Freezing botanical specimens

On 8 Mar 2005, at 19:39, Mary-Lou Florian wrote:

>The following are important points:
>
>    1.  The moisture content of the materials when exposed to
>        reduced temperature is important. If it is high enough to
>        support condensed moisture--above the fiber saturation
>        point--ice crystals will form in the materials and cause ice
>        damage. If the moisture content is near the fiber saturation
>        point the small amount of moisture it adsorbs during reduced
>        temperature could reach the saturation point and ice
>        crystals form. ...

I think we are justifying the same point from different angles, but
I will elaborate on what I said.

The moisture content of the material will not be important providing
that the specimens are being stored in the appropriate recommended
ambient conditions set out in the US ansi standard for paper and in
the UK as BS5454.

These standards set out an exacting environmental RH to ensure that
the specific moisture content of the material will always be below
the fibre saturation point so long as the material remains within
the RH fluctuation limits set out in the same standards. Therefore,
providing that the environmental conditions are kept at, or below
the limits set out in the standards prior to the freezing process,
the specific moisture content of the item will remain below the
fibre saturation limit during the process and consequently will not
be an important factor.

The most serious threat to to stability in this process is the
atmospheric moisture content because as the temperature decreases,
the RH will rise. This can be absorbed into the material and push
the specific moisture content over the fibre saturation level.
Consequently, the surrounding environmental conditions are the most
important factor to consider during the freezing process, not the
moisture content of the specimen. This is why we both acclimatise
specimens in a lower RH atmosphere prior to freezing (both around
50%), thus reducing the possibility of reaching the saturation level
and concomitant crystallisation.

>    2.  If the bag is sealed and contains ambient room temp and RH,
>        and excess air removed there is very little moisture present
>        and during temperature reduction the amount available to the
>        materials for adsorption is small compared to their(plant
>        specimens) adsorptive ability.

I would like to add to this that we do not remove so much air that
there is a negitive pressure within the bag as we have found that
this contributes to the deterioration of the specimen which becomes
increasingly desiccated, the longer the specimen is kept frozen.

The first noticeable symptom is brittle fracture of the more fragile
areas of the specimens, followed by brittle fracture of more sturdy
leaf material as it progresses and finally a breakdown in the paper
size of the herbarium sheet.

Most of the negitive pressure freezing tests I have performed,
resulted in significant proportions of the specimen breaking away,
and had they not been expendable specimens, the most serious would
have required re-mounting.

As a consequence, we ensure that there is as little air in the
bagging as possible, whilst also ensuring that the bag itself is not
acting as a reduced pressure container.

>    3.  Stress has to be clarified--during temperature reduction and
>        increase in moisture content--material stress is reduced.
>        Dry materials become more flexible.

I can not agree with this. Dry materials may appear to be more
flexible in comparison to a similar material containing moisture
under the same conditions, however this is merely a perception. In
reality, they are not, and they are certainly not in relation to the
same material at ambient conditions, all materials increase rigidity
as their temperature reduces, this is not dependant on moisture
content, it is the second law of thermodynamics. To be flexible,
there must be available energy in the system, and as entropy
increases, available energy decreases. Consequently gases become
liquids, liquids become solids and the lower the temperature (the
higher the entropy) the harder (less flexible) the solid becomes.
All matter is subject to this law, even anhydrous matter, such as
dried paper and plants.

As evidence, these are fold-endurance score averages over 10 tests
for our 1998 batch of Kew Herbarium Sheet (Specific Moisture Content
percentages are w/w):

     5% at 21 deg. C =  90
    30% at 21 deg. C = 180
    60% at 21 deg. C = 230

     5% at  0 deg. C  = 47
    30% at  0 deg. C  = 40
    60% at  0 deg. C  =  3

Although the paper with the lower percentage SMC lasted longer at
lower temperatures than the others, the score was still
significantly lower than its score at ambient conditions because it
was becoming less flexible with the reduction in temperature.

>    4.  The purpose of subjecting the specimens to a reduced
>        temperature is to kill insect structures. The moisture in
>        their protoplasm must freeze- ice crystals must form.
>        Freezing the protoplasm causes condensation effect by
>        withdrawal of water causing damage to proteins and DNA and
>        an increase in pH, enzymes, etc. These along with the
>        physical ice damage causing membrane leakage are the cause
>        of death of insect structures- eggs, larvae, pupae  and
>        adults. The initial blast freezing may prevent ice formation
>        and protect the insect but it is likely that during increase
>        in temperature ice crystals will form which will kill the
>        insect. Has any research been done on the effect of the
>        blast freezer on the insects? The blast freezer may be good
>        but the simple chest freezer is also suitable for many
>        materials.

This also is not exactly correct, but is generally what is taught on
conservation courses. There are many insect species that can
withstand this kind of treatment. A wide variety of sub-arctic
insects have evolved to cope with extended  periods of freezing (six
months+), then thaw out and continue to live. You will also find
that there are many Saharan insects which can withstand total
dehydration for similar periods, rehydrate and continue unaffected.
More importantly, most of the insect eggs we are likely to encounter
in herbaria are equipped with their own natural anti-freeze and do
not get killed off by freezing, except through being exposed to
desiccating wind-chill and sometimes, not even then.

Crystallization of water or total dehydration has very little effect
on enzyme structure or protein structure and has absolutely no
effect on DNA. Don't forget that viable DNA has been removed from
mammoths that have been frozen in the Siberian permafrost for 4
million years! What is our paltry seven days compared to that? The
pH variation is marginal and not enough to cause serious damage to
most insects, at most it is simply a mild kind of anoxia to them.

The key to killing insects is not the freezing but the temperature
of the freezing. All eradication freezing process must go below -30
deg. C.

-30 deg. C is an interesting temperature, it is the temperature at
which a process known as 'The Water Event' happens. Essentially
water is a polar molecule and at -30 deg. C the energy vibration of
the molecules reduces enough to allow inter molecular attractions to
take hold and the molecules polarise. It is this event that actually
causes the degeneration of enzyme structures, non-cellulose based
cell walls and protein structures, all of which are largely reliant
on ionic bonds for their structure and function and therefore can
not survive this catastrophic event. On the other hand DNA can
withstand this because of its bonds are covalent. It is not the
method by which this temperature is achieved that matters, but that
the temperature is achieved, and achieved throughout the whole
material.

As a consequence of this, the freezing method is not relevant to the
killing process, merely an expedient to ensure that all the material
is reduced in temperature in the most consistent way. I have found
that a regular chest freezer is perfectly adequate to reduce single
items and small collections in a speedy manner (5 to 30 minutes).
Larger collections, such as whole boxes, require a more aggressive
method to ensure that the centre of the mass reduces in temperature
with as little variation as possible to the speed of the exterior's
reduction. This is purely because large variations across the mass
will result in stress damage.

The centre of one of our green boxes (approximately 100-150
specimens) reduced past freezing with a maximum of 3 deg. C
variation to the exterior over a half hour period using the blast
freezer I have access to, whereas a similar box in the chest freezer
showed the centre of the mass still being at ambient temperature
while the exterior was approaching zero after one hour.  In the
blast freezer, the whole mass had acquired the target temperature of
-35 deg. C after a two hour period whereas a similar collection in
the chest freezer showed the centre of the mass acquiring the target
temperature 30 hours after initiating freezing and 28 hours after
the exterior of the mass had acquired the target temperature. The
result of this was serious cockling of the specimen's sheets and
damage to the specimens.

I broke two thermal probes finding that out, and got laughed at for
my 1970s Parka, but I was nicely comfortable going into the freezer
to take the measurements!

Most importantly, it is necessary to quickly and evenly reduce your
material's temperature through certain temperature zones which may,
if passed through more slowly have a deleterious effect on the
specimen:

              4 deg. C = maximum expansion point of water
              0 deg. C = freezing point of water (point of
                         crystallization)
        0 to 11 deg. C = re-tack zone for some acrylic adhesives
     -12 to -30 deg. C = no discernible deleterious effects noted
                         (so far)
            -30 deg. C = the water event

Jonathan S Farley MA ACR MIPC
Senior Conservator
Royal Botanic Gardens
Kew
+44 208 332 5419
Fax: +44 208 332 5430


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                  Conservation DistList Instance 18:43
                  Distributed: Monday, March 14, 2005
                       Message Id: cdl-18-43-002
                                  ***