Ligustrum spp. TS safranin-o stain

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mazo4033
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Ligustrum spp. TS safranin-o stain

#1 Post by mazo4033 » Thu Jul 29, 2021 10:42 pm

Hi all,

Recently I began botanical sectioning, and I give my thanks to forum members mrsonchus and MicroBob for their excellent and very helpful posts on this subject matter. After experimenting with various plants nearby, I found that the privet bush (Ligustrum spp.) happens to be a particularly excellent plant for hand sectioning. Thus begins my little experiment in hand sectioning and staining transverse sections of Ligustrum vulgare (?)

1 - background
Ligustrum is a tall and resilient hedge often used in gardens, but is sometimes a small tree depending on the species. They flower and produce berries (unfortunately I missed the flowering period this year and did not get a chance to section the flowers :( ... next summer!)

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An image of Ligustrum vulgare which I sectioned.

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The branches of Ligustrum have multiple stages of growth. Prior to becoming a tough and woody branch once it is fully grown (see image above - branch 3), the young, sprouting branches have a green color and are strong, but much softer than the fully-grown branch (see image above - branches 1 and 2). I have found that the privet branch at this sprouting stage is optimal for hand sectioning; it is soft enough that a razor blade cuts through without much effort, but thick and strong enough that thin sections and the pressure of cutting still allow the section to retain its shape and not get crushed.

2 - procedures
A single edge razor blade was utilized for sectioning (a method that requires much practice and patience! :lol:) and as I was making a temporary mount because my main objective was to test staining, I opted for a pva glue and glycerin mounting medium. It is a 1:1 mixture of a pva-based glue (Elmer's clear glue, for example) and glycerin, along with some water. This recipe is derived from Richard H. Zander's 2014 paper linked here: http://www.mobot.org/plantscience/ResBo ... ia2014.pdf. It is a useful mountant: easy to make and use, and preserves the slides for many months.

I tested 2 different staining protocols to see the difference in results. Protocol 1, derived from mrsonchus' post here: https://www.microbehunter.com/microscop ... 10&t=7889&: 1% safranin-o aqueous for 5 seconds, transfer sections to water for a bit to remove excess stain, then transfer sections to 70% ethyl alcohol for 30 minutes. Mrsonchus did 50% alcohol to 95% alcohol - I only had 70% alcohol on hand and it seemed to still work. Protocol 2, derived from mrsonchus' post here: https://www.microbehunter.com/microscop ... 1&p=44944&: sections in a weak (diluted) safranin-o stain for 1 hour.

3 - results
These were my most successful sections and staining so far - for a beginner like me this makes me very happy! :D

Protocol 1:

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40x - quite possibly the best section in this batch - I think it took to the stain well, the xylem are particularly resolvable and so are the medullary parenchyma in the middle.

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xylem, 100x

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medullary parenchyma, 100x

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xylem, 400x

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40x, protocol 1 but a different section - it took to the stain differently despite enduring the same conditions - not sure why

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100x, medullary parenchyma

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collenchyma, 100x and 400x

Protocol 2:

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40x, a much deeper red than resulted in protocol 1, still gorgeous though. But the difference in protocols definitely shows

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100x, medullary parenchyma and xylem

4 - conclusion
That concludes my little experiment. I had a lot of fun and consider this to be one of my first bigger successes with botanical sectioning. Happy to see the safranin working well and my hand sectioning skills improving. I look forward to investing in other stains - possibly fast green or alcian blue to be able to follow more complex staining procedures. I did this all with very simple materials - hopefully this shows that botanical sectioning is not as daunting as it seems and even beginners can achieve some decent sections - go out and try it if you haven't, it's tons of fun! I won't be forgetting about Ligustrum, but I look forward to my next adventure: pollen! :D

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jimur
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Re: Ligustrum spp. TS safranin-o stain

#2 Post by jimur » Fri Jul 30, 2021 9:19 am

Good job. Waiting to see more from you.
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perrywespa
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Re: Ligustrum spp. TS safranin-o stain

#3 Post by perrywespa » Fri Jul 30, 2021 1:52 pm

Nice work on your first attempts! I started doing the same about a year ago. Below in a section of sweet gum petiole stained with Toluidine Blue:
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mrsonchus
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Re: Ligustrum spp. TS safranin-o stain

#4 Post by mrsonchus » Fri Jul 30, 2021 2:03 pm

Excellent sections, love the stining too - Safranin's been a favourite of mine since I started back in 2015.

Both Safranin and Toluidine blue (TBO) are 'metachromatic' and this property is especially noticeable I find in hand-sections - probably because they're a lot thicker than microtome-sections, that plus the 'unscrubbed' (chemically that is) nature of the tissue - most of the original chemistry is very likely still in the tissue, in it's 'correct' location too I think...

Metachromasia (exhibited by stains that are metachromatic) is the stain's ability to stain in usually several different tones (not simply intensities but actual tonal shifts) and colours. This can be seen in both sections and gives a nice 'all in one' stain for starting-out with staining and sectioning....

Fast-green as a stain is better suited to permanently-mounted (and of course fully processed) sections together with another complementary stain such as Safranin, rather than for hand sectioning. At the first glimpse of water for example - fast-green will disappear from the tissue!

Great start and lovely colourful results!
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mazo4033
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Re: Ligustrum spp. TS safranin-o stain

#5 Post by mazo4033 » Fri Jul 30, 2021 6:15 pm

Thank you jimur, perrywespa, and John!

Perry: very nice result with the sweet gum section!! I like the tonal shifts with the blues and the section came out very nicely.

John: The metachromatic aspect of safranin is my favorite part; it creates these layers of colors that are really nice (especially for the simplicity to use the stain!)
Fast-green as a stain is better suited to permanently-mounted (and of course fully processed) sections together with another complementary stain such as Safranin, rather than for hand sectioning. At the first glimpse of water for example - fast-green will disappear from the tissue!
Great advice, thank you, I had no idea fast-green was that fickle! I suppose toluidine blue is a better option for the next stain to acquire - I haven't started processing tissues or anything of the sort - just temporary sections so far.

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mrsonchus
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Re: Ligustrum spp. TS safranin-o stain

#6 Post by mrsonchus » Fri Jul 30, 2021 6:25 pm

Definitely - TBO is a superb stain, especially for live (i.e. unprocessed as per hand-sectioned) tissue. Make sure you rinse it well after staining to get the very best from TBO - take the time (only a matter of say 10 minutes really) to rinse well with water (preferable deionised - whatever water make sure it's pH is neautral) to get the very best metachromasia from this superbly easy stain. Same goes really for Safranin, proper rinsing to fully exploit metachromasia. Incidentally two other water-based metachromatic stains are the old favourite methylene-blue (which I find just too powerful and not as good as TBO which is similar) and another familiar, gentian-violet (AKA crystal-violet) which again is very potent. Lastly alcian-blue is an excellent stain and can be mixed with safranin to make a very easy to use one-shot bi-stain..... All the above are water-based stains - no alcohol need apply!
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Re: Ligustrum spp. TS safranin-o stain

#7 Post by perrywespa » Fri Jul 30, 2021 6:26 pm

I will say that mounting these sections for permanent mounts in clear nail polish or PVA glue (clear Elmer's) results in some bleeding of the stain into the mounting medium.
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mrsonchus
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Re: Ligustrum spp. TS safranin-o stain

#8 Post by mrsonchus » Fri Jul 30, 2021 7:19 pm

perrywespa wrote:
Fri Jul 30, 2021 6:26 pm
I will say that mounting these sections for permanent mounts in clear nail polish or PVA glue (clear Elmer's) results in some bleeding of the stain into the mounting medium.
Yes, the stains will all bleed to some extent in anything but a resin-based mountant I think. Don't think that resin-mounting is only for processed microtome-cut slides, hand-sections can also be permanently-mounted into resinous mountants such a Histomount or NuMount, they just need to be dehydrated via a water/alcohol series ending in pure (as you can get anyway) alcohol, which can then be removed by several applications of an agent that will displace and remove the alcohol, and be compatible with resinous mountants. This leads us onto either Xylene or Histomount as the resin's antemedium. A lot easier than it sounds......
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iconoclastica
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Re: Ligustrum spp. TS safranin-o stain

#9 Post by iconoclastica » Fri Jul 30, 2021 7:54 pm

A very interesting discussion, this is. Thanks.

I use methylene blue in the 1% aq solution, but I find it much easier to handle when diluted much more. To a lesser extent the same applies to safranine. 1-3 Drops in a 3cm pertri dish with water gives all the time I need to judge the staining intensity vs. time.

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Re: Ligustrum spp. TS safranin-o stain

#10 Post by mrsonchus » Fri Jul 30, 2021 9:14 pm

iconoclastica wrote:
Fri Jul 30, 2021 7:54 pm
A very interesting discussion, this is. Thanks.

I use methylene blue in the 1% aq solution, but I find it much easier to handle when diluted much more. To a lesser extent the same applies to safranine. 1-3 Drops in a 3cm pertri dish with water gives all the time I need to judge the staining intensity vs. time.
Indeed, and of course stain regression back to the desired point is easy with these 'regressive' stains - a little alcohol to remove any overstaining and back to water-rinse if just right. Water removes excess (in this sense 'unused') stain, alcohol removes stain that has actually taken to the tissue and so remains after excess stain is water-rinsed.... This may be termed 'overstain' rather than excess - not however a good method with TBO, very good with safranin, alcian blue or gentian violet. Gentian violet stains really strongly but may, after moving to alcohol, be regressed with clove-oil very effectively, then back to alcohol and if desired then back to water. The methodology/protocol can become as easy or difficult as desired really.....

Staining that is not regressed rather stained/examined then again until just right is the other side of the coin as it were, 'creeping up to' the desired staining then stopping, know as progressive staining. The establishment of 'borders' between two or more stains in a tissue, to minimise 'stacked stains' I may call it, will achieve differentiation of the tissue structures, each stain ideally 'sticking to' those structures for which within a protocol it has greatest affinity....
Staining is mostly heuristic and very hard to give catch-all protocols for. I rather explain the technique rather than attempt to formularise, the heuristic nature is then to be taken on by the fellow staining the tissue. This approach is the road to good staining and great versatility born of understanding the process rather than learning protocols unti your brain bursts!

Here's an old post of mine hand-sectioning an apple twig that may give you an idea...
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mazo4033
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Re: Ligustrum spp. TS safranin-o stain

#11 Post by mazo4033 » Fri Jul 30, 2021 11:38 pm

Definitely - TBO is a superb stain, especially for live (i.e. unprocessed as per hand-sectioned) tissue. Make sure you rinse it well after staining to get the very best from TBO - take the time (only a matter of say 10 minutes really) to rinse well with water (preferable deionised - whatever water make sure it's pH is neautral) to get the very best metachromasia from this superbly easy stain.
Thank you for the tips! I think that I will explore TBO next and also alcian blue - I'll keep this in mind.
Incidentally two other water-based metachromatic stains are the old favourite methylene-blue (which I find just too powerful and not as good as TBO which is similar) and another familiar, gentian-violet (AKA crystal-violet) which again is very potent.
I too find both methylene-blue and crystal violet to be very potent stains, though as I think about it now, I realize that I have not tried the regression technique and/or applying alcohol like I had done with safranin. It would be interesting to try those stains again but with a different approach and perhaps diluting the stains as well. I did have success once with methylene-blue on part of an onion, but when applying it to something like the privet it was too potent.
the heuristic nature is then to be taken on by the fellow staining the tissue. This approach is the road to good staining and great versatility born of understanding the process rather than learning protocols unti your brain bursts!
I agree, it is important to learn basic protocols and the behaviors/compatibility of different stains, but the true understanding does come from the doing and observing :D So easy to get carried away even with the simple stains. Sometimes I think I'll sit down and prepare a few quick sections...2 or 3 hours pass :lol: I love the way the apple twig turned out. Safranin & alcian blue is a good and pretty combination. How are the sections holding up over a year later?
I will say that mounting these sections for permanent mounts in clear nail polish or PVA glue (clear Elmer's) results in some bleeding of the stain into the mounting medium.
Yes! I have noticed this also. Although I found that using water to rinse excess stain and letting the sections spend some time in alcohol as I did with the sections in this post lessens the bleeding greatly. For protocol 1 I did not notice this problem at all and for protocol 2 I did, though I forgot to rinse that section after taking out of the stain. Doing so would have probably helped. Although I don't know how this changes once slides are kept for a longer time. The oldest slide I have using this pva glue-based mountant is from early Feb 2021 and it is in perfect shape, but the sections are not stained. I wonder if the stain bleeds more heavily as the slide becomes older; I don't have a slide with stained sections that I've kept for long enough to observe.

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