Brown-Hopps Method For Gram Positive And Gram Negative Bacteria

EMS26106

Components

Crystal Violet Staining Solution, 1% Aqueous

Gram's Iodine Solution

Basic Fuchsin Staining Solution, 0.5% Aqueous

Gallego’S Differentiating Solution

Cellosolve

Tartrazine, 1.5% Aqueous

Fixation

10% Buffered Neutral Formalin

Section

Paraffin, @ 6µm

Staining Procedure

1. Deparaffinise and hydrate to distilled water.
2. Place slides on staining rack and pour on Crystal Violet Solution, 1%, Aqueous for two minutes. Rinse in distilled water.
3. Mordant in Gram's Iodine for five minutes. Rinse in distilled water.
4. Differentiate in Cellosolve, until blue color no longer streams away from the section (approximately 5-10 seconds). (See note)
5. Quickly rinse in distilled water. (See note)
6. Stain in Basic Fuchsin Solution, 0.5%, Aqueous for five minutes. Rinse in distilled water.
7. Gallego's Solution , for five minutes. (Differentiates and "fixes" the Basic Fuchsin).
8. Rinse thoroughly in distilled water and blot lightly to remove excess water (not to dryness).
9. Stain for three seconds in Tartrazine Solution . Immediately blot away excess, but not to dryness).
10. Cellosolve three changes, for six quick dips in each. (See note)
11. Xylene, three changes, for 10 dips each. Slides may remain in Xylene until ready for mounting.
12. Mount with Permount.

Results

 Notes:

• Preferably, all stains and solutions are applied to the slide while it is in a horizontal position, except for steps 4, 10 and 11, where the slide should be dipped into the solutions contained in coplin jars.
• Steps 4, 5, 9 and 10 are critical with regard to timing.
• This procedure does not work well on Bouin's fixed tissues.

References

AFIP Manual of Histologic Staining Methods; ed. G. Luna, McGraw-Hill Publications, 3 rd . ed.; New York, c. 1968, p.224.

Brown, R. C. and Hopps, H.C., Geographic Pathology Division Armed Forces Institute Of Pathology, Washington, D.C. 20305