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Reverse (India Ink) Stain
This is somewhat a different staining technique than other staining procedures in that the sample is not air dried but is rather "live" when India ink is introduced with the sample while viewing under the microscope. 

 
 Reverse (India Ink) Stain Procedure
Preparation:
 
     Solution 1: India Ink (aqueous suspension of carbon black particles)
 
 
Procedure:
 
  1. Mix one drop of India Ink and one drop of activated sludge sample on a microscope slide.  Depending on the ink used, the sample volume may need to be adjusted
  2. Place the cover slip on top of the mixture and apply slight force to squeeze out excess liquid. Use paper towel to soak up liquid
  3. In "normal" activated sludge, the India ink particles penetrate the flocs almost completely, at most leaving a clear center
  4. In activated sludge containing large amounts of exocellular polymeric substance (EPS), there will be large clear areas which contain low density of cells.

 

 

 Video of Reverse (India Ink) Stain