We also have problems with the light green, so we add extra acetic acid to
the working solution and get much better results.
Director, Histotechnician Training Program
Mt. San Antonio College
1100 N. Grand Ave.
Walnut, CA 91789
(909) 594-5611 ext. 4884
Sent by: firstname.lastname@example.org
04/13/2007 07:29 AM
yvan lindekens , Histonet
Re: [Histonet] plant anatomy - sectioning problems
The procedure we followed is listed below. We had pretty good results as
far as sectioning went. We are staining with safranin and fast green or
hematoxylin and safranin. Neither comes out as bright as I would like to
see it. I would welcome any suggestions you have for staining.
Processing Plant Tissues for Paraffin Sections
On January 5 and 6, 2007 various plant organs were collected in the SJU
Greenhouse. They were fixed in FAA and kept in this solution until
13 and 14, 2007.
The composition of FAA is as follows:
95% ethanol 50 cc
glacial acetic acid 5 cc
formalin 10 cc
distilled water 35 cc
Wiley, R. L. 1971 Microtechniques: A Laboratory Guide, The Macmillan
Company, New York
Dehydration and clearing was carried out according to the following
1. Jar #1: 30:50:20 water:alcohol:tert-butyl-alcohol
2. Jar #2: 15:50:35 water:alcohol:tert-butyl-alcohol for 1 hour.
3. Jar #3: 25:75 alcohol:tert-butyl-alcohol for 1 hour.
4. Jar #4: 100% Tert-butyl-alcohol for 1 hour.
5. Jar #5: 100% Tert-butyl-alcohol for 1 hour.
6. Jar #6: 100% Tert-butyl-alcohol + paraffin chips, ON at 45oC.
7. Jar #7: Pure paraffin 30 min, 60oC under vacuum
8. Jar #8: Pure paraffin in 60oC oven 1hr.
9. Jar #9: Third change of pure paraffin in 60oC oven 1hr.
This schedule is a modified version of the protocol we followed in 1997. I
Grey, P. 1964 Handbook of Basic Microtechnique 3rd. ed, McGraw-Hill Book
Company, New York.
Ruzin, S. E. 1999 Plant Microtechnique and Microscopy, Oxford University
Press, New York
On 4/13/07 4:17 AM, "yvan lindekens" wrote:
> Hi all,
> I'm trying to cut some plant samples but I have lots
> of problems with the samples containing even the
> smallest amount of wood (f.e. young white deadnettle
> stems - Lamium album L.).
> A thickness of 15 micron is the thinnest I can get,
> without the ribbon scattering longitudaly after only a
> few sections. Whiping the blade with a cloth moisted
> with some xylene solves the problem, but only for
> another 2 or 3 sections... I use a Leica-Jung Autocut
> 1140 and Feather S/R/N 35 blades.
> Cutting at veeeeeeryyyyy looooow speed gives slightly
> better results but still not good enough!
> When sectioned on a sliding microtome(Reichert-Jung
> Hn-40, Feather S/R/N 35 blades, declination angle +/-
> 130°), the sections are okay, but I would like to use
> a rotary.
> Samples were fixed in AFA, processed by hand using an
> ETOH/IPA/xylene protocol. On the other hand I
> processed some samples using a tert. butyl alcohol
> schedule. There's hardly any difference between the
> two regarding sectioning properties...
> By the way: is there something as a "standard
> thickness" for plantanatomical sections?
> Thanks in advance for every suggestion!
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