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Gram stains 2021
Margie A. Morgan, PhD. D(ABMM)
Gram Stain
Assess for 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
Prepare a thin smear of material onto the glass slide.
Air dry and then heat fix specimen on slide prior to staining.
Stain approximately one minute per step and rinse with sterile
water between each step. Air dry before scope reading.
Presence of large amount of peptidoglycan in the Gram positive cell wall,
traps the crystal violet making for Gram positive staining with the primary
stain crystal violet and the organism appearing blue in color.
Cellular Material – Gram stains are not able to differentiate
many cell types. Best for identifying polymorphonuclear leukocytes
(PMNs), mononuclear cells, and epithelial cells. For other cell types,
differential stains must be used, such as Wright-Giemsa)
Artifacts in Gram stains
Safranin
crystals
Crystal Violet crystals
When reading a Gram stain, check for the uniformity of the cell borders
of suspected organisms, there should be repeating/consistent size and
shape of the cells that is supportive of a micro-organism. Artifacts such
as stain crystals are usually not consistent in shape or size.
The most common
Gram Positive Bacteria
Staphylococcus species
Gram positive cocci in random clusters
Cells are very round and deeply stained,
0.5 – 1 µm in diameter
Streptococcus / Beta hemolytic
 Gram positive cocci in chains of variable length
 Cocci are 0.5 wide by 2.0 µm in length (oval)
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 – which appears as
clearing around the stained organism
Viridans Streptococcus
 Gram positive cocci in short chains and pairs
 Chains are usually not as long as the Beta hemolytic
Streptococcus, size is 0.5 µm – 1.5 µm.
Enterococcus species
 Gram positive cocci in short chains and pairs
 Can be difficult to differentiate from viridans Streptococcus, size is
also size is 0.5 µm – 1.5 µm.
Bacillus species
Gram positive rods – large (1.0 X 4.0 µm)
Box car shaped, sometimes spores are visible. Spores will not
stain with gentian violet.
Can be over-decolorized and appear Gram negative on Gram stain
Over-decolorized Spores
Listeria monocytogenes
Gram positive short rods (0.5 X 1.5 µm)
Do not appear in chains and they do not branch.
Corynebacterium species
Gram positive rods in that group in Chinese
letter forms and palisade cell arrangement (0.5 X 4 µm)
Palisade cell
arrangement
Cutibacterium (Propionibacterium)
acnes
Small pleomorphic Gram positive rods that branch.
Nocardia species
Delicate Gram positive
branching rods, sometimes
staining quite poorly and
appearing speckled.
Nocardia stain red
with modified acid fast stains
Actinomyces species
Gram positive rods with antler like branching, can be seen in sulfur
granules in tissue
Sulfur Granule
Formation
Gardnerella vaginalis
Gram variable (not quite red or blue) rods
Associated with “clue cells” in bacterial vaginosis
Lactobacillus species
Gram positive rods, thin, appear sometimes in short chains
Clostridium species
Gram positive rods, large, with spores sometimes visible.
Spores will not stain with the Gram stain and appear as colorless areas
in the cell.
Spores are located throughout the length of the rod-shaped organism
Mycobacterium species
Do not stain well with Gram stain due to lipid in the cell
wall, they stain beaded and barely visible.
Acid Fast stains are superior for the staining of
Mycobacteria
Gram negative bacteria
Enteric Gram negative rods
(Enterobacterales)
Plump rectangular in shape(1 x 3 µm)
Pseudomonas aeruginosa
thinner and a bit longer than enteric Gram negative rods
0.5 X 3µm
Haemophilus influenza
pleomorphic gram negative rods, thin and faintly staining
Vibrio species
curved or comma shaped Gram negative rods
Fusobacterium species – faintly staining
unusually shaped Gram negative rods
F. nucleatum F. necrophorum
Spindle shaped, with pointed Chaining pleomorphic rods
ends
Campylobacter
sea gull shaped, faintly staining Gram negative rods
Capnocytophyga spp –
pleomorphic Gram negative bacilli, variable in size and shape
Bacteroiodes spp
Pleomorphic Gram negative bacilli, variable in length
Yersinia pestis
Safety pin like with bipolar staining
---
(0.5 X 1.5 µm)
Neisseria species (meningitidis and gonorrhoeae)
Gram negative diplococci
Kidney bean shaped in pairs
Yeast – Candida species
 Large Gram positive oval globules (@ 8 um) that produce
a smaller attached cell known as a bud
 Sometimes pseudo-hyphae can be seen which are
extensions of the yeast cells into long hyphae like forms
Cryptococcus neoformans/gatti
Due to the polysaccharide
capsule of C. neoformans/gatti
the yeast appears somewhat
like a round blob on Gram
stain
 An India ink prep can be used
to visualize the polysaccharide
capsule
Mycelial Fungi
Variably staining of hyphae on Gram stain

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Gram Stains 2021

  • 1. Gram stains 2021 Margie A. Morgan, PhD. D(ABMM)
  • 2. Gram Stain Assess for 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. Prepare a thin smear of material onto the glass slide. Air dry and then heat fix specimen on slide prior to staining. Stain approximately one minute per step and rinse with sterile water between each step. Air dry before scope reading.
  • 4. Presence of large amount of peptidoglycan in the Gram positive cell wall, traps the crystal violet making for Gram positive staining with the primary stain crystal violet and the organism appearing blue in color.
  • 5. Cellular Material – Gram stains are not able to differentiate many cell types. Best for identifying polymorphonuclear leukocytes (PMNs), mononuclear cells, and epithelial cells. For other cell types, differential stains must be used, such as Wright-Giemsa)
  • 6. Artifacts in Gram stains Safranin crystals Crystal Violet crystals When reading a Gram stain, check for the uniformity of the cell borders of suspected organisms, there should be repeating/consistent size and shape of the cells that is supportive of a micro-organism. Artifacts such as stain crystals are usually not consistent in shape or size.
  • 7. The most common Gram Positive Bacteria
  • 8. Staphylococcus species Gram positive cocci in random clusters Cells are very round and deeply stained, 0.5 – 1 µm in diameter
  • 9. Streptococcus / Beta hemolytic  Gram positive cocci in chains of variable length  Cocci are 0.5 wide by 2.0 µm in length (oval)
  • 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 – which appears as clearing around the stained organism
  • 11. Viridans Streptococcus  Gram positive cocci in short chains and pairs  Chains are usually not as long as the Beta hemolytic Streptococcus, size is 0.5 µm – 1.5 µm.
  • 12. Enterococcus species  Gram positive cocci in short chains and pairs  Can be difficult to differentiate from viridans Streptococcus, size is also size is 0.5 µm – 1.5 µm.
  • 13. Bacillus species Gram positive rods – large (1.0 X 4.0 µm) Box car shaped, sometimes spores are visible. Spores will not stain with gentian violet. Can be over-decolorized and appear Gram negative on Gram stain Over-decolorized Spores
  • 14. Listeria monocytogenes Gram positive short rods (0.5 X 1.5 µm) Do not appear in chains and they do not branch.
  • 15. Corynebacterium species Gram positive rods in that group in Chinese letter forms and palisade cell arrangement (0.5 X 4 µm) Palisade cell arrangement
  • 17. Nocardia species Delicate Gram positive branching rods, sometimes staining quite poorly and appearing speckled. Nocardia stain red with modified acid fast stains
  • 18. Actinomyces species Gram positive rods with antler like branching, can be seen in sulfur granules in tissue Sulfur Granule Formation
  • 19. Gardnerella vaginalis Gram variable (not quite red or blue) rods Associated with “clue cells” in bacterial vaginosis
  • 20. Lactobacillus species Gram positive rods, thin, appear sometimes in short chains
  • 21. Clostridium species Gram positive rods, large, with spores sometimes visible. Spores will not stain with the Gram stain and appear as colorless areas in the cell. Spores are located throughout the length of the rod-shaped organism
  • 22. Mycobacterium species Do not stain well with Gram stain due to lipid in the cell wall, they stain beaded and barely visible. Acid Fast stains are superior for the staining of Mycobacteria
  • 24. Enteric Gram negative rods (Enterobacterales) Plump rectangular in shape(1 x 3 µm)
  • 25. Pseudomonas aeruginosa thinner and a bit longer than enteric Gram negative rods 0.5 X 3µm
  • 26. Haemophilus influenza pleomorphic gram negative rods, thin and faintly staining
  • 27. Vibrio species curved or comma shaped Gram negative rods
  • 28. Fusobacterium species – faintly staining unusually shaped Gram negative rods F. nucleatum F. necrophorum Spindle shaped, with pointed Chaining pleomorphic rods ends
  • 29. Campylobacter sea gull shaped, faintly staining Gram negative rods
  • 30. Capnocytophyga spp – pleomorphic Gram negative bacilli, variable in size and shape
  • 31. Bacteroiodes spp Pleomorphic Gram negative bacilli, variable in length
  • 32. Yersinia pestis Safety pin like with bipolar staining ---
  • 33. (0.5 X 1.5 µm)
  • 34. Neisseria species (meningitidis and gonorrhoeae) Gram negative diplococci Kidney bean shaped in pairs
  • 35. Yeast – Candida species  Large Gram positive oval globules (@ 8 um) that produce a smaller attached cell known as a bud  Sometimes pseudo-hyphae can be seen which are extensions of the yeast cells into long hyphae like forms
  • 36. Cryptococcus neoformans/gatti Due to the polysaccharide capsule of C. neoformans/gatti the yeast appears somewhat like a round blob on Gram stain  An India ink prep can be used to visualize the polysaccharide capsule
  • 37. Mycelial Fungi Variably staining of hyphae on Gram stain