PMID:14273671

Citation	LECHTMAN, MD , BARTHOLOMEW, JW , PHILLIPS, A and RUSSO, M (1965) RAPID METHODS OF STAINING BACTERIAL SPORES AT ROOM TEMPERATURE. J. Bacteriol. 89:848-54
Abstract	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.
Links	PubMed PMC277547
Keywords	Acids; Bacillus subtilis; Hot Temperature; Hydrochloric Acid; Indicators and Reagents; Microscopy, Electron; Nitrates; Phosphates; Research; Spores; Staining and Labeling; Sulfates; Temperature
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Main Points of the Paper

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Materials and Methods Used

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• Spores tested
  • Bacillus subtilus var. niger (main)
  • B. megaterium
  • B. cereus
  • B. coagulans
  • B. polymyxa
  • B. stearothermophilus

• Heat treatment of spores
  • spore smear on a glass slide
  • hot plate adjusted to various temperatures: 215˚, 225˚, 240˚, 260˚C
  • slide heated to various temperatures for various amounts of time: 10s, 1,2, 4, and 6 minutes
  • slide air-cooled to room-temperature

• Mechanical pressure treatment of spores
  • spore smear on a glass slide
  • 10 0.2mm glass beads placed on smear
  • No. 1 coverslip or a glass slide was placed over the beads
  • Thumb pressure (estimated at 8 tons/sq. inch) was applied and coverslip was used roll the beads

• Acid hydrolysis treatment of spores
  • tested various acids (consistent 12N concentration) in Coplin dishes at various temperatures and times
  • expose to acid for various times and wash
  • neutralize with 1N NaOH for 30s and wash

• Spore Staining
  • spore smear on a glass slide
  • stained for 2 minutes at room temperature with 2% crystal violet in 1% phenol and 26% ethyl alcohol

Phenotype Annotations

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Phenotype of	Taxon Information	Genotype Information (if known)	Condition Information	OMP ID	OMP Term Name	ECO ID	ECO Term Name	Notes	Status
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References

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