Parchment preparation
The skins from four lambs (Ovis aries) were taken from the Ovine Center of the University of Namur (natural deaths); the animals were cared for according to procedures conforming to the European requirements on farm animals (EC directive 86/609). The skins - two from stillborn animals and two from animals less than 2 weeks old (approximately a rectangular shape and size of 16 cm by 20 cm or lower) were manufactured into parchment (Figure 2). Recipes for parchment production exist from the 8thC [14], the first well documented example being John Beale in “The Art of making Parchment, Vellum, Glue etc” read to the Royal Society in 1664 [24]. Here, we used the later description of Diderot & D’Alembert [16], see Box 1.
Preparation
Fresh skins were washed in water then left in a light lime solution (3g/L) for five days.
Dehairing
Two dehairing processes were used (each treatment was applied on skins from one born dead animal and from one animal less than 2 weeks old), both at ambient temperature, either:
- the flesh side of a skin was uniformly covered with a milky lime solution and left for one week folded in two, flesh side together (pancake method), or
- the full skin was put into a lime bath and left for one week (bath method).
Skins were dehaired using successively stronger lime Ca(OH)2 solutions (5 g Ca(OH)2 /l, 15 g/l and 30 g/l), equivalent to ‘plein mort’, ‘plein gris’ and ‘plein vif’ for 1 week each, with a wash in clean water for one day in between each different solution.
Scraping
The hair was removed by rubbing the skin with hands and finished with a semi lunar knife. The skins were then put on a framed, scraped, chalked and pounced, to produce the final parchment.
Box 1
Aussitôt que les peaux ont été pelées, on les lave à la rivière afin de les nettoyer, on les laisse ensuite égoutter quelque temps; après quoi on les met dans un mort-plein, c'est-à-dire dans un plein qui a servi et dont la chaux a presque perdu toute sa force;
on les laisse dans ce mort-plein environ vingt-quatre heures;
d'où on les retire ensuite pour les mettre égoutter sur le plein....
Deux jours après que les peaux sont sorties du mort-plein, on les plonge dans un autre plein dont la chaux est moins usée, on les y laisse environ deux ou trois jours, après lesquels on les retire pour les mettre en retraite égoutter comme auparavant…
s'il en est besoin, on les replonge ensuite dans le plein,
on réitère cette opération pendant six semaines ou deux mois seulement, pendant les chaleurs de l'été ; mais en hiver il faut les faire passer successivement de plein en plein au-moins pendant trois mois.
Diderot & D’Alembert (1765)
Once the skins are peeled, they are washed in the river, then allowed to drain for some time; next they are put into ‘dead-lime’ that is to say, lime which has lost almost all its strength.
Skins are left in baths of dead-lime for about twenty-four hours;
They are removed and allowed to fully drain again.
Two days after the skins are removed from the dead-lime, they are immersed in another bath with less aged lime and left there for about two or three days with agitation, after which they are removed put to drain…
If necessary, they are then plunged back in the lime bath again, this operation is repeated for between six weeks to two months during the heat of summer, but in winter in the open it is necessary for the skins to soak for at-least three months.
Loose translation, Matthew Collins
Two subsamples of skins were subsampled at each step of the manufacturing processes described by Diderot & D’Alembert [16] and placed immediately in formaldehyde for subsequent histological analyses: one for the paraffin embedding and the other one for the frozen section to enable alternative histochemical staining to be used. Samples of approximately one square centimeter were taken in periphery of the skin, after specific steps of the manufacturing process (Figure 2): fresh skin (sample #1), skin after hair removal (sample #2), skin after the 3rd lime bath (sample #3), parchment (sample #4).
Method: Histological procedures
The sheepskin and parchment samples were fixed in acetified formalin (formol 4%, acetic acid 1%). Six-micron thick sections of the paraffin and of the frozen blocks were placed on SuperFrost + glass slides.
Sections from the paraffin blocks were dewaxed, rehydrated in graded alcohols and stained using the five following methods (the two first being generalized topographic stains, while the three others are specific for specific macromolecules):
1) Hemalun, Erythrosin and Saffron stain (HES) stains nucleic acid blue/purple, cytoplasmic proteins red and collagen yellow-orange;
2) Green trichrome stains nucleic acid in blue/purple, cytoplasmic proteins red and collagen green;
3) Orcein and hematoxylin stains elastin fibers red brown and nuclei blue/purple;
4) Picro-Sirius Red and hematoxylin stains collagen I fibers and nuclei blue/purple;
5) Wilder and green light stains reticulin fibers, mainly collagen III fiber, black with a green background.
Sections from the frozen blocks were stained with Oil Red O (ORO) and hematoxylin which stains lipids red and nuclei blue.