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From thomas_borell@msgw.mayo.edu Fri Aug 4 05:04:51 1995 Subject: To: Aaron Hicks <ahicks@prism.nmt.edu>
AJ,
technique for counting chromosomes: Not difficult to master if approx. counts suffice; although accurate counts are necessary in sci. research, counts of +/- 1 chrom. are usually satisfactory for practical purposes, esp. when chrom numbers are at 3X and higher polyploidy levels (NB: there may be a demand for services that require this kind of work- Aaron). The microscope: Except for Paph. plants (which have mondo chroms that can be counted with a high, dry objective (40X), orchids require a 'scope equipped with an oil-immersion lens (90 to 100X), and a 10-15X ocular. The optical system should be clean and perfectly aligne to give a clear image. Equipment and supplies that follows are also needed: 'Scope illuminating lamp
Prep. of Aceto-Orcein Stain Prep. 100 ml of 45% acetic acid by pouring 45 ml of glacial AA in 55 ml of DI. Place 1 g of orcein in the acetic acid, heat to dissolve. Cool, filter and transfer into a medicine dropper bottle; it is now ready to use. Prep. of 9-Hydroxyquinoline Solution Pretreatment of root tips with 8-HQ will cause contraction and may improve spreading of chroms and thereby facilitiate counting. Prep .002 M solution of 8-HQ (mw=145.15) by dissolving .029 g of 8-HQ in 100 ml of DI.
Prep of fixing fluids The fixing fluid will kill cells rapidly with a minimum of shinkage swelling, distortion, and production of artifacts. Any of the following should suffice: 1) Modified Carnoy's 2 parts 95% EtOH
2) Carnoy's 3 parts EtOH 3) 45% acetic acid
Prep of HCl-Alcohol mixture: HCl is used to soften the tissues to let them spread easily. One can use 1N HCl at 140F (60C), or 1:1 con HCl (12N) and 95% EtOH at room temp. Prep of paraffin-gum mastic mixture Heat 1 part gum mastic with 9 parts paraffin until they melt and mix. Cool to solidify. This mix is used to seal cover slips.
Root Smear Technique for Counting Chroms of Orchids (oooo, the moment we have been waiting for!). 1) Sever root tips, about 1mm long, and place in a small vial with .002M 8-HQ sol'n for 3-5 hours at about 65F (18C). 2) Wash with water. 3) Fix with 2:1:1 mix of 95% EtOH, chloroform, and glacial AA for at least 10 mins at about 50F(10C). 4) Hydolize with 1N HCl at 140F (60C) for 3-5 mins. 5) Wash with water. 6) Place in 45% acetic acid for 10 mins (may also be stored several days in refrigerator). 7) Transfer root to a clean slide and cover with 45% acetic acid to keep roots from drying. 8) Remove root cap under dissecting scope. 9) Separate cells with needles. 10) Blot out acetic acid, add 1-2 drops of aceto-orcein, and place slide in a chamber sat. with 45% acetic acid for 10-30 mins. 11) Place cover glass, tap cover with point of needle or a slide tapper, and remove excess stain. 12) When cells are well-spread, heat slide for a few seconds. Prevent boiling. 13) Place bibulous paper ove rslide and press firmly with thumb to flatten cells and remove excess stain. 14) Seal edges of cover glass with paraffin-gum mastic mixture or dental wax. 15) Count chroms with a compound microscope under an oil immersion lens.
Oh, MY how SIMPLE that all sounds.... =-D
Pollen Mother Cell (PMC) Smear Technique for Counting Choms of Orchids 1) Carefully slit open a young bud with a surgical knife, and remove a small portion of a pollinum to determine the stage of development 2)Place on slide, fix with 45% acetic acid at 50F for 5 mins. 3) Replace acid with 1% aceto-orcein and stain for a few minutes. 4) Place a cover glass, heat, and press firmly to remove excess stain.
5) Examine under a scope. If the stage of meiosis is too early to sample, seal the bud with scotch or masking tape to prevent drying, and sample at a more appropriate time. 6) If the PMCs are at metaphase division, remove pollinia and treat with a 45% acetic acid solution at 50F for 10 minutes. 7) Place a portion of the pollinia on a slide and add a drop of aceto-orcein. Keep in chamber for 5 minutes. Prep add'l slides to ensure adequate numbers of analyzable figures. 8) Place a cover glass, tap gently, remove excess stain. 10) Press firmly, seal with dental wax. 11) Count chroms with a compound scope, under an oil immersion lens.
PHEW! Sounds like a blast; a great weekend project! There are several pages of tables with chrom counts, most from the 60s and 70's; nothing recent- and little between 1980 and 1984, but I can cite some stuff if you care to know it that badly. Otherwise, sounds like classic cookbook chemistry; for someone who piddles with the human component (and, knowing Mayo, has access to 'scopes that most cell biologists would donate a kidney to have access to!), this may well be trivial. If any abbrevs are unclear, or some techniques need to be expounded upon, I can quote them verbatim; I thought that doing so would take too much time, on top of insulting your intelligence (I don't know much about the stains, and although there are zillions of them- you're probably far and away further in the know with respect to orcein than I am!). Best of luck; now is the time to do it, with root-tips in the growing phase right now. Enjoy!
Date: Wed, 29 May 1996 07:26:48 +0000
Carson W. asked about Labs that can do chromosome counting:
Well, I have considered offering that service many times. I am working on my new lab, and have been thinking about a lot of things including chromosome counting again. There are a few points that should be clarified:
1) Chromosome counting on orchids (and other plants) is not as easy as one might think.
a) most orchids are periodical in their mitosis, so it will have to be established first at what time of the day the root tips are to be taken. If the tips are taken at the wrong time, no results b) The root tips have to be fixed and stored in a fresh mixture of alcohol and glacial acetic acid. If not, no results. c) There are a number of other steps in the procedure that are critical. And to get decent results one needs a temperature cabinet and a microscope with phase contrast. It may work at room temperature and a simple microscope but the results would be questionable.
Therefore we come to point 2
2) Costs. No-one is going to get himself such a setup to do one or two counts for amateurs (this is not meant to be insulting and negative, but realistic). After all, we are talking about an investment (German prices ) of 10,000 to 15,000 DM or more (divide by 1.5 and you have the US $ prices). And that is a low estimate, calculating for second class materials (catalogues are on my desk) and
3) There are too many critical factors. Therefore, the lab can never give any guarantee.
4) There is the psychological aspects. I have found most hobbyists to be very reluctant to cut one single growing root of their plant, even when the plant has hundreds. And you need more than one root tip. The first experiment would involve to cut and fix two or three root tips every two hours to establish the right time to harvest the root tips.
Hope we can discuss this further
Regards
Dr. Guido J. Braem ---- Plant Taxonomist Tel. [+49](6441]65333 Fax [+49](6441)65334
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