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Gram Stains 2021
This document provides information on performing and interpreting Gram stains, including: - The steps of the Gram stain procedure and how to assess quality. - That Gram positive bacteria appear blue due to their thick peptidoglycan layer trapping crystal violet. - Common Gram positive and Gram negative bacteria and their morphologies under the Gram stain. - Potential artifacts like stain crystals.
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Gram Stains 2021
- 1. Gram stains 2021 MargieA. Morgan, PhD. D(ABMM)
- 2. Gram Stain Assess forGram 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 thinsmear 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 largeamount 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 Gramstains 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 GramPositive Bacteria
- 8. Staphylococcus species Gram positivecocci in random clusters Cells are very round and deeply stained, 0.5 – 1 µm in diameter
- 9. Streptococcus / Betahemolytic Gram positive cocci in chains of variable length Cocci are 0.5 wide by 2.0 µm in length (oval)
- 10. Streptococcus pneumoniae Grampositive 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 Grampositive 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 Grampositive 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 positiverods – 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 positiveshort rods (0.5 X 1.5 µm) Do not appear in chains and they do not branch.
- 15. Corynebacterium species Gram positiverods in that group in Chinese letter forms and palisade cell arrangement (0.5 X 4 µm) Palisade cell arrangement
- 16. Cutibacterium (Propionibacterium) acnes Small pleomorphicGram positive rods that branch.
- 17. Nocardia species Delicate Grampositive branching rods, sometimes staining quite poorly and appearing speckled. Nocardia stain red with modified acid fast stains
- 18. Actinomyces species Gram positiverods 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 positiverods, thin, appear sometimes in short chains
- 21. Clostridium species Gram positiverods, 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 notstain 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
- 23.
- 24. Enteric Gram negativerods (Enterobacterales) Plump rectangular in shape(1 x 3 µm)
- 25. Pseudomonas aeruginosa thinner anda bit longer than enteric Gram negative rods 0.5 X 3µm
- 26. Haemophilus influenza pleomorphic gramnegative rods, thin and faintly staining
- 27. Vibrio species curved orcomma 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 – pleomorphicGram negative bacilli, variable in size and shape
- 31. Bacteroiodes spp Pleomorphic Gramnegative bacilli, variable in length
- 32. Yersinia pestis Safety pinlike with bipolar staining ---
- 33. (0.5 X 1.5µm)
- 34. Neisseria species (meningitidisand gonorrhoeae) Gram negative diplococci Kidney bean shaped in pairs
- 35. Yeast – Candidaspecies 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 tothe 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 stainingof hyphae on Gram stain