Gerbera, just 3 days in a vase *

[Leo van Griensven]

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 . obj.10x
Diseased vessel obj 10x
And diseased vessel at 20x obj.
Technovit 7100 and toluidine blue stain.
I did not enjoy the flower's fate. But the pathology is impressive.

Regards,
Leo

[Fahrenheit]

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

[Florian Stellmacher]

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):



An additional photograph in higher magnification/x100 oil immersion would be interesting!

Best regards,
Florian

[Leo van Griensven]

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 . In between parenchymous cells  I found a better collection and .

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

[Leo van Griensven]

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

[Florian Stellmacher]

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

[Ronald Schulte]

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

[Leo van Griensven]

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

[Leo van Griensven]

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