Why 2% acetocarmine is widely used for meiotic chromosome studies? what is the peculiarity of carmine stain in meiotic chromosome squash? Can 2% acetoorcein do the same wonders? please provide me literature. I need a lucid / easy to do classroom practical method of chromosome preparation from leaf tip tissue in plants to show my PG students.
Hi
Attached file is for practical protocols, source: BHU.
Regarding Orcein, both the versions are used in a protocol here, do not know much, but am hurriedly sharing the manual now. See protocol for Polytene chromosome I think some explanation is given.
Best regards
The stains: 2% acetocarmine and orcein are chromatin-specific dyes. They bind permanently to the nucleoprotein component of chromatin. Thats why chromatin can be visualized by treating it with the tw dyes. The dyes are very active and small quantities of them can be very efective to observe chromatine material. The binding of the 2 dyes is gradual and improves with time; and can be facilitated by applying gentle heating of the glass slide over a flame. Both orcein and acetocarmine can do the same "wonders". They work well on most biological samples. They stain both euchromatine and heterochromatin alike; but a stain like Giemsa binds euchromatin and heterochromatin differentially. The differential staining by Giemsa is what produces the bands of stain on chromosomal structures. Though carmine is pinkish while orcein is purplish. It is important to note that both can be dissolved in acetic acid, acitic and propionic acid or propionic acid. A solution of 1:1:1:1 formic acid: Lactic acid: Propionic acid : water can give excellent orcein staininability results. You can find some literature by googling my name to find some of my works online. The practical manual attached by Sonal Rajiv Bakshi is also very informative.
nice information. Giemsa will stain differentially only following trypsin and similar treatment otherwise it gives solid stain purple blueish, useful for cytogenetic endpoints other than karyotyping.
After reviewing the excellent recommendations provided in the above article and responses, copies of the protocols for staining chromosomes of plants, insects, and mammals, should be held in readiness by teachers/instructors, to provide copies to students of all ages the moment they express the desire to view their own chromosome preparations. Once they’ve seen a well spread metaphase, the image can, as in my case, serve as the starting point towards achieving academic goals.
If I'm not mistaken, two items were not mentioned in the above references regarding the preparation of acetocarmine. (1) the addition of several eyedrops of the mordant, ferric acetate, prepared earlier by simply adding an iron tack/nail to age in 25 ml of 45% acetic acid, to an eyedropper bottle-full of stock acetocarmine. The mordant assists in attaining a deeper staining of chromosomes particularly useful when viewing preparations with light microscope lenses, and (2) the substitution of 45% propionic acid in place of 45% acetic acid to lighten the staining of cytoplasm.
I believe the first published compilation covering the staining of mitotic and meiotic chromosomes in a single article was when Alexander Hollaender, Director of the Biology Department at Oak Ridge National Laboratory, invited K.S. Lavappa and me to submit the chapter entitled "Procedures for culturing diploid cells and preparation of meiotic chromosomes from dwarf species of hamsters, published In: "Chemical Mutagens. Volume 2. Principles and Methods for their detection". A. Hollaender, Ed. Plenum Press, New York, pp.387-410, 1971.
Hello
Aceto-carmine is more safer than aceto-orcein because it is lightly toxic by ingestion and should be avoid all body tissue contact.
But technically. both of them could be helpful depend on your samples. sometimes 2% concentration is too much and it is staining all tissue even in seconds. So optimizing of concentration and duration is necessary.
Best wishes
Dear colleagues, I am about to prepare 2% Acetocarmine and ferric acetate reagent for my first attempts on karyology. Let me know if this protocol works fine:
1: Add 1g of iron fillings or rust to 50mls 45% Glacial acetic acid solution
2: Filter to remove debris
3: add 1g Carmine powder stain and boil for 30-60mins (reflux heating to avoid evaporation)
4: Filter again, close in reagent bottle, store at 5Celcius
I also have orcein acetic solution (la cour) from Timstar Lab (UN1760 / OR4430). Do you know if this is ready to use for karyology work or if it should be pretreated/modified ?
This is also useful:
https://home.cc.umanitoba.ca/~frist/PLNT3140/lab/CytoLabManual_v
Should Acetocarmine dye be fresh prepare for use or the stored one can also use? If it can be stored for later use, how should it (Acetocarmine dye) be stored for future use?
George do you have the book chapter if so let have a copy!
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