Leaf TS stain-trial, Safranin vs Fast-green - the battle for supremacy!
Posted: Sun Apr 03, 2016 11:46 pm
Hi all, as you may know I'm currently working to improve my staining technique; at this time I'm concentrating on the stain/counter-stain combination of Safranin (pink/red) with Fast-green. This is a very, very well-known combination and one that I particularly like for the differentiation of tissues comprising my Botanical sections.
The ideal result generally, is to use the 'basic' (in that it stains acid-tissue elements) Safranin (pink-red sometimes orange-red) to stain nuclei, chromosomes when condensed during cell-division and other elements also acid in their exposed (to incoming stains) tissue-chemistry, then to 'counter-stain' the basic (chemically speaking) elements with the acid-stain Fast-green.
Practically this means with perfect differentiation of the stain/s that the Fast-green will stain only cytoplasm and not any acidic structures etc that the Safranin has already stained... ideally that is!
Anyway, the balance isn't an easy one to get right let alone perfect, and like all staining techniques in my limited experience is variable across different tissue types, thicknesses etc - many factors effect staining results. This makes staining a very difficult thing to learn without a lot of experimentation, and these pictures are from tonight's 'session' in my teeny lab (dining-room )..
I just bought a new cheap condenser for £15 with some of my slide-income and have seen a noticeable improvement of in particular images taken with my Toupcam camera. They seem a lot sharper and of a better resolution than they were with my 'old' rather tatty condenser. The new one is similarly a very basic abbe 1.25 but I think is of a better quality or maybe condition - hard to be sure though as these are the first and only images I have of these particular slides. Time and more pictures of different slides will tell I suppose. Anyway, enough talk, here are some pictures..
This is a section stained primarily with fast-green and a tiny (unintentionally so I hasten to add ) measure of Safranin which is all but invisible. I still like this result as the Fast-green has made a fine and rather delicate image to my eyes, with pretty good detail despite the rather low contrast of this stain when used alone... This is a 2-picture stitch of a Daffodil (again!) leaf TS and has most leaf-morphology within.. Here's a rather better single image of the same tissue with the same objective, and a few labels, The labels are:
p = 'parenchyma' - a 'filler tissue' in the center of the leaf that also stores substances, large volume cells..
vb = 'vascular bundle' AKA leaf-vein running parallel to long-axis of leaf and so seen in TS...
sm = 'spongy mesophyll' - (mesophyll is another name for parenchyma, when found in a leaf) a hardly if at all photosynthetic type of parenchyma with an open 'spongy' cell arrangement, facilitates gaseous exchange within leaf tissue by virtue of this structure also...
pm = 'palisade mesophyll' = 'long' chloroplast-filled and therefore photosynthetic cells found immediately below the epidermis...
s = 'stomate' (pl stomata), a pore that opens & closes to regulate gaseous exchange - often has an large chamber behind it, the 'sub-stomatal chamber'...
I think the very-predominant Fast-green has made a reasonable job of staining, many structures & tissues are readily discernible it seems...
Now, here's a picture of how the tissue looks when the Safranin & Fast-green work together in their intended stain/counter-stain roles - to me a far superior result.... The Safranin has stained the cuticle very nicely (the red strip on the epidermis' surface) and shows quite clearly the way this cuticle is continuous within the stomate-linings that form the pore itself.. The Safranin has just started to catch the chloroplasts also.
In the following picture we see how the chloroplasts may also be made to stand-out clearly with a little extra Safranin.... The chloroplasts (strange to think they are naturally green ) are really easy to see here, together with the way they 'pack' the palisade cells to maximize photosynthesis - the palisade cells' nuclei have also appeared as if by magic - their acidic contents stained by the basic Safranin - lovely to see. Also the cells (parenchyma/mesophyll) behind the palisade layer may be seen to have small amounts of what I think are chloroplasts only, and these are thinly lining the cell-walls rather than filling the cell (see left-edge of image just above the text-box)! Amazing what stains are able to show when they're feeling cooperative!
Well, that's about all I've time for tonight, I hope this hasn't been a little long-winded, it's a habit of mine I'm afraid. I just thought that this small example of one of my explorations of a couple of slides may give folks an insight into the reasons that I love Botany and histology so much! The mysteries are endless, the surprises constantly appearing, the adventures always exciting!
Anyway, I'd better get some sleep, the dog wants to go out also!
I hope you enjoy this little adventure as much as I have!
The ideal result generally, is to use the 'basic' (in that it stains acid-tissue elements) Safranin (pink-red sometimes orange-red) to stain nuclei, chromosomes when condensed during cell-division and other elements also acid in their exposed (to incoming stains) tissue-chemistry, then to 'counter-stain' the basic (chemically speaking) elements with the acid-stain Fast-green.
Practically this means with perfect differentiation of the stain/s that the Fast-green will stain only cytoplasm and not any acidic structures etc that the Safranin has already stained... ideally that is!
Anyway, the balance isn't an easy one to get right let alone perfect, and like all staining techniques in my limited experience is variable across different tissue types, thicknesses etc - many factors effect staining results. This makes staining a very difficult thing to learn without a lot of experimentation, and these pictures are from tonight's 'session' in my teeny lab (dining-room )..
I just bought a new cheap condenser for £15 with some of my slide-income and have seen a noticeable improvement of in particular images taken with my Toupcam camera. They seem a lot sharper and of a better resolution than they were with my 'old' rather tatty condenser. The new one is similarly a very basic abbe 1.25 but I think is of a better quality or maybe condition - hard to be sure though as these are the first and only images I have of these particular slides. Time and more pictures of different slides will tell I suppose. Anyway, enough talk, here are some pictures..
This is a section stained primarily with fast-green and a tiny (unintentionally so I hasten to add ) measure of Safranin which is all but invisible. I still like this result as the Fast-green has made a fine and rather delicate image to my eyes, with pretty good detail despite the rather low contrast of this stain when used alone... This is a 2-picture stitch of a Daffodil (again!) leaf TS and has most leaf-morphology within.. Here's a rather better single image of the same tissue with the same objective, and a few labels, The labels are:
p = 'parenchyma' - a 'filler tissue' in the center of the leaf that also stores substances, large volume cells..
vb = 'vascular bundle' AKA leaf-vein running parallel to long-axis of leaf and so seen in TS...
sm = 'spongy mesophyll' - (mesophyll is another name for parenchyma, when found in a leaf) a hardly if at all photosynthetic type of parenchyma with an open 'spongy' cell arrangement, facilitates gaseous exchange within leaf tissue by virtue of this structure also...
pm = 'palisade mesophyll' = 'long' chloroplast-filled and therefore photosynthetic cells found immediately below the epidermis...
s = 'stomate' (pl stomata), a pore that opens & closes to regulate gaseous exchange - often has an large chamber behind it, the 'sub-stomatal chamber'...
I think the very-predominant Fast-green has made a reasonable job of staining, many structures & tissues are readily discernible it seems...
Now, here's a picture of how the tissue looks when the Safranin & Fast-green work together in their intended stain/counter-stain roles - to me a far superior result.... The Safranin has stained the cuticle very nicely (the red strip on the epidermis' surface) and shows quite clearly the way this cuticle is continuous within the stomate-linings that form the pore itself.. The Safranin has just started to catch the chloroplasts also.
In the following picture we see how the chloroplasts may also be made to stand-out clearly with a little extra Safranin.... The chloroplasts (strange to think they are naturally green ) are really easy to see here, together with the way they 'pack' the palisade cells to maximize photosynthesis - the palisade cells' nuclei have also appeared as if by magic - their acidic contents stained by the basic Safranin - lovely to see. Also the cells (parenchyma/mesophyll) behind the palisade layer may be seen to have small amounts of what I think are chloroplasts only, and these are thinly lining the cell-walls rather than filling the cell (see left-edge of image just above the text-box)! Amazing what stains are able to show when they're feeling cooperative!
Well, that's about all I've time for tonight, I hope this hasn't been a little long-winded, it's a habit of mine I'm afraid. I just thought that this small example of one of my explorations of a couple of slides may give folks an insight into the reasons that I love Botany and histology so much! The mysteries are endless, the surprises constantly appearing, the adventures always exciting!
Anyway, I'd better get some sleep, the dog wants to go out also!
I hope you enjoy this little adventure as much as I have!