Gram Stain Testing
Document #6/Version #04
Effective Date: 09/18/09
GRAM STAIN
Principle
A crystal violet-iodine complex forms in the protoplast (not the cell wall)
of all organisms stained by this procedure. Organisms able to retain this
dye complex after decolorization are classified as gram positive while
those that can be decolorized with alcohol/acetone and counterstained with
safranin are classified as gram negative.
The gram stain is used to differentiate bacteria into two groups based on
cell color after staining. In addition, cell form, size and structural details
are evident. Such preliminary information provides important clues to the
type of organism(s) present and the further techniques required to
characterize them.
Sample
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Materials
Urethral discharge collected with a cotton swab or a urethral specimen
collected with a calcium alginate swab rolled onto a glass slide.
Bacterial colony from a plate medium suspended in saline on a glass
slide. Colonies may be either from a control (stock) culture plate or
from a patient specimen plate.
Equipment
 Incinerator
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Reagents
Gram Stain Reagent
Kit
 water
 QC organisms
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Quality
Control
Supplies
Sterile cotton
swabs or sterile
applicator sticks
microscope
glass slides
pencil
staining rack
blotting paper or
paper towels
immersion oil
clock or timer
Gram positive (S. aureus) and gram negative (E. coli) organisms are tested
each week of patient testing and when each new bottle of reagent is
opened, at least monthly. Results are recorded on the gram stain QC log.
A control slide is made by placing a small drop of bacterial suspension of
each of the control organisms on a glass slide. Appropriate gram reaction
must be obtained on the control slide before patient results are reported.
Columbus County Health Department Laboratory, Whiteville NC 28472
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Gram Stain Testing
Document #6/Version #04
Effective Date: 09/18/09
Test
Procedure
Step
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Action
Specimen is allowed to air dry, then heat fixed by passing the
slide by the front of the incinerator, slow enough to fix the
cells onto the slide but not so slow as to incinerate them. Cool
to room temperature before staining.
Place the fixed slide on the rack across the sink and flood with
crystal violet. Set timer for 1 minute.
Gently rinse with tap water.
Flood the slide with iodine and set the timer for 1 minute.
Gently rinse with tap water.
Decolorize until solvent running from the slide is colorless
(30-60 seconds).
Note: conservative use of decolorizer may be accomplished by
holding the slide with a gloved hand, placing 2-4 drops of
decolorizer on the slide, swirling it around, then tilting the
slide so decolorizer runs off, then repeating the process until
the runoff is clear. If the smear is thick, it may not decolorize
entirely.
Gently rinse with tap water.
Flood the slide with safranin and set timer for 1 minute.
Gently rinse with tap water.
Blot slide with blotting paper or paper towel (do not wipe) or
allow to air dry.
Examine the smear under 100x oil immersion lens.
Interpretation
Gram Reaction
Purple/blue cells mean the
crystal violet is retained by
the bacteria. They are gram
positive.
Red/pink cells mean that the
safranin has stained the
bacteria because crystal
violet was removed during
decolorization. They are
gram negative.
Result Reporting
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Morphology and Configuration
Morphology refers to the natural shape of
the bacterial cells when viewed
microscopically. Neisseria sp. have a
characteristic round shape and are found in
pairs, known as diplococci.
Configuration refers to the relationship of
bacterial cells to each other. Neisseria sp.
usually occur in pairs with a slightly
flattened plane where the cells touch. This
configuration is referred to as diplococci.
GC culture specimen – only the presence of gram negative diplococci
(GNC) are reported for patients.
Male urethral smears – record results on lab log and enter into EMR.
Columbus County Health Department Laboratory, Whiteville NC 28472
2
Gram Stain Testing
Document #6/Version #04
Effective Date: 09/18/09
If there is
Gram-negative intracellular
diplococci inside the
leukocytes
No intracellular gram
negative diplococci seen.
Expected
Values
Limitations
References
Then
 Enter a report of Intracellular GNID
Found.
 Enter a report of NF.
 Enter the number of WBCs observed
as <2 WBCs/oif or ≥2 WBCs/oif.
There are no critical values. There are no panic values.
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Excessive heating of slide with cause atypical staining.
Use of 18-24 hour cultures is advisable for best results since fresh cells
have a greater affinity than old cells for most dyes.
Smears too thick may not decolorize and should be recollected.
Smears too thin may make it difficult to find cells/organisms.
Excessive rinsing may wash the specimen off the slide.
Failure to dry the specimen may cause organisms to wash off in the oil.
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Manufacturer’s package insert for BD Gram Stain Kits and Reagents.
Related Documents
 Policy #2, Specimen Rejection Policy.
 Policy #14, EMR Policy
Author
Karen H. Wall, BSMT (ASCP), Technical Consultant
Approval
Signature
_____________________________
Name
Columbus County Health Department Laboratory, Whiteville NC 28472
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Date
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