Rapid Methods of Staining Bacterial Spores at Room Temperature.

Lechtman, M. D. (University of Southern California, Los Angeles), J. W. Bartholomew, A. Phillips, and M. Russo. Rapid methods of staining bacterial spores at room temperature. J. Bacteriol. 89:848-854. 1965.-Spores of Bacillus subtilis var. niger were stained in 2 min at room temperature, after suitable pretreatment, with a dye reagent composed of 2% crystal violet in 1% phenol and 26% ethanol. Pretreatments included heat fixation to 260 C, mechanical rupture, and hydrolysis at room temperature in 44 n H(3)PO(4) for 5 min, 33.4 n H(3)PO(4) for 10 min, 12 n HCl for 5 sec, 6 n HCl for 2 min, 12 n HNO(3) for 5 sec, and 6 n HNO(3) for 60 sec. Acid hydrolysis at 60 C enabled the lowering of both acid concentration and time: 33.4 n H(3)PO(4) for 15 sec, 25.9 n H(3)PO(4) for 60 sec, 2 n HCl for 30 sec, 1 n HCl for 30 sec, 2 n HNO(3) for 15 sec, and 1 n HNO(3) for 30 sec. After acid treatment, 1 n NaOH was used as a neutralization agent. The cytological manifestations of these pretreatments, examined in an electron microscope after replication, showed definite degradation of spore coats, which probably explains the increase in dye permeability. The pretreatments were evaluated for use in a differential staining procedure for spores and vegetative cells. They were found to be too drastic in that they resulted in replacement of the primary dye by the 0.25% safranine counter stain in both vegetative cells and endospores. Less drastic pretreatments, such as 6 n HNO(3) for 10 sec at room temperature, gave good differential stains, but failed to stain some free spores. The staining techniques above were evaluated with six species of Bacillus and were found to apply to all.