1. Gram stains Just a few to remember
2. Gram Stain Stain Procedure: – – – – Crystal violet – primary stain for 1 min/rinse Gram’s iodine – mordant for 1 min/rinse Acetone/Alcohol – decolorizer for 5 seconds Safranin – counterstain for 1 min/rinse – Gram negative (red) and Gram positive (blue) – Assess Gram reaction and morphology 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. Gram Positive Bacteria
4. Staphylococcus Gram positive cocci in clusters Cells are very round and deeply stained 1-2 µm in size
5. Beta hemolytic Streptococcus • Gram positive cocci in long chains • Organisms are oval with variable staining with size range of 1 – 1.5µm
6. Streptococcus pneumoniae • Gram positive cocci in pairs • Cocci are lancet or bullet shaped /@ 1µm size • Sometimes can visualize a capsule (clearing around the cells)
7. Viridans Streptococcus • Gram positive cocci in short chains and pairs – cocci around 1 – 1.5 µm in size • Chains are not as long as Beta hemolytic Streptococcus
8. Enterococcus species • Gram positive cocci in short chains and pairs with size of @ 1.5 µm • Difficult to impossible to differentiate from viridans Streptococcus
9. Yeast – Candida species • Large Gram positive globules (@ 8 um) with buds • Sometimes hyphae can be seen
10. Cryptococcus neoformans Due to the capsule of C. neoformans the stain appears somewhat like a blob Size range from 8 – 12 µm • India ink can be used to visualize the capsule
11. Aspergillus species Gram stain Mycelial Fungi do not stain well with the gram stain
12. Bacillus species Gram positive rods – large , box car shaped, sometimes spores are visible – 1.2 X 4µ in size Sometimes they over-decolorize and appear Gram negative Over-decolorized – clear areas are spores
13. Listeria monocytogenes Gram positive rodshort rods, deeply stained – size 1.2 X 2.5µm
14. Diphtheroids (Corynebacteria)gram positive pleomorphic rods in Chinese letter forms 1.4 X 3 µm
15. Nocardia species Delicate Gram positive branching rods - faintly staining Nocardia stain red with modified acid fast stains
16. Actinomyces Antler-like branching rods
17. Lactobacillus species Gram positive thin and long rods that appear sometimes in chains
18. Clostridium species Gram positive rods with spores @1.2 X 4 µm - the spores can be located throughout the organism
19. Mycobacterium species do not stain well with gram stain due to the lipid in the cell wall Kinyoun stain – AFB positive
20. Gram negative bacteria
21. Enteric Gram negative rods Plump rectangular in shape @ 1.4 X 2.5 - 3µm
22. Pseudomonas thinner than the enteric rods @ 1 x 3 µm
23. Haemophilus influenza pleomorphic small faintly staining gram negative rods
24. Vibrio species curved or comma shaped gram negative rods
25. Fusobacterium delicate fusiforme shaped faintly staining gram negative rods
26. Campylobacter sea gull shaped faintly staining gram negative rods
27. Yersinia pestis Safety pin bipolar staining ---
28. Neisseria species Gram Negative Diplococci Kidney bean shaped in pairs
29. Fastidious Gram negative rods such as the HACEK group, Brucella, Bordetella Small cocco – bacilli rods
30. Legionella does not stain well with Gram stain Does stain with Carbol Fuchsin used as a counterstain – pleomorphic Gram negative rod
31. Human cells PMNs Epithelial cells Mononuclear cells Mononuclear cells