Hoechst 33258/ bisbenzimide stain for PPLO

Pigmented cells give false positives with some PPLO tests, owing to nonspecific binding of nucleic acids etc by melanin. However this test is ok for melanocytes.

A simple stain. Available as bisbenzimide from Sigma.

Cells are plated on dishes or coverslips (not flasks).

Fix after at least 2-3 days' growth but preferably not yet confluent. PPLO can be washed away at subculture, so more sensitive after a few days.

Fix/ permeabilize with fresh methanol-acetic acid, 3:1 and rinse 2 x in ethanol. Air-dry. (Can store at this stage).

Bisbenzimide stock solution can be made at 20-500 µg/ml and stored in frozen aliquots if you snap-freeze in liquid nitrogen. Don't freeze slowly, or it precipitates and will not redissolve. Always handle dye in subdued light.

Use-solution of stain is at 0.2 µg/ml in PBS. No higher, otherwise mitochondria stain too. Dilute solution can be kept in refrigerator, but for no more than a few days, wrapped in foil.

Cover cells with stain solution; leave about 15 min (not critical).

Remove solution and rinse briefly 2x in distilled water. To view, either use water-immersion objective (in water), or mount with coverslip in UV-transparent mountant. View with fluorescence microscope, epi-illumination, and filters that transmit blue light, at high magnification. 20x objective or more.

This is a DNA stain. Nuclei should be bright blue.

Negative : bright, sharp, blue nuclei and cytoplasm totally invisible.

Positive : any kind of blue speckling in cytoplasm: some cells or all cells.