Additional observations on the preparation of R banded human chromosomes with acridine orange

A number of technical factors which affect acridine orange R banding (RFA banding) were studied. These variables included age of slide, timing of fixation, details of incubation mounting and the use of sequential technics. Optimal RFA banding was obtained between 15 and 20 days but good or very good preparations were obtained between 7 days and 2 months. Improved results were obtained in slides that were 3-4 months old by refixing the slides in ethanol acetic acid. Intermittent movement of slides during incubation in buffer as well as the details of mounting and removal of cover slips were found to be important. The best sequential banding was obtained with the sequence of Q to R but good results were obtained with the sequence G to R using ASG banding. Satisfactory results with the sequence R to C were not obtained. With careful attention to these variables good RFA binding can be obtained over a period of several months.