The Gram staining method

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The Gram staining method

• 1. A small sample of a bacterial culture is
  removed from a culture. In this example it is
  being taken from a broth culture of the pure
  microbe but it could be removed from a culture on
  solid medium .

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The Gram staining method

• 2. The bacterial suspension is smeared onto a
  clean glass slide. If the bacteria have been
  removed from a culture on solid media it will
  have to be mixed with a drop of distilled water.

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The Gram staining method

• 3. The bacterial smear is then dried slowly at
  first and then, when dry, heated for a few
  seconds to the point when the glass slide is too
  hot to handle. This fixes ie kills the bacteria
  making the slide safe to handle. Care must be
  taken not to overheat.

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The Gram staining method

• 4. Once cool, the slide is transferred to a
  support over a sink and flooded with a stain
  called Gentian Violet. The stain is left on the
  slide for about 1 minute. This stains all the
  bacteria on the slide a dark purple colour.

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The Gram staining method

• 5. The Gentian Violet is gently washed off the
  slide with running water

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The Gram staining method

• 6. The bacterial smear is then treated with
  Gram's iodine. This iodine solution reacts with
  the Gentian Violet turning it a very dark shade
  of blue. It also causes it to be retained by
  certain types of bacteria in a way which is not
  really understood.

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The Gram staining method

• 7. After about 30 seconds the slide is gently
  rinsed with ethyl alcohol (just let it flow over
  the slide) which causes the dye-iodine complex to
  be washed out of some bacteria but not others.
  This is called decolourisation.
• Do not overdo this stage!

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The Gram staining method

• 8. We now treat the slide a compound which stains
  the Gram-negative cells a colour which contrasts
  markedly with the blue-black colour of the
  Gram-positive cells. The stain common used for
  this is fuchsin which is red. This is called the
  counterstain. Bacteria in the smear which are
  Gram-positive are unaffected by the counterstain.

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The Gram staining method

• 9. The counter stain is left on the smear for
  about 30-60 seconds and then gently rinsed away
  with running water.

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The Gram staining method

• 10. After the counterstain has been rinsed off,
  the slide is placed between some absorbent paper
  and the excess water gently blotted off. Care
  must be taken not to rub the slide with the
  blotting paper because this would remove the
  adhering bacteria.