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Gram Stain Update 2019

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Gram Stain Update 2019

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Gram Stain Update 2019

  1. 1. Gram stains 2019 UPDATE
  2. 2. Gram Stain Assess Gram reaction and morphology of organism  Gram negative (red) /Gram positive (blue)  Crystal violet – primary stain  Gram’s iodine – mordant  Acetone/Alcohol – decolorizer  Safranin – counterstain  How to judge quality of gram staining – is everything blue?  If so, slide is under-decolorized.  Are the polys and other cells washed out in appearance?  If so, the slide is over-decolorized  Poor quality stains must be repeated
  3. 3. Presence of Peptidoglycan in the cell wall (Gram positive cell wall) traps the crystal violet making for Gram positive staining (purple)
  4. 4. Prepare thin smear of material onto the glass slide Heat fix prior to staining
  5. 5. Cellular Material – Gram stains are not able to differentiate many cell types. Best for PMNs, mononuclear cells, and epithelial cells. For other cell types, differential stains must be used.
  6. 6. 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 should be repeating/consistent size and shape of the cells that is suggestive of a micro-organism.
  7. 7. Gram Positive Bacteria
  8. 8. Staphylococcus Gram positive cocci in random clusters Cells are very round and deeply stained, 0.5 – 1 µm in diameter
  9. 9. Streptococcus / Beta hemolytic  Gram positive cocci in long chains  Cocci are 0.5 wide by 2.0 µm in length (oval)
  10. 10. Streptococcus pneumoniae  Gram positive cocci in pairs – 0.5 – 1.5 µm in size  Cocci are lancet or bullet shaped with flattened ends together  Sometimes can visualize a capsule on gram stain – clearing around the stained organism
  11. 11. 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)
  12. 12. Enterococcus species  Gram positive cocci in short chains and pairs  Difficult to impossible to differentiate from viridans Streptococcus
  13. 13. Bacillus species Gram positive rods – large (1.0 X 4.0 µm) Box car shaped, sometimes spores are visible Can over-decolorized and appear Gram negative Over-decolorized Spores
  14. 14. Listeria monocytogenes Gram positive short rods (0.5 X 1.5 µm)
  15. 15. Corynebacterium spp. (diphtheroids) Gram positive rods in Chinese letter forms and palisades (0.5 X 4 µm) palisades
  16. 16. Propionibacterium acnes Small branching Gram positive rod
  17. 17. Nocardia species Delicate Gram positive branching rods Nocardia are red stained with modified acid fast stains
  18. 18. Actinomyces species Gram positive rods Antler like branching Sulfur Granule Formation
  19. 19. Gardnerella vaginalis Gram variable (not quite red or blue) rods
  20. 20. Lactobacillus species Gram positive long rods that appear sometimes in chains
  21. 21. Clostridium species Gram positive rods with spores, Spores will not be stained with the Gram stain Spores can be located throughout the organism
  22. 22. Mycobacterium species Do not stain well with Gram stain due to the lipid in the cell wall – stain weakly and beaded
  23. 23. Gram negative bacteria
  24. 24. Enteric Gram negative rods (Enterobacteriaceae) Plump rectangular in shape(1 x 3 µm)
  25. 25. Pseudomonas aeruginosa thinner and a bit longer than enteric Gram negative rods 0.5 X 3µm
  26. 26. Haemophilus influenza pleomorphic thin faintly staining Gram negative rods
  27. 27. Vibrio species curved or comma shaped Gram negative rods
  28. 28. Fusobacterium species – faintly staining unusually shaped Gram negative rods F. nucleatum F. necrophorum Spindle shaped, pointed Chaining pleomorphic GNR ends
  29. 29. Campylobacter sea gull shaped faintly staining gram negative rods
  30. 30. Capnocytophyga spp – pleomorphic Gram negative bacilli, variable in size and shape
  31. 31. Bacteroiodes spp Pleomorphic Gram negative bacilli, variable in length
  32. 32. Yersinia pestis Safety pin / bipolar staining ---
  33. 33. (0.5 X 1.5 µm)
  34. 34. Neisseria species (meningitidis and gonorrhoeae) Gram negative diplococci Kidney bean shaped in pairs
  35. 35. Yeast – Candida species  Large Gram positive oval globules (@ 8 um) with buds  Sometimes pseudo-hyphae can be seen
  36. 36. Cryptococcus neoformans/gatti Due to the polysaccharide capsule of C. neoformans/gatti the yeast appears somewhat like a round blob  An India ink prep can be used to visualize the polysaccharide capsule
  37. 37. Mycelial Fungi on Gram stain Fungi stain +/- with Gram stain

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