Gram Staining Instructions

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Gram Staining Instructions

  1. 1. The Gram staining method <ul><li>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 . </li></ul>
  2. 2. The Gram staining method <ul><li>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. </li></ul>
  3. 3. The Gram staining method <ul><li>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. </li></ul>
  4. 4. The Gram staining method <ul><li>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. </li></ul>
  5. 5. The Gram staining method <ul><li>5. The Gentian Violet is gently washed off the slide with running water </li></ul>
  6. 6. The Gram staining method <ul><li>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. </li></ul>
  7. 7. The Gram staining method <ul><li>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. </li></ul><ul><li>Do not overdo this stage! </li></ul>
  8. 8. The Gram staining method <ul><li>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. </li></ul>
  9. 9. The Gram staining method <ul><li>9. The counter stain is left on the smear for about 30-60 seconds and then gently rinsed away with running water. </li></ul>
  10. 10. The Gram staining method <ul><li>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. </li></ul>

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