Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Acridine orange method for DNA and RNA
Can acridine orange be used to stain DNA and RNA in different fluorescent colors in sections as well as in smears of cells?
In the late sixties, early seventies, I used to use the original method (Bertalanffy F.D. A new method for cytological diagnosis of pulmonary cancer. Ann. New York Acad. Sci. 84: 225-238) for screening cytology slides fixed in alcohol for malignant cells, and I thought it worked quite well, as did my pathologist at the time. The DNA of the nucleus fluoresces brilliant green, and RNA in the cytoplasm of malignant cells is brilliant orange. However, I have never met a
cytotechnologist who liked the method, so, when I was forced to hire one because of work load, she quickly relegated this technique to the garbage bin of history.
I don't know of anyone who is currently using the technique. However, as we found it very useful at the time, I worked out a method for using it on paraffin sections, that gives very similar results to the alcohol fixed smears.
1. Bring paraffin sections to water in usual manner.
2. Stain sections in acridine orange stain for 30 minutes.
3. Rinse sections briefly in 0.5% acetic acid in 100%
4. Rinse sections in two additional changes of 100% alcohol.
5. Rinse sections in two changes of xylene.
6. Mount sections in a non-fluorescent resinous medium.
Results: DNA brilliant green. RNA brilliant orange.
Most gram positive microorganisms brilliant orange. Most
gram negative microorganisms (including helicobacter) green to
Acridine orange stain
Acridine orange (C.I. 46005)
(Note, some batches of the acridine orange dye
work better than others.)
Kelowna General Hospital
Kelowna B.C. Canada