[Histonet] Re: Dioxane questions (Long,with extras)


Dear Maria Mejia,

Dioxane is a liquid with a 60-year
histotechnical history. Your boss's potential
application for attaching cells to slides
is not part of the story, and may be misguided.
Read on.

Dioxane is a cyclic diether that boils at 101C.
It is inflammable (slightly less so than ethanol)
and yes, it can form explosive peroxides on
prolonged exposure to air. If you buy a small bottle
and don't store it when almost empty, peroxide
formation should not be a risk. You can test for
peroxides by adding 1 ml of freshly made 10% aqueous
potassium iodide to about 10 ml of dioxane, in a small,
stoppered container. Wait one hour. Formation of
iodine (yellow-brown) indicates the presence of
organic peroxides.

The danger of explosion exists if the solvent is
evaporated to dryness, because the organic peroxides
do not evaporate and therefore  become concentrated.
With small volumes of "young" dioxane that are not
being boiled to recover solutes, peroxides do not
present a risk.

Disposal of dioxane is the same as for other
non-chlorinated flammable liquids - collection in
drums for taking away and burning. If you detect
peroxides in a bottle of dioxane, add some ferrous
sulphate crystals (reduces peroxides to non-explosive
substances) before pouring the dioxane into the
waste solvents container.

Having said all this, I must question your
modus operandi in connection with dioxane.

Dioxane will not "coat" glass (except perhaps with
organic peroxides if you use old stock). As a solvent
it will remove any grease that's present and it will
then simply evaporate.

If your "conA" is concanavalin A (the lectin from
jack beans), that won't really attach cells to glass
either; the lectin sticks to the surfaces of many
types of cells and microorganisms by virtue of its
affinity for alpha-D-mannosyl and alpha-D-glucosyl
units in cell surface glycoproteins. This lectin,
like many others, is bivalent, so it can make cells
or bacteria clump together.

Lectins are also called phytohaemagglutinins - proteins
from plants that agglutinate blood cells. Some are
specific for particular human blood types, notably
Groups B and O[H]. Concanavalin A is a nonspecific
phytohaemagglutinin; it can bind to nearly all cell
surfaces and also to extracellular carbohydrates
(eg collagen), glycogen, and some types of mucus.
Fluorescently tagged concanavalin A was one of the
first lectins to be used as a reagent in carbohydrate
histochemistry, in the early 1970s.

A digression follows. Back to dioxane.

The chief use of dioxane in histotechnology has been
as a solvent that mixes well with both water and
paraffin wax. I have used it occasionally. It is toxic
(drowsiness, skin irritation, renal failure, possible
carcinogen etc, according to the Merck Index, which
gives references). Its rather weak (quite pleasant)
smell could easily go unnoticed, so this is not a liquid
to use on a large scale in paraffin processing.
Tetrahydrofuran can also be used as a single
dehydrating+clearing solvent, but it has disadvantages
similar to those of dioxane and a lower boiling point
(66C), so it's more of a fire hazard than dioxane.
Tertiary butyl alcohol (tert-butanol; 2-methyl-2-propanol)
is an altogether safer single solvent for transition
from water to wax. It's often used in processing for
plant histology. The only practical drawback with
t-butanol is that the 100% liquid freezes at 26C.
This is not one for general use in processing machines!

John Kiernan
Dept of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada
Quoting Maria Mejia :

> Hello Everyone, in the next few weeks, we plan to use very small amounts
> of (dioxane)
> to coat coverslip - 10ml -20ml at one time - 2-4 times /month.
> In previous experiments, the PI has tried other chemical substitutes -
> without success.
> Without the use of dioxane, a protein called conA that attaches cells to
> the glass will not
> bond properly with the coverslips and instead will wash away during
> testing.
> This question is for anyone (past or current) using dioxane, please
> advise, on the following
> procedure:
> I understand in order to prevent peroxidizable compounds in the dioxane, it
> is necessary to store the liquid material - inerting the atmosphere in
> storage containers
> with nitrogen to inhibit peroxide formation.
>  >When using dioxane, is there another alternative to storing in
> nitrogen or argon
>    atmosphere?
> For example,  adding an inert high boiling substance like mineral oil to
> prevent the
> peroxide from concentrating to a dangerous level? Has anyone tried this?
>  >What type of containers (metal or plastic) should be used for
> cryogenic process?
>  >What type of container should be used in disposal of dioxane?
> Polyethylene
>    plastic?
>  > Can I assume that -20C is a good temperature for storage?
>  > How long should I keep working dioxane? I read 3-12 months.
>  > How long should I keep waste dioxane waiting for disposal pick-up?
>  > Disposal of peroxides - should I dilute with water - what
> concentration of H20?
>  > How can I detect & determine peroxides? I notice in the Laboratory
> Safety that they
>    sell peroxide check paper strips. Does anyone know if these strips
> are reliable and
>    accurate?
>  > How often should we have the fume hood where dioxane will be
> use...checked?
>    Every six months? Once a year (currently doing)?
> Nitrile (heavy weight - 8mil-12 mil) and butyl gloves will be used.
> I believe that's it for now - please let me know if I have forgotten
> anything else! Any
> help or information (resources, references, companies etc) anyone can
> provide will be
> greatly appreciate.
> regards
> Maria Mejia
> neurohistologist
> Smith-Kettlewell Eye Res. Inst.
> San Francisco, CA 94115
> Email: maria@ski.org

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