Re: [Histonet] plant anatomy - sectioning problems

From:Jennifer MacDonald



We also have problems with the light green, so we add extra acetic acid to 
the working solution and get much better results.

Jennifer





Jennifer MacDonald
Director, Histotechnician Training Program
Mt. San Antonio College
1100 N. Grand Ave.
Walnut, CA 91789
(909) 594-5611 ext. 4884
jmacdonald@mtsac.edu



"Wurdak, Elizabeth"  
Sent by: histonet-bounces@lists.utsouthwestern.edu
04/13/2007 07:29 AM

To
yvan lindekens , Histonet 

cc

Subject
Re: [Histonet] plant anatomy - sectioning problems






Hi Yvan,
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.
Liz

 
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 
February
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
schedule:
 
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.
 
10. Embed.
 
This schedule is a modified version of the protocol we followed in 1997. I
also consulted
 
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!
> 
> Yvan.
> 
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