The Canada balsam we're using consistently starts "crystallizing" after a few months, completely obscuring the smaller detailed of the specimens on the slides. The specimens have been cleared either by KOH followed by acetic acid, or through DNA extraction. In both cases, the specimens (insect exoskeletons) are then placed in 95% ethanol, absolute ethanol, and then oil of cloves before being placed in the balsam.
Without fail, 3–5 moths after mounting, something in the balsam starts congealing and crystallizing, often looking like small water drops on the underside of the cover glass, and then just a jumble of small crystal-like things take over the balsam until the specimen is entirely obscured. We've tried around half a dozen different brands (Chinese or Indian, can't get hold of other brands here as far as we can tell), and it makes no difference. This happens uniformly throughout the preparation, and happens even if we have no specimens on the slide, so it seems to be independent of the specimen preparation method.
Attached is a photo of what it looks like.
Anyone have any suggestions for what we're doing wrong? And what we can do to overcome this problem?
Do you know what/if any solvent is mixed with the balsam? The only time I think anything like this happened to me was when I did not leave the specimens in the last dehydration step (in my case xylen) long enough. But I'm wondering if it is a reaction of some kind between the solvent in the basam and the oil of clove or residues of ethanol? Otherwise I have no idea.
Only xylene is mixed with the balsam. So unless there is something we don't know about, there should be only oil of clove, canada balsam, and xylene there.
I use the exact same method as I've used in Sweden and the US, and never had this problem there, but with these Chinese/Indian brands of balsam, it seems to happen all the time. We thought it had to do with the ambient humidity, but putting the slides in a drying cabinet just sped up the process and the slides were ruined in a few weeks instead of a few months.
Ok, then I do not have any more suggestions. Hope someone else have any ideas.
I have replaced formalin-fixed samples with 80%-90% to absolute ethanol, transparented with methyl salicylate (98%) instead of xylene and then sealed with balsam dissolved in methyl salicylate (98%). No such phenomenon has been observed with this method.
I do not have a solution, either, only a few ideas:
Yes, Canada Balsam has xylene as solvent but this evaporates completely with the hardening process (which can take months). So if the are crystals, the most probably explanation is that the product is not of high quality.
In such products, age may play a role, which would explain that previously you did not have such problems with this product.
A German provider (Waldeck) offers "Canada Balsam, reinst = purest", so there seem to be quality differences.
I would just buy from a different source and make a test.
It may also have to do with the clove oil - some constituents do not evaporate, as far as I know, and may interact with balsam consitutents - but I have no experience with clove oil, my specimens go from 100 xylene into the Balsam - so this is only a speculation.
Anyway, best would be to start with a completely new set of "ingredients".
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