Friends,
I always thought that bacterial infection of flower stems led to clogged vessels. No water transport and wilting as a consequence. It took these Gerberas just 3 days to wilt. Of course dirty, bacterially infected water. Histology showed a surprise: all the phloem had disappeared.
Normal vessel (https://www.mikroskopie-forum.de/pictures002/73753_17059733.jpg). obj.10x
Diseased vessel (https://www.mikroskopie-forum.de/pictures002/73753_28360416.jpg) obj 10x
And diseased vessel(https://www.mikroskopie-forum.de/pictures002/73753_21926403.jpg) at 20x obj.
Technovit 7100 and toluidine blue stain.
I did not enjoy the flower's fate. But the pathology is impressive.
Regards,
Leo
Dear Leo,
thank You for the interesting sections!
I asume, that the saccharid concentration in the Phloem is responsible for the growth of bacteria and the following destruction of the tissue.
Regards
Jörg
Dear Leo,
very impressive - and quite similar to several things happening to human tissue in infectious disease.
I tried to identify the causative organism by enlarging your photograph, and there seams to be a mixed flora of rod shaped and coccoid bacteria.
Presuming your accordance, here an enlarged detail of your photograph (of cause in reduced resolution):
(https://www.mikroskopie-forum.de/pictures002/73777_15517527.jpg)
An additional photograph in higher magnification/x100 oil immersion would be interesting!
Best regards,
Florian
Dear Florian,
Thank you for your picture. Our immune system is quite a bit better than that of a (cut) plant, usually.
I did the 100xoil. In the xylem the form may not be representative because of sitting in a tube (https://www.mikroskopie-forum.de/pictures002/73782_32238868.jpg). In between parenchymous cells I found a better collection(https://www.mikroskopie-forum.de/pictures002/73782_46725179.jpg) and (https://www.mikroskopie-forum.de/pictures002/73782_36684149.jpg).
The latter 2 seem to reflect rod-shaped bacteria, because this is a section where the rods are seen in all directions from circle to rod. Could however be different to what's present in the xylem.
Kind regards,
Leo
Dear Jorg,
I think you are completely right. But they seem also to eat extracellular matrix and hate lignin or lignocellulose. Anyway, we need a disinfectant in the water of Gerbera. That's a lesson.
Best regards,
Leo
Dear Leo,
thank you. To estimate whether there are morphologically different bacteria it would further be interesting how thick your specimen were sliced.
Kind regards,
Florian
Leo,
My first reaction of your first picture was that I saw a thyroid but that was not correct.
Nice pictures and embedded in Technovit I believe.
Could you describe some more of your embedding and cutting techniques?
Greetings Ronald
Dear Ronald,
Technovit 7100 protocol.
Fixate tissue in 4% buffered formaldehyde for 48 hours. Alternative: Fixate in same , but use Microwave lowest setting 2 x 10 min. on ice. Then alcohol range 50 % overnight. 75 % for 4 hrs followed by 96 % 2 x >4 hrs.
Change for Technovit + harder1/alcohol mix 50/50 for 24 hours. Then Technovit +harder1 for 24 hours. Change for same fresh and continue for >4 hrs. After that you can safely do the polymerization.
I polymerize in 2 ml Eppendorf tubes in which I first make a solid cushion of 0.2 ml polymerized Technovit7100. I put the pieces on this cushion, preventing air bubbles, and polymerize in 0.5 ml Technovit. After polymerization I add 0.5 ml Technovit 3040. A few hours later I cut off most of the cushion using a small (1.5 cm) electrical circle saw (Dremel) and punch the resulting embedded material from the tube.
I then cut slides using an AO800 microtome, applying a Kultzer knife and set the thickness as low as I can possibly do. (The thing's setting is inaccurate: at 4 micron I get no material sliced off, so I put it at 6 microns or higher, if necessary).
I spread slides on tapwater, dry at app. 45 oC on a plate for some minutes and stain 5-20 sec. in 0.5 % toluidine blue, 0..5 % borax, which is filtered on Millipore 0.20 um. Differentiate in tapwater and clean finally in MilliQ. Drying again at 45 oC on the plate.
Important: the changes for Technovit 50-Technovit-Technovit before polymerizing is important to prevent fragmentation of the tissue.
I use the Eppendorf tubes because the Kultzer moulds are terribly expensive, and sofar I have not found them on eBay.
Groeten,
Leo
Dear Florian,
I do not know exactly. The AO800 microtome I use is not exact in its settings; it has been repaired and at setting 4 um I get nothing. So I regularly cut at 6 um or 8 and assume to get 2 resp. 4 um. This tissue is very labile because of bacterial enzyme effects so I had to cut at 8um, i.e. at assumed 4 um.
Hope it helps.
Leo