Gram Stains

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
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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
Mononuclear cells
Mononuclear cells
Epithelial cells