Products

Safranin O Stain 0.25%, Aqueous

Newcomer Supply Beaker

(use: Counterstain in Gram methods for microbiology smears.)

Product Options:

Part # 1360A 500 ml $26.80

SOLUTION:

500 ml
Safranin O Stain 0.25%, Aqueous Part 1360A

 

Additionally Needed:

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.

 

APPLICATION:

Newcomer Supply Safranin O Stain 0.25%, Aqueous is 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.

 

METHOD:

Technique:  Flat staining rack method.
Solutions:  All solutions are manufactured by Newcomer Supply, Inc.

 

STAINING PROCEDURE:    

    1. Prepare a well-made smear with a focus on uniform distribution of material.
    2. Allow slides to thoroughly air-dry before staining.
    3. Fix slides according to laboratory protocol.
        1. See Procedure Note #1.
    4. Place slides on flat staining rack suspended over sink.
        1. See Procedure Note #2.
    5. Flood fixed smears with Crystal Violet Stain 1%, Aqueous, Brown-Hopps (Part 1041) for 45-60 seconds.
    6. Drain off Crystal Violet Stain and rinse well in distilled water.
    7. Mordant smears in Iodine, Gram, Aqueous (Part 1140) for 45-60 seconds.
    8. Rinse well in running tap water to remove excess iodine.
    9. Blot one slide at a time and individually decolorize in Acetone, ACS (Part 10014) until color stops running; 5-10 seconds.
        1. See Procedure Note #3.
    10. Quickly rinse in distilled water to remove excess acetone.
    11. Counterstain in Safranin O Stain 0.25%, Aqueous for 30-60 seconds.
        1. See Procedure Note #4.
    12. Wash well in distilled water.
    13. Allow slides to air-dry.
    14. Examine microscopically with oil immersion.

 

RESULTS:

Gram-positive bacteria Blue to blue/black
Gram-negative bacteria Pink to red
Background Yellow

 

PROCEDURE NOTES:

    1. Gentle heat or methanol are accepted methods of smear fixation.
    2. Do not allow smears to dry out at any point during procedure.
    3. To decolorize slower; dip slides in Alcohol, Ethyl Denatured, 95% (Part 10842) in Coplin jar for 10-60 seconds or until color stops running.
    4. For enhanced counterstaining;
        1. Safranin O Stain 0.25%, Aqueous 10 ml
        2. 95% Ethyl Alcohol (Part 10842)   1 ml
        3. Combine; mix well and stain for 30-60 seconds.
        4. Rinse well in distilled water.

       

REFERENCES:

    1. Kidd, Larry. “Histology vs. Microbiology – The Gram Stain the Easy Way.” The Journal of Histotechnology 7.2 (1984): 85-86.
    2. Lillie, R. D., and Harold Fullmer. Histopathologic Technic and Practical Histochemistry. 4th ed. New York: McGraw-Hill, 1976. 726-727.
    3. McPherson, Richard and Matthew Pincus. Henry’s Clinical Diagnosis and Management by Laboratory Methods. 22nd ed. Philadelphia: Elsevier Saunders, 2011. 1080.
    4. Sheehan, Dezna C. and Barbara B. Hrapchak. Theory and Practice of Histotechnology. 2nd ed. St. Louis: Mosby, 1980. 235.
    5. Modifications developed by Newcomer Supply Laboratory.