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Gram Stain Procedure
Principle:
The differential staining seen with the Gram stain occurs because the two types of bacteria differ
significantly in their cell envelope composition. The cell envelopes of both gram-positive and gram
negative bacteria contain a cell wall (also known as the murein layer) and a cytoplasmic membrane.
In addition, the cell envelopes of gram-negative bacteria contain a periplasmic space (between the
cell wall and cytoplasmic membrane) and an outer membrane.
It is the murein layer, or cell wall, that causes the differential staining seen with the Gram stain. The
murein layer, which is composed of a peptidoglycan macromolecule, is much thicker in gram-positive
bacteria than in gram-negative bacteria. Also, the cell walls of gram-positive bacteria contain teichoic
acids and mycolic acids which fortify the murein layer. The thin cell walls of gram-negative bacteria,
on the other hand, contain lipopolysaccharides which are extracted during decolorization.
In the Gram stain procedure the slide is flooded with crystal violet (the primary stain), followed by
Gram’s iodine (the mordant), which chemically bonds the alkaline crystal violet to the cell wall. The
slide is then decolorized most commonly with a mixture of acetone and absolute alcohol. The
decolorizer substantially damages the thin cell walls of gram-negative bacteria and allows the crystal
violet-iodine complex to wash out, whereas the thicker cell walls of gram-positive bacteria are more
resistant to damage and therefore retain the stain complex. In the final step, the slide is flooded with
safranin (the counterstain), which stains the decolorized gram-negative bacteria pink or red.
Specimen Collection and Preparation
The Gram stain is used routinely and as requested in the clinical microbiology laboratory for the
primary microscopic examination of specimens submitted for smear and culture. It is ideally suited for
those specimen types in which bacterial infections are strongly suspected, but it may be used to
characterize any specimen. Cerebrospinal fluid, sterile fluids, expectorated sputum or
bronchoalveolar lavages, and wounds and exudates are routinely stained directly. Urine specimens
may not be routinely stained directly. Stool specimen for gram stain is performed only to evaluate the
presence or absence of polymorphonuclear cells. Samples sent for focused screening cultures
usually are not stained. The Gram stain is regularly used to characterize bacteria growing on culture
media.
Specimen Gram stain preparation:
1. Roll swab, taken from representative areas of specimen, gently across slide to avoid destruction
of cellular elements and disruption of bacterial arrangements.
2. If only one swab received, roll on sterile swab to sterile slide for gram stain inoculate media
plates, then place swab in the broth last.
3. Biopsies and tissue specimens. A “touch prep” preparation may be used. Homogenizing or
grinding tissue specimens before preparing smear will often destroy characteristic cellular entities
and bacterial arrangements.
• Mince with sterile scissors or scalpel.
• Prepare a “touch prep.” Use sterile forceps to hold pieces, and touch the sides of one or more
of the minced fragments to a sterile glass slide, grouping touches together for easier
examination.
Colonies from media preparation:
1. Place a drop of sterile saline on slide.
2. Transfer a small portion of colony with a sterile applicator stick, wire needle, or loop.
3. Gently mix to emulsify.
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Reagents and Supplies
1. Gram Crystal Violet Solution: Approximately 0.4% crystal violet in aqueous alcohol/acetone (3:1
ratio, respectively) solution
2. Gram Iodine Solution (stabilized): Approximately 13% polyvinylpyrrolidone-iodine complex in
1.9% aqueous potassium iodine
3. Gram Decolorizer Solution: Denatured ethyl alcohol and acetone, approximately one part to one
part
4. Gram Safranin Solution: Approximately 0.25% safranin in 20% ethyl alcohol
5. Glass slides
6. Slide warmer
Storage
1. Keep all reagents at room temperature.
2. Protect the Gram’s iodine reagent from the light when not in use.
Quality Control
Run controls daily using 18-24 hour cultures of known gram-positive and gram-negative
microorganisms.
1. The following QC organisms should be used: Positive control – Staphylococcus aureus
Negative control – Escherichia coli
2. Expected results:
Staphylococcus aureus – stains as gram positive cocci (dark blue to blue-black)
Escherichia coli – stains as gram negative bacilli (pink-red)
Procedure
1. Using either heat or methanol, fix the specimen onto the slide.
Heat fixation: Place on a slide warmer for 1 minute.
Caution: Do not overheat. Overheating can cause over-decolorization.
Methanol fixation: Flood the slide with 70%-95% methanol for 1 minute. The methanol is then
drained off, and the slides are air-dried.
2. Place the slide on a slide holder rack at the sink. You may wish to wear latex gloves to protect
your skin from the stain reagents.
3. Flood the slide with crystal violet. Allow to remain 30 seconds. Rinse with water.
Caution: Avoid excessive rinsing. This can cause over-decolorization because crystal violet is
not bound to the cell until Gram’s iodine is added.
4. Flood the slide with Gram’s iodine. Allow to remain 30-60 seconds, then rinse with water.
5. Decolorize the slide with the acetone-alcohol mixture. Pick up slide and carefully apply
decolorizer solution (1-2 quick squirts) just until no more color is being washed from the smear.
Quickly rinse off any remaining decolorizer with tap water.
Caution: Avoid prolonged decolorization or excessive rinsing. These can wash the crystal
violet-iodine complex from gram-positive cells.
6. Flood the slide with safranin for 30-60 seconds. Rinse gently with water.
Caution: Do not leave the counterstain on longer than 1 minute. This can make gram positive
organisms appear gram-negative.
7. Blot slide dry with paper towel. Carefully wipe excess stain from the back of the slide.
8. Let the slide dry completely before adding immersion oil and examining microscopically.
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Reading Gram stains:
• Scan on low power (10x) to locate correctly stained areas
• Read gram stain on oil immersion
• Note the cellular morphology, arrangement and Gram stain reaction.
Interpretation of Test
Gram-positive bacteria stain dark blue to blue-black. All other elements stain safranin red. Individual
structures absorb a different amount of safranin, so some will have prominent staining (strong avidity)
and others will be weakly stained (low avidity). Among the gram-negative bacteria the enterics have
strong avidity and stain a bright red; pseudomonads are less avid and stain moderately well.
Anaerobic bacilli and other thin-walled gram-negative organisms, such as Borrelia, Legionella, and
Spirillum, stain weakly. Always check the quality of the stain before moving to interpretation.
Reporting Gram stains: (NOTE: scale may vary at your clinical site)
• Gram reaction (gram positive = GP, gram negative = GN, gram variable = GV)
• Morphology (bacilli or rod = R, cocci = C, coccobacilli = CB)
• Arrangement
GNR
GPC
GPR
GNC
large
single
spores
diplococci (DC)
thin
pair
large
fusiform
chains
palisading
tiny
clusters
branching
tetrads
• Other descriptive terms (i.e. pleomorphic, lancet-shaped, etc.)
When looking at SPECIMEN Gram Stains, also include the following:
• Sputum
>25 squamous epithelial cells per low power field – REJECT specimen and do not
do gram stain.
• Type and amount of cells (if a specimen gram stain)
None/NCS
no cells seen
Rare (1+)
< 1 per oil immersion field
Few (2+)
1-5 per oil immersion field
Moderate (3+)
5-10 per oil immersion field
Many (4+)
>10 per oil immersion field
• Enumerate the types of organisms as follows (if a specimen gram stain):
None/NOS
no organisms seen
Rare (1+)
<1 per oil immersion field
Few (2+)
1-10 per oil immersion field
Moderate (3+)
11-25 per oil immersion field
Many (4+)
>25 per oil immersion field
Modifications
Common modifications of the classic Gram stain procedure involve variations in fixation method,
reagents, and timing. Fixation, which attaches the specimen to the slide before staining, can be done
with heat or methanol. In heat fixation, the slide is gently warmed so that all moisture evaporates from
the material. In methanol fixation, the slides are flooded with 70%-95% methanol for 1 minute. The
methanol is then drained off, and the slides are air-dried. Although more labs use heat fixation,
methanol fixation is superior for 5 reasons:
1. It preserves bacterial and human cell morphology.
2. It preserves red blood cells, which makes it especially useful with bloody specimens.
3. It provides greater control over the decolorization process, because organisms fixed with
methanol are more resistant to decolorization.
4. It prevents liquid specimens from washing off the slide.
5. It leaves a clearer background.
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Common variations in reagents involve the decolorizer and the counterstain. Slides can be
decolorized with acetone, absolute alcohol, or a mixture of the two. Acetone works more rapidly, but
the slide must be rinsed with water as soon as the purple color disappears to avoid overdecolorization. Slides are usually counterstained with safranin, but neutral red or dilute carbolfuchsin,
which better stains anaerobes, are used sometimes. Some microbiologists also advocate adding a
fast green and tartrazine step before the final counterstain with safranin to give a better contrast
between gram-negative organisms and background material.
Finally, the duration of the steps in the Gram stain procedure may vary somewhat. Because
anaerobes tend to stain weakly, they may stain better if the counterstain is left on for 1 minute instead
of 30 seconds.
Precautions
Pitfalls in the Gram stain include both inherent limitations and technical errors. The Gram stain will not
detect organisms which exist within host cells (e.g., Chlamydia spp), organisms with no cell wall (e.g.,
Mycoplasma spp and Ureaplasma spp), and organisms too small to be seen with light microscopy
(e.g., spirochetes). Mycobacteria usually will not stain, and Legionella spp stain only when taken
directly from culture. Gram-negative bacteria that stain poorly with safranin include Campylobacter
spp, Legionella spp, Bacteroides spp, Fusobacterium spp, and Brucella spp.
Certain conditions are known to damage the cell wall, causing gram-positive bacteria to falsely
appear gram-negative or gram-variable. These include antibiotic treatment, cultures more than 48
hours old, inflammatory responses in the host, and autolytic enzymes (e.g., S. pneumoniae). To
minimize ambiguous results, specimens should be collected before the patient begins antibiotic
therapy. Also, Gram stains should be performed on colonies taken from culture media that do not
contain antibiotics, preferably on colonies that are 18-24 hours old.
Finally, correct interpretation of Gram stains requires a theoretical background of bacteria and their
morphology, because improper technique or suboptimal reagents can cause unreliable results. Errors
in technique which can alter Gram stain results include the following:
• Fixation with excessive heat alters cell morphology and makes organisms more susceptible
to over-decolorization.
• Low concentrations of crystal violet make gram-positive organisms more susceptible to
overdecolorization.
• Insufficient exposure to iodine and lack of available iodine can prevent crystal violet from
bonding firmly with the cell wall, thus making gram-positive organisms more susceptible to
overdecolorization. To ensure reliable Gram stain results, only fresh iodine should be used.
• Prolonged decolorization, especially with acetone, can cause gram-positive bacteria to
appear gram-negative. Insufficient decolorization can make gram-negative organisms
falsely appear gram positive.
• Insufficient counterstaining can fail to stain gram-negative bacteria and background
material, whereas excessive counterstaining will leach the crystal violet-iodine complex
from gram positive bacteria and stain them with safranin, thus making them falsely appear
gram-negative.
• Prolonged washing between any of the steps can cause over-decolorization.
References:
Mahon & Manuselis, Textbook of Diagnostic Microbiology, Second edition, 2000. Pages 267-268.
http://www.api-pt.com/pdfs/2004Bmicro.pdf