Safranin O Stain 0.25%, Aqueous
(use: Counterstain in Gram methods for microbiology smears.)
|Safranin O Stain 0.25%, Aqueous||Part 1360A|
|Crystal Violet Stain 1%, Aqueous, Brown-Hopps||Part 1041|
|Iodine, Gram, Aqueous||Part 1140|
|Acetone, ACS||Part 10014|
For storage requirements and expiration date refer to individual bottle labels.
Newcomer Supply Safranin O Stain 0.25%, Aqueous provides the preferred counterstain for Gram staining of microbiology smears. The Gram Stain technique, is used for differential staining of gram-positive and gram-negative bacteria in both smears and tissue sections.
Technique: Flat staining rack method.
Solutions: All solutions are manufactured by Newcomer Supply, Inc.
- Prepare within an accepted time frame, a well-made smear(s) per laboratory protocol, with a focus on uniform distribution of material.
- Allow slides to thoroughly air-dry before staining.
- Fix slides according to laboratory protocol.
- See Procedure Note #1.
- Place slides on flat staining rack suspended over sink.
- See Procedure Note #2.
- Flood fixed smears with Crystal Violet Stain 1%, Aqueous, Brown-Hopps (Part 1041) for 45-60 seconds.
- Drain off Crystal Violet Stain and rinse well in distilled water.
- Mordant smears in Iodine, Gram, Aqueous (Part 1140) for 45-60 seconds.
- Rinse well in running tap water to remove excess iodine.
- Blot one slide at a time and individually decolorize in Acetone (Part 10014) until color stops running off the smear; 5-10 seconds.
- See Procedure Note #3.
- Quickly rinse in distilled water to remove excess acetone.
- Counterstain in Safranin O Stain 0.25%, Aqueous for 30-60 seconds.
- See Procedure Note #4.
- Wash well in distilled water.
- Allow slides to air-dry.
- Examine microscopically with oil immersion.
|Gram-positive bacteria||Blue to blue/black|
|Gram-negative bacteria||Pink to red|
- Gentle heat or methanol are both accepted methods of smear fixation.
- Do not allow smears to dry out at any point during staining procedure.
- To decolorize slower, dip slides in Alcohol, Ethyl Denatured, 95% (Part 10842) in Coplin jar for 10-60 seconds or until color stops running off the smear.
- For enhanced counterstaining;
- Safranin O Stain 0.25%, Aqueous 10 ml
- 95% Ethyl Alcohol (Part 10842) 1 ml
- Combine; mix well and stain for 30-60 seconds.
- Kidd, Larry. “Histology vs. Microbiology – The Gram Stain the Easy Way.” The Journal of Histotechnology 7.2 (1984): 85-86.
- Lillie, R. D., and Harold Fullmer. Histopathologic Technic and Practical Histochemistry. 4th ed. New York: McGraw-Hill, 1976. 726-727.
- McPherson, Richard and Matthew Pincus. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia: Elsevier Saunders, 2011. 1080.
- Sheehan, Dezna C., and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 235.
- Modifications developed by Newcomer Supply Laboratory.