INING: Salts of colored compounds, the ionized form either is basic (positive) or acidic

(negative):
Stains usually dissolved in an alcohol or water solution,
SIMPLE STAINS
Basic dyes: are positive when ionized, stain negatively charged materials such as
bacteria
examples: crystal violet
methylene blue
safranin
Acid stains: are negative when ionized, stain positively charged materials (zB: glass)
examples: eosin
nigrosin (spirochete)
results in negative staining because background is usually positive, and so is stained
Fluorescence microscopy: stain with fluorochromes:
auramine O glows yellow in UV, absorbed by Mycobacterium tuberculosis
fluorescein isothiocyanate apple green for Bacillus anthracis
DIFFERENTIAL STAINS: Usually four steps: primary stain, mordant, decolorize, secondary stain)
Gram Stain (p 70): by Hans Christian Gram (1884): Hucker=s stain, Iodine, 95% EtOH,
safranin O( Fankhauserser's page)
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Acid fast: (p 70) 1) primary stain: steam carbolfuchsin (fuchsin is a red dye)
on specimen, several min.
(Ziehl-Neelsen) 2) decolorize acid-alcohol, removed color if not acid fast.
3) counter stain methylene blue
If red (p 69), may be either Mycobacterium tuberculosis or leprae. (or Nocardia, a closely related
bacterium)
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Negative stain (p 71): demonstrate capsules, usually not stainable, add India ink (or
other acid dyes?), capsule shows up as halo (stains background)
Endospore staining: five genera of bacteria make spores.
(P 71) Very difficult to stain, although easily seen due to different refractive
index.
Schaeffer-Fulton endospore stain: (p 71)
malachite green steamed for five minutes wash 30 seconds with water (spores stay
green)
safranin counter stain
Gram Stain
The Gram stain is a technique for staining and detecting bacteria and yeast.
It is one of the most commonly performed procedures in the clinical microbiology
laboratory.

Gram Stain: Procedure

Four reagents are used to
perform a gram stain: crystal
violet, Gram's Iodine, acetone
- alcohol, and safranin.


Direct Smear Preparation

If the specimen is received on a
swab, gently role the swab on a
clean glass slide to avoid rupturing
host cells. Allow to air dry.

Direct Smear Pap

If the specimen is a fluid,
place a drop of fluid on a
clean glass slide and allow to
air dry.
*In both cases, the specimen
is fixed to the glass slide by
passing it a few times over a
flame.

Control Slide

Fishers Band Gram Slide
has control Gram positive
cocci and Gram Negative
rods.

Staining Procedure

Step 1. Flood the slide with crystal violet
for 1 minute. Rinse with water.
Step 2. Flood the slide with Gram's
iodine for 1 minute. Rinse with water.

Staining Procedure
Step 3. Decolorize the slides by gently rinsing with an acetone - alcohol solution
for 1 to 10 seconds dependent on content of acetone in solution. Rinse with
water.
step4. Flood the slide with saffranin, the counterstain, for 1 minute. Rinse with
water and dry air.


Theory: Cell Wall Construction
Gram positive and gram negative bacteria stain differently because of the
structure of their cell walls.
Gram - positive bacteria and yeasts stain purple.
Gram negative bacteria and host cells stain pink.

Microscopy

Proper adjustment of the microscope is essential.

Microscopy
Scan slide on low power (10X) to find the best area of the slide.
Than go to oil immersion (100X)


Scan slide on low power
10X: Many Neutrophils seen
Oil (100X): Neutrophils
with
gram positive cocci

Focusing Technique
Focusing techniques: Often it is necessary to focus through a specimen since
organisms, and cells can be found on different planes.

Oil (100X): Slide shows neutrophils and
gram positive cocci.

Morphology
Bacteria
Host Cells: WBC's
Macrophages, RBC's, Epithelial Cells
Yeast
Artifact

Gram Positive Rods
Gram positive rods can be found in found in 4 clinically significant forms
o long, wide
o lonf, narrow
o coccobacillus
o Branched

Gram Positive Rods



Gram Positive Rods
click on icon


Gram Negative Rods
Gram negative rods can be present in 5 Clinically significant forms:
o Long and narrow
o Coccobacillus
o Curved
o Fusiform
o Spiral


Gram Negative Rods
click on icon


Gram Negative Rods
click on icon


Mixed Populations
Avian Stool, Gram stained
80% Gram positive rods
20% Gram negative rods




Find the Gram positive and Gram negative rods in this field.
click on icon

Gram negative spiral rods (spirochetes)
click on icon

Gram positive Cocci: a spherical bacterium
Gram positive Cocci may be present as:
diplococci - pairs
Chains of cocci
Tetrad appear as a cluster of exactly four cocci
Cluster - Groups of cocci with variable numbers


Bacteria: Gram positive Cocci
Gram positve Cocci

Diplococci

Clusters

Gram positive Cocci
click on icon

Gram positive Cocci




Gram positive cocci in
cluster
(top black arrow)



Gram positive cocci in
tetrad formation
(botton black arrow)

Gram Negative Cocci

Diplococci
Do not form typically chains or clusters



Host Cells
Epithelial Cells
Stain Gram Negative
White Blood Cells




Yeast

Stains Gram Positive
Can be budding or in
branching hyphae form






Artifact
Crystal violet precipitate on epithelial
cell:
May be confused with Gram positive
cocci.

Crystal violet precipitate
crystal on gram stain.


Underdecolorization
Neutrophils that should stain pink are underdecolorized at acetone alcohol step
and stained purple









underdecolorized neutrophils


Overdecolorization

Part of this slide
(pink area) has
been
overdecolorized
with the acetone
alcohol giving
the false
impression of
gram negative
rods being
present.

Reporting Results

Use systematic, descriptive
terminology to report gram
stain results.
* For example, this Gram stain
is properly described as
"Gram - positive cocci
(clusters) and white blood
cells present.

Reporting Results: Avian Stool
Avian Gram Stain
Report percentages of Gram positive,
Gram negative bacteria and yeast.

Normal Psittacine stool consist of 95% gram positive rods and cocci and
up to 5% gram negative rods.
Occasional yeast is normal.


Gram Stain: Normal
Avian (Psittacine) Stool

Normal Psittacine:
100% gram positive
rods and cocci.
No yeast seen.

Avian Stool: Increased numbers of Gram negative rods





Abnormal Avian
gram stain:
50% Gram
negative rods
50% Gram
positive rods

Reporting Results
* It is NOT possible to determine the species of bacteria from Gram stain results
alone!
Difinitive identification requires culture and biochemical testing.

Acid fast stain: Ziehl Neelson Method
Procedure
Morphology

Acid fast Organisms
Contains waxlike lipoidal material affecting staining quality.
Carbolfuchsin is primary stain.
Acid fast organisms resist decolorization with acid alcohol.
After decolorization, methyelene blue is added to organisms to counterstain any
material that is not acid fast.


Ziehl Neelsen Acid fast Stain method



Acid Fast Organisms on wrights stain


Acid Fast Stain: Ziehl neelsen method

Acid fast organisms
stain red.
Non acid fast
organisms and tissue
cells stain blue.

Acid fast stain: Cryptosporidium