Since this is fiction maybe that is enough.

Read the abstract and check out the bibliography:

For the case where you have multiple cells, not just a single (this is a bovine sperm example ):
https://www.sciencedirect.com/science/article/abs/pii/0093691X94902801 Hypoosmotic swelling test
 

BillTre

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Generally speaking, the thawed egg cell (or cells) would be unlikely to survive for a few days in a tube at body temperature (inside someone's pocket of clothes they are wearing. Normally the cell would be in a tissue culturing setting where they would have well controlled temperature, O2 and CO2 gas exchange, and media changes to keep their nutrients at high concentration and their wastes at low concentrations.
If there is only one a single cell in a large tube, these limitations may not be so limiting since a single cell will not have enough metabolic activity to use up all the nutrients and O2 or produce enough wastes to cause itself problems in a few days.

However, successful freezing of cells is done with preservatives in the freezing media. They keep more of the cells intact in the face of the freezing conditions, but they are somewhat toxic to the cells and have to be removed after thawing. This is usually done by thawing the cells rapidy, dumping them in a tube with a larger volume of media (to dilute the preservatives), spinning the cells out of solution in a centrifge, pouring off the supernatant (fluid), and resuspending the cells in new media with no preservatives in them. In this condition a single cell might survive for a few days in a relatively large volume of clean media.

There are many ways to determine if cells are live or dead. A popular method now is use specialized live-dead stains. These are fluorescent dye sets that will differentially label (fluorescently) live or dead cells so that they can be identified with a fluorescent microscope (or by other techniques).

@jim mcnamara is correct in saying that you should expect cells to die during their thawing. Successful freezing and thawing of cells is generally done following very specific procedures to maximize the successful recovery of viable cells. Vials of frozen cells often contain many cells. Thawing kills a percentage, but if you start with hundreds, thousands or millions of cells, it doesn't really matter that much.
 

DaveC426913

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Whether or not it is perfectly accurate in the timeline, it is certainly plausible that the ovum could lose its cellular composure and begin to break down. This would be a fairly obvious visual indication of death.

It certainly fits the needs of the plot. And it is just short of details enough that readers won't be able to call foul.
 

DaveC426913

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AFAIK: Tests for thawed cell viability require samples that contain multiple cells. You sacrifice some cells for testing.
Heh. It's a quantum mechanics thing: you can't observe the subject without altering the subject irreversibly.

So you can't have a cell that is both alive and has been tested.

You test other cells (which are altered and killed) and then infer the condition of the untested ones.
 

rbelli1

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liquid hydrogen
Liquid nitrogen is generally used. Liquid hydrogen would be rather dangerous to have around boiling and getting explody.

BoB
 

DaveC426913

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Liquid nitrogen is generally used. Liquid hydrogen would be rather dangerous to have around boiling and getting explody.
Haha that's probably what andrew meant.
 

andrewkirk

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Thank you all for your helpful answers. I passed them on and they enabled my friend to complete her story. I'll post the news here if it becomes a hit!
 

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