The invention of differential staining.

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Differential staining is a staining process that uses more than one chemical stain as using multiple stains can better differentiate between different microorganisms or structures/cellular components of a single organsim. Gram stain and Ziel-Neelson stain are well-known examples of differential stains. White blood cell differential is an important medical laboratory test that uses differential stains[1].


The German physician and microbiologist Heinrich Hermann Robert Koch invented[2] the process of differential staining in 1882 while trying to identify Mycobacterium tuberculosis bacilli in the tissues of animals infected with tuberculosis. Here is the method he used in creating the differential stain:

  • He first prepared microscope slides with the tissues that were to be examined.
  • He then mixed 200 millilitres of distilled water with 1 millilitre of a concentrated alcoholic methylene blue solution and placed the slides into the solution.
  • He then shook the solution with the slides and added a solution of 10% potassium hydroxide to it.
  • Then, he kept the solution heated at 40°C for 1 hour in a water bath (with the slides still inside).
  • He then took the slides out and poured a concentrated aqueous solution of Bismarck brown Y (a dye) onto them. They were then rinsed with distilled water after 2 minutes.

Upon observing the slides, cell organelles (namely the nuclei) appeared as pale brown and the Mycobacterium tuberculosis bacilli as blue. Here is a photomicrograph[3] of a differential stain (Ziehl-Neelsen stain with the bacilli in red):

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Here is a photograph[4] of Robert Koch:

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  1. staining
  2., pages 428-429.
  3. 4-afb-stain-acid-fast-bacilli-stain-procedure-and-interpretation/