Why do we need to make fixative "fresh"?

[julie]

Having been trained in blood harvests I was instructed to always make
the fixative fresh and usually just before use as this was very
important. I was given no reason why this was important and could only
assume that some change occurs in the fixative over time.I am new to
cytogenetics and my knowledge is relatively poor. Does anyone know why
fresh fixative is best for harvesting?

[Random lawce]

The word I have is that the mixture forms acetates that make spreading
more difficult. Perhaps that is why we usually replace the fixative
in a stored tube before making more slides from it, and why really old
pellets get that cytoplasmic background. Also, both methanol and
acetic acid will absorb water from the air. In our lab, unless there
is a problem with slidemaking, we feel that the fixative lasts for a
few hours up to an 8-hr. work day before it needs to be made again.
Oregon is not that humid most of the time (50% relative humidity is
considered high!) so the fixative may stay "dry" longer here than it
does in say, Florida. Helen

[vantuinen]

Remember High school chemistry when you added an acid to an alcohol?
You get an ester - in this case methyl acetate. It is unclear how
fast this occurs and what the equilibrium is between the starting
ingredients and the ester. We make fresh daily and save leftovers in
a bottle for destaining Giemsa from slides, Coplin jars, etc.

[Fatma SILAN]

Alcohol evaporate easily, so if fixative waits it become less alcohol and more acetic acid relatively.... Fresh is the best. If you have to prepare early, you have to close the cap and you can put it in the fridge..... --- On Tue, 6/15/10, julie <julie...@gmail.com> wrote:

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[trypanblue]

julie it all comes down to the fact that the fixative will change over
time.
At first the fixative becomes more acidic and eventually is
contaminated with acetates which are by-products of a methanol &
acetic acid reaction. Light and temperature speedup the reaction, so
keep the fixative in a brown smoky glass bottle away from light and
keep the mixture cool. At the sametime the mixture absorbs water from
the atmosphere which further hampers the fixative in doing its job so
always keep the bottles well topped up with little air space. my
advice to you is make the fixative as soon as you require it. Only use
the highest quality(purity) methanol & acetic acid...dont give in to
the temptation to by cheaper quality stuff.

[Mary Emanuel]

Fresh fix: because you do not want moisture i.e. water in the harvesting process and you are using anhydrous methanol (next to no water) and glacial acetic acid.  Water will ruin any harvest every time.  A great source is the Association of Genetics Technology Manual, now in the 3rd edition. 

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