1.
Gram stains
2017 UPDATE

2.
Gram Stain
Assess Gram reaction and morphology
 Gram negative (red) /Gram positive (blue)
 Crystal violet – primary stain for 1 min/rinse
 Gram’s iodine – mordant for 1 min/rinse
 Acetone/Alcohol – decolorizer for 5 secs
 Safranin – counterstain for 1 min/rinse
 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.
What can go wrong?
If the stain is all blue at the
end of the staining process
You did not decolorize
the slide for enough time.
If the stain is all red and washed
out appearing at the
end of the staining process
You over decolorized with the
acetone.

4.
Cellular Material – Gram stains are
not able to differentiate many cell types. Best for
PMNs, Mononuclear cells, and epithelial cells.

5.
Possible Artifacts in Gram
stains
Safranin
crystals
Crystal Violet crystals
When reading a Gram stain, you need to check for the uniformity of the
cell borders of the suspected organisms, there needs to be repeating/consistent
size and shape that is suggestive of a micro-organism.

6.
Gram Positive Bacteria

7.
Staphylococcus
Gram positive cocci in clusters
Cells are very round and deeply
stained, 0,5 – 1 µm in diameter

8.
Streptococcus
Beta hemolytic
 Gram positive cocci in chains (long chains)
 0.5 wide by 2.0 µm in length (oval)

9.
Streptococcus
pneumoniae
 Gram positive cocci in pairs – 0.5 – 1.5 µm in size
 Cocci are lancet or bullet shaped with flattened sides
together
 Sometimes can visualize a capsule on gram stain –
clearing around the stained organism

10.
Viridans Streptococcus
 Gram positive cocci in short chains and pairs
 Chains are not as long as Beta hemolytic
Streptococcus (0.5 µm – 1.5 µm)

11.
Enterococcus species
 Gram positive cocci in short chains and pairs
 Difficult to impossible to differentiate from viridans
Streptococcus

12.
Yeast – Candida species
 Large Gram positive globules (@ 8 um) with buds
 Sometimes pseudo-hyphae can be seen

13.
Cryptococcus
neoformans/gatti
Due to the capsule of
C. neoformans/gatti
the stain appears
somewhat like a blob
 An India ink prep can be used
to visualize the capsule

14.
Mycelial Fungi Gram stain
Fungi stain +/- with the Gram stain

15.
Bacillus species
Gram positive rods – large , box car shaped, sometimes
spores are visible (1.0 X 4.0 µm)
Sometimes they over-decolorize and appear Gram negative
Over-decolorized
Spores

16.
Listeria monocytogenes
Gram positive short rods (0.5 X 1.5 µm)

17.
Corynebacterium spp. (diphtheroids)
gram positive rods in Chinese
letter forms and palisades (0.5 X 4 µm)
palisades

18.
Propionibacterium acnes – small
branching Gram positive rod

19.
Nocardia species
Delicate Gram positive
branching rods
Nocardia are red when stained
with modified acid fast stains

20.
Actinomyces species
Antler like branching
Sulfur Granule Formation

21.
Gardnerella vaginalis
Gram variable rods

22.
Lactobacillus species
Gram positive long rods that appear
sometimes in chains

23.
Clostridium species
Gram positive rods with spores
the spores can be located throughout the organism

24.
Mycobacterium species
do not stain well with gram stain due to
the lipid in the cell wall – stain weakly
and beaded
Kinyoun AFB stain

25.
Gram negative bacteria

26.
Enteric Gram negative rods
(Enterobacteriaceae)
Plump rectangular in shape
(1 x 3 µm)

27.
Pseudomonas aeruginosa
thinner than the enteric rods
0.5 X 3µm

28.
Haemophilus influenza
pleomorphic thin faintly staining Gram
negative rods

29.
Vibrio species
curved or comma shaped gram negative rods

30.
Fusobacterium species –
F. nucleatum F. necrophorum

31.
Campylobacter
sea gull shaped faintly staining gram
negative rods

32.
Capnocytophyga spp –
pleomorphic Gram negative bacilli, variable in size and
shape

33.
Bacteroiodes spp
Pleomorphic Gram negative bacilli, variable in length

34.
Yersinia pestis
Safety pin bipolar staining
---

35.
(0.5 X 1.5 µm)

36.
Neisseria species (meningitidis and gonorrheae)
Gram Negative Diplococci
Kidney bean shaped in pairs