Revised Gram Staining


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Revised Gram Staining

  2. 2. GRAM STAINING <ul><li>Gram's Stain is a widely used method of staining bacteria as an aid to their identification. It was originally devised by Hans Christian Joachim Gram, a Danish doctor. </li></ul>
  3. 3. GRAM STAINING <ul><li>Gram's stain differentiates between two major cell wall types. </li></ul>
  4. 4. GRAM STAINING <ul><li>Bacterial species with walls containing small amounts of peptidoglycan and, characteristically, lipopolysaccharide, are Gram-negative </li></ul>
  5. 5. GRAM STAINING <ul><li>Bacteria with walls containing relatively large amounts of peptidoglycan and no lipopolysaccharide are Gram-positive. </li></ul>
  6. 6. GRAM STAINING <ul><li>It's a mystery </li></ul><ul><li>Although it may seem strange, the reason why bacteria with these two major types of bacteria cell walls react differently with Gram's stain appears to be unconnected with the wall structure itself. The exact mechanism of the staining reaction is not fully understood, however, this does not detract from its usefullness. </li></ul>
  7. 7. GRAM STAINING <ul><li>In may Gram-negative species the lipopolysaccharide acts as an endotoxin. </li></ul><ul><li>These cause inflamation in the human body. </li></ul>
  8. 8. GRAM STAINING <ul><li>Many antibiotics penetrate Gram positive walls much more easily than Gram-negative. </li></ul><ul><li>Gram-negative infections are much more difficult to treat with antibiotics. </li></ul>
  9. 9. GRAM STAINING <ul><li>Gram-negative bacteria are much less prone to the action of lysozyme , an anti-bacterial enzyme secreted in tears, sweat and saliva. </li></ul>
  10. 10. 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>
  11. 11. 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>
  12. 12. 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>
  13. 13. 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>
  14. 14. The Gram staining method <ul><li>5. The Gentian Violet is gently washed off the slide with running water </li></ul>
  15. 15. 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>
  16. 16. 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>
  17. 17. 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>
  18. 18. 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>
  19. 19. 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>
  20. 20. The Gram staining method <ul><li>Typical Gram-positive bacteria </li></ul><ul><li>staphylococci such as Staphylococcus epidermidis and Staphylococcus aureus which is a common cause of boils </li></ul><ul><li>streptococci such as the many species of oral streptococci, Streptococcus pyogenes which causes many a sore throat. </li></ul>
  21. 21. The Gram staining method <ul><li>Typical Gram-negative bacteria </li></ul><ul><li>the bacilli that cause </li></ul><ul><li>1 whooping cough, Bordetella pertussis </li></ul><ul><li>2. typhoid, Salmonella typhi </li></ul><ul><li>3.the normally benign, ubiquitous, gut-dwelling Escherichia coli </li></ul>
  22. 22. Gram Staining – Gram +’ve
  23. 23. Gram Staining – Gram -’ve