Characterisation by staining: Capsular staining


Sometimes it is convenient to determine overall bacterial morphology without the use of harsh staining or heat-fixing techniques that change the shape of cells. This might be the case when the bacterium does not stain well (e.g., some of the spirochetes) or when it is desirable to confirm observations made on the shape and size of bacteria observed in either a wet-mount or hanging drop slide. Negative staining is also good for viewing capsules.

Negative, indirect, or background staining is achieved by mixing bacteria with an acidic stain such as nigrosin, India ink, or eosin, and then spreading out the mixture on a slide to form a film. The above stains will not penetrate and stain the bacterial cells due to repulsion between the negative charge of the stains and the negatively charged bacterial wall. Instead, these stains either produce a deposit around the bacteria or produce a dark background so that the bacteria appear as unstained cells with a clear area around them.


  1. Bacterial culture (Klebseilla)
  2. Clean microscope slides
  3. Inoculating loop
  4. Immersion oil
  5. Wax pencil
  6. Bunsen burner
  7. India ink
  8. Bright field microscope


  1. With a wax pencil, label the left-hand corner of three glass slides with the names of the respective bacteria.
  2. Use an inoculating loop to apply a small amount of bacteria to one end of a clean microscope slide.
  3. Add 1 to 2 loops of India ink solution to the bacteria and mix thoroughly.
  4. Spread the mixture over the slide using a second slide. The second slide should be held at a 45° angle so that the bacteria-nigrosin solution spreads across its edge. The slide is then pushed across the surface of the first slide in order to form a smear that is thick at one end and thin at the other. This is known as a thin smear.
  5. Allow the smear to air dry. Do not heat-fix!
  6. With the low-power objective, find an area of the smear that is of the optimal thickness for observation.
  7. Use the oil immersion lens to observe.
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