2. Gram Stain
Stain Procedure:
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–
–
–
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
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
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