Gram Churukian–Schenk
for Gram Positive & Negative Bacteria

Solutions

Stock basic fuchsin
Basic fuchsin 0.5 g
Distilled water 100 mL
Solution A
Crystal violet 10% in ethanol 2 mL
Ammonium oxalate 1% aqueous 98 mL
Solution B
Iodine 2 g
Potassium iodide 4 g
Distilled water 400 mL
Solution C
Absolute ethanol 1 volume
Acetone 1 volume
Solution D
Stock basic fuchsin 5 mL
Distilled water 45 mL
Solution E
Picric acid 0.1 g
Acetone 100 mL
Solution F
Acetone 1 Volume
Xylene 1 volume

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Method

  1. Bring sections to water via xylene and ethanol.
  2. Place in solution A for 2 minutes.
  3. Rinse with tap water.
  4. Place in solution B for 1 minute.
  5. Rinse well with tap water.
  6. Blot the slide, but not the tissue.
  7. Decolorise with solution C until no more blue floods off.
  8. Wet section with solution D then apply for 1 minute.
  9. Rinse with distilled water.
  10. Blot the slide, but not the tissue.
  11. Place in acetone for 3 seconds.
  12. Differentiate in solution E for 10 seconds.
  13. Quickly dip a few times in solution F.
  14. Clear with xylene and mount with a resinous medium.

Expected results

Notes

  1. Picric acid should be handled with care. Solution E may be made by taking 12 mL of a saturated solution of picric acid in ethanol and diluting to 1 litre with acetone.
  2. Basic fuchsin homologues with CI numbers of 42500 (pararosanilin) or 42510 (rosanilin) were specified. It was also noted that new fuchsin (CI 42520) was satisfactory, but not recommended because it was not certified by the Biological Stain Commission.
  3. The authors note that sections must not be allowed to dry out after being stained with basic fuchsin. Doing so makes it difficult or impossible to properly differentiate the red counterstain.

 

Reference
Churukian, C. J. & Schenk, E. A. (1982)
A method for demonstrating Gram-positive and Gram-negative bacteria.
Journal of Histotechnology, v.5, No.3, p.127

 


 

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