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Gram Stains Update 2017



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Gram Stains Update 2017

  1. 1. Gram stains 2017 UPDATE
  2. 2. Gram Stain Assess Gram reaction and morphology  Gram negative (red) /Gram positive (blue)  Crystal violet – primary stain for 1 min/rinse  Gram’s iodine – mordant for 1 min/rinse  Acetone/Alcohol – decolorizer for 5 secs  Safranin – counterstain for 1 min/rinse  How to judge quality of a gram stain- are the polys blue?  If so, slide is under-decolorized.  Are the polys washed out?  If so, the slide is over-decolorized  Poor quality stains must be repeated
  3. 3. What can go wrong? If the stain is all blue at the end of the staining process You did not decolorize the slide for enough time. If the stain is all red and washed out appearing at the end of the staining process You over decolorized with the acetone.
  4. 4. Cellular Material – Gram stains are not able to differentiate many cell types. Best for PMNs, Mononuclear cells, and epithelial cells.
  5. 5. Possible Artifacts in Gram stains Safranin crystals Crystal Violet crystals When reading a Gram stain, you need to check for the uniformity of the cell borders of the suspected organisms, there needs to be repeating/consistent size and shape that is suggestive of a micro-organism.
  6. 6. Gram Positive Bacteria
  7. 7. Staphylococcus Gram positive cocci in clusters Cells are very round and deeply stained, 0,5 – 1 µm in diameter
  8. 8. Streptococcus Beta hemolytic  Gram positive cocci in chains (long chains)  0.5 wide by 2.0 µm in length (oval)
  9. 9. Streptococcus pneumoniae  Gram positive cocci in pairs – 0.5 – 1.5 µm in size  Cocci are lancet or bullet shaped with flattened sides together  Sometimes can visualize a capsule on gram stain – clearing around the stained organism
  10. 10. Viridans Streptococcus  Gram positive cocci in short chains and pairs  Chains are not as long as Beta hemolytic Streptococcus (0.5 µm – 1.5 µm)
  11. 11. Enterococcus species  Gram positive cocci in short chains and pairs  Difficult to impossible to differentiate from viridans Streptococcus
  12. 12. Yeast – Candida species  Large Gram positive globules (@ 8 um) with buds  Sometimes pseudo-hyphae can be seen
  13. 13. Cryptococcus neoformans/gatti Due to the capsule of C. neoformans/gatti the stain appears somewhat like a blob  An India ink prep can be used to visualize the capsule
  14. 14. Mycelial Fungi Gram stain Fungi stain +/- with the Gram stain
  15. 15. Bacillus species Gram positive rods – large , box car shaped, sometimes spores are visible (1.0 X 4.0 µm) Sometimes they over-decolorize and appear Gram negative Over-decolorized Spores
  16. 16. Listeria monocytogenes Gram positive short rods (0.5 X 1.5 µm)
  17. 17. Corynebacterium spp. (diphtheroids) gram positive rods in Chinese letter forms and palisades (0.5 X 4 µm) palisades
  18. 18. Propionibacterium acnes – small branching Gram positive rod
  19. 19. Nocardia species Delicate Gram positive branching rods Nocardia are red when stained with modified acid fast stains
  20. 20. Actinomyces species Antler like branching Sulfur Granule Formation
  21. 21. Gardnerella vaginalis Gram variable rods
  22. 22. Lactobacillus species Gram positive long rods that appear sometimes in chains
  23. 23. Clostridium species Gram positive rods with spores the spores can be located throughout the organism
  24. 24. Mycobacterium species do not stain well with gram stain due to the lipid in the cell wall – stain weakly and beaded Kinyoun AFB stain
  25. 25. Gram negative bacteria
  26. 26. Enteric Gram negative rods (Enterobacteriaceae) Plump rectangular in shape (1 x 3 µm)
  27. 27. Pseudomonas aeruginosa thinner than the enteric rods 0.5 X 3µm
  28. 28. Haemophilus influenza pleomorphic thin faintly staining Gram negative rods
  29. 29. Vibrio species curved or comma shaped gram negative rods
  30. 30. Fusobacterium species – F. nucleatum F. necrophorum
  31. 31. Campylobacter sea gull shaped faintly staining gram negative rods
  32. 32. Capnocytophyga spp – pleomorphic Gram negative bacilli, variable in size and shape
  33. 33. Bacteroiodes spp Pleomorphic Gram negative bacilli, variable in length
  34. 34. Yersinia pestis Safety pin bipolar staining ---
  35. 35. (0.5 X 1.5 µm)
  36. 36. Neisseria species (meningitidis and gonorrheae) Gram Negative Diplococci Kidney bean shaped in pairs