safranin and toluidine blue solution prep

[mete]

I found them as dry powder and will prepare solutions for plant sections. I plan to make 0.1% safranin and 0.05% toluidine blue aq. solutions. Is there anything I should be aware of or any other recommendation ?

[ldflan]

For botanical work with safranin, definitely lay hands on Fast Green FCF if you can, and prepare it in 100% alcohol (ETOH or IPA); use it as the counterstain to your aqueous safranin. You should be fine with the safranin at 0.1% for most botanical work. They key to using safranin well is timing how long sections stay in the stain, and then how long you destain them. Safranin sections in my experience destain best in about 70% alcohol. Once destained to the level you want (determined by experiment) take the section up to 100% ETOH, then into Fast Green FCF in ETOH or IPA for a very short time as your counterstain, then rinse in 100% ETOH (or IPA), then coverslip in Permount, Canada balsam, or similar non-aqueous mountant. The Safranin / Fast Green combination is a great technique to master for plant sections. Can be extraordinarily beautiful.

Toluidine Blue-O ("TBO") - A great choice for staining botanical sections on its own, with metachromatic effects from pinkish through purple and blue. I would prepare it at a somewhat higher concentration than .05% (.1%, say), though I do frequently us it at .05% dilution. Sometimes even less (.025%). Experiment with different dilutions and times from a stock solution, and also try preparing it at different pH levels. pH can make a big difference in the metachromatic effect of the dye on the final slide Be sure to filter it. Re-filter it as needed when scum forms on top.

This is important with TBO: If you are using Permount or a similar water-free mountant with TBO-stained botanical sections, once you have dehydrated your stained sections back up through the alcohols, try breathing on the slide to fog it, then let the fog evaporate from the glass, and once the fog is gone immediately drop the mountant on the sample and coverslip it. The metachromatic effect of TBO seems to be highly dependent on just a little bit of water being present in the sample! Your breath is usually enough water to suffice for a thin section.

Also, aqueous basic fuschin can make an interesting combination with TBO, depending on what you are working with.

Leonard

[mete]

Many thanks Leonard.

Is there an alternative to Fast Green FCF ? Would Light Green SF work similarly since I dont plan to make permanent mounts ? I dont know how quick it fades.

[mrsonchus]

Many thanks Leonard.

Is there an alternative to Fast Green FCF ? Would Light Green SF work similarly since I dont plan to make permanent mounts ? I dont know how quick it fades.

There's a very good one - Alcian-Blue. For high-res high-mag detail I go for FG as it's more inclined to stain with delicacy.
For impact at scanning level and sheer beauty and intensity of colour the safranin/a-blue combo is very eyecatching and detailed also.
Alcian blue can be rather too bright and vivid however for more intense observation of finest detail - glaring almost.

Fast green has a subtle ability also to be reduced to blueish tones when used with safranin, and to achive a level of tranlucency that is able to reveal underlying safranin-stained structure well.

[ldflan]

I agree with John's comments above. Alcian Blue 8G will indeed work quite well with Safranin-O. It's also a very good counterstain for Nuclear Fast Red, so a good one to have in your arsenal. But Fast Green is the way to go if you can find it. I haven't tried Light Green in this application, so I can't comment on that. I can't recall anyone mentioning it as a particularly good choice, though...

Another pretty useful botanical / fungal stain (suspected carcinogen, if I am remembering right) is Chlorazol Black. If interested in lipids, and not making permanent mounts anyway, the Sudan Black stains can be interesting to work with.

And, since you aren't making permanent mounts, you might consider making up a batch of Lugol's solution. Diluted about 1:10 with water and you have an effective and very inexpensive temporary stain for differentiating amylose (blue black) and amylopectin (brick red). Observe while wet. Inverted scope recommended for this kind of stuff.

Leonard

[mete]

There's a very good one - Alcian-Blue. For high-res high-mag detail I go for FG as it's more inclined to stain with delicacy.
For impact at scanning level and sheer beauty and intensity of colour the safranin/a-blue combo is very eyecatching and detailed also.
Alcian blue can be rather too bright and vivid however for more intense observation of finest detail - glaring almost.

Fast green has a subtle ability also to be reduced to blueish tones when used with safranin, and to achive a level of tranlucency that is able to reveal underlying safranin-stained structure well.

Sounds good, I will keep an eye on Alcian-Blue, thanks.

[mete]

I haven't tried Light Green in this application, so I can't comment on that. I can't recall anyone mentioning it as a particularly good choice, though…

I always see Safranin together with Fast Green in the documents. I think Light Green is a chemically similar dye to Fast Green, and Fast Green is recommended instead because it does not fade. So I thought it might function same, but the result might not be as good. Light Green seems to me a safer dye, that is why I asked.

Another pretty useful botanical / fungal stain (suspected carcinogen, if I am remembering right) is Chlorazol Black. If interested in lipids, and not making permanent mounts anyway, the Sudan Black stains can be interesting to work with.

SDS of Chlorazol Black looks scary to me

And, since you aren't making permanent mounts, you might consider making up a batch of Lugol's solution. Diluted about 1:10 with water and you have an effective and very inexpensive temporary stain for differentiating amylose (blue black) and amylopectin (brick red). Observe while wet. Inverted scope recommended for this kind of stuff.

Right on time, I added this today to my todo list after seeing it on a book.

May I ask why I do not see much (or not at all) mention of Toluidine Blue in ~pre-60s books ? Is it relatively a new invention ?

[ldflan]

No, I think Toluidine Blue-O has been around a long time - 1850s or so. I looked in my copy of Gray's formulary from 1954, and he lists formulations from at least 1905, and probably before. Didn't do an exhaustive search. Lugol's iodine is one of the oldest stains used in microscopy, and one of the first (if not the first) histochemistry reagent.

Leonard