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Standard Operating Procedure
Subject Gram Stain
Index Number Lab-8683
Section Laboratory
Subsection Regional/Affiliates
Category Departmental
Contact Sarah Stoner
Last Revised 5/29/2019
References
Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers
for Medicare and Medicaid Services (CMS) and/or COLA.
Applicable To
Employees of the Gundersen Health System clinical laboratories, Gundersen Tri-County Hospital
laboratories, St. Joseph’s Health Services laboratories, Gundersen Boscobel Area Hospital laboratories,
Gundersen Moundview Hospital laboratories, and Gundersen Palmer Lutheran Hospital and Clinics
laboratories.
Detail
INTENDED USE:
The Gram Stain Reagents and Kit are recommended for the differential staining of bacteria in primary
specimens and from culture.
SUMMARY AND EXPLANATION OF THE TEST
The Gram stain is well established as an important aid in the differentiation and identification of isolated
microorganisms. A correctly performed and interpreted Gram stain of certain clinical specimens also
can be a rapid source of presumptive diagnostic information. Numerous studies and reports have
established the value and importance of this technique in the examination of sputum and transtracheal
specimens, cerebrospinal, and other normally sterile body fluids, uncontaminated abscess fluids and
specimens from soft tissue infections, and exudates from the male urethra. The Gram stain examination
of uncentrifuged urine also is a procedure recommended to provide rapid preliminary information.
Gram stain examinations should not be used, however, as a substitute for complete and careful culture
studies.
PRINCIPLES OF THE PROCEDURE:
The gram stain serves to divide bacteria into two main groups: gram positive organisms that retain the
primary crystal violet dye and appear deep blue or purple, and gram negative organisms which can be
decolorized easily, take up the counterstain safranin and appear red or pink. Gram positive organisms
have increased cross linked teichoic acids and a lower lipid content in their cell wall that cause
decreased permeability to organic solvents and they retain the crystal violet stain. Gram negative
organisms have a higher lipid content in their cell wall, which increases permeability to decolorizer so
they lose the crystal violet and take up the counterstain.
REAGENTS/MATERIALS:
Affiliate Laboratories
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Standard Operating Procedure
1. Gram Crystal Violet Solution, BD
a. Approximately 0.4% crystal violet in an aqueous alcohol solution.
2. Gram Iodine Solution (Stabilized), BD
a. Approximately 13% polyvinylpyrrolidone-iodine complex in 1.9% aqueous potassium
iodine
3. Gram Decolorizer Solution, BD
a. Denatured ethyl alcohol and acetone, approximately three parts to one part,
respectively
4. Gram Safranin Solution, BD
a. Approximately 0.25% safranin in 20% ethyl alcohol
GHS Core Microbiology
1. Crystal violet stain, Fischer Scientific
2. Gram's Iodine, Fischer Scientific
a. Add contents of iodine concentrate vial to the large bottle labeled Iodine.
b. Mix well before use.
3. Decolorizer
a. 500 ml Ethanol, 95%
b. 500 ml Acetone
c. Store excess working stock in the cabinet under the stainer in microbiology. The large
stock container is located in the explosion proof room in chemistry.
4. Safranin stain, Fischer Scientific
PRECAUTIONS:
For invitro diagnostic use.
As with all techniques involving pathogenic and potentially pathogenic microorganisms, established
aseptic practices should be consistently applied throughout his procedure.
These reagents are harmful or fatal if swallowed and can cause eye irritation if contact is made. In event
of eye contact, flush eyes with an eye wash system or tap water for 15 minutes. Gram decolorizer
solution is flammable and its vapors; may be harmful; use in a well-ventilated area away from open
flames. Directions for use and interpretation should be read and followed carefully.
STORAGE
On receipt, store at 15-30°C.
The expiration date is for product in unopened bottles. Store as directed.
DO NOT OPEN UNTIL READY TO USE.
PRODUCT DETERIORATION
Some precipitation may occur in the Crystal Violet Solution upon prolonged storage. If this appears to
affect the quality of staining, with the dispensing closure open, briefly warm the bottle or quantities
dispensed into the bottle in a 37°C water bath, then close and shake until the precipitate is dissolved.
The working Gram Iodine Solution from unstabilized concentrate with diluent deteriorates rapidly,
especially when exposed to light and/or heat. Working solution may remain stable for up to 3 months
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Standard Operating Procedure
under normal conditions of daily use. It should be discarded when color loss becomes significant or
when adequate results are no longer obtained (see User Quality Control).
SPECIMEN COLLECTION AND PREPARATION:
The Gram stain may be performed on smears prepared from clinical specimens or samples containing
mixed flora or pure cultures or on smears of microbial growth from laboratory cultures.
QUALITY CONTROL:
As a test of both reagent integrity and correct reading and staining technique, the daily (or with each
new shipment of stain) performance of quality controls is required. This is especially important when
clinical specimens are being examined to provide presumptive diagnostic information or a guide for
antimicrobial therapy. Overnight (18-24 hour) cultures of Escherichia coli (Gram negative) and
Staphylococcus aureus (Gram positive) are suitable control organisms. More subtle deficiencies in
reagent quality and techniques can be detected by the use of weakly reactive bacteria such as Bacillus
subtilis (Gram positive) and Moraxella catarrhalis (Gram negative). Record results in BEAKER on
OUTSTANDING LIST.
Implementation
PROCEDURE:
METHANOL FIXED GRAM STAIN
NOTE: Methanol fixation preserves the morphology of red blood cells as well as bacteria and is
especially useful for examining blood specimens and blood cultures.
1. Flood slide with crystal violet for 5 seconds.
2. Rinse gently with tap water.
3. Flood with Gram’s iodine for 10 seconds.
4. Rinse gently with tap water.
5. Decolorize with alcohol-acetone decolorizer for 5 seconds or until blue color stops running.
6. Rinse gently with tap water.
7. Flood with safranin counterstain for 5 seconds.
8. Rinse, air dry and examine.
PROCEDURAL NOTES:
Gram stain slides may be decolorized and restained if necessary. Remove oil with KimTech wipes, flood
smear with decolorizer until smear appears colorless, and then re-stain.
INTERPRETATION:
When the differential Gram procedure is performed correctly, organisms which retain the primary stain-
mordant complex will appear microscopically blue to purple and are termed “Gram-positive”;
organisms which are decolorized and therefore take up the counterstain microscopically will appear pink
to red and are termed “Gram-negative”.
Morphology:
1. Rods: Elongated organisms, longer than wide
2. Cocci: Round organisms, may be in singles, pairs, chains or clusters.
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Standard Operating Procedure
3. Yeast: Organisms that may be oval in shape, longer than bacteria, may exhibit “budding” or
pseudohyphae.
4. Coccobacillus: Organisms that appear somewhat elongated, but not enough to call bacillus.
REPORTING:
Direct smears from specimens other than sputum and blood cultures:
1. For wound cultures only: report number of epithelial cells per 10x field:
a. Rare: Rare: (< than 1 per 10x field)
b. Small # (1-5 per 10x field)
c. Moderate # (5-10 per 10x field)
d. Large # (> than 10 per 10x field)
2. Report number of WBC per oil immersion field:
a. Rare: (< than 1 per oil immersion field)
b. Small # (1-5 per oil immersion field)
c. Moderate # (5-10 per oil immersion field)
d. Large # (> than 10 per oil immersion field)
3. Report number and morphology of organisms present per oil immersion field:
a. Rare: (< 1 per oil immersion field)
b. Small # (1-5 per oil immersion field)
c. Moderate # (5-10 per oil immersion field)
d. Large # (> 10 per oil immersion field)
Sputum:
1. Gram stains should be examined as soon as possible to determine the quality of the specimen.
a. Examine at least 10 fields under low power (10X) to determine the number of WBC’s and
epithelial cells present.
b. Report per low power field as >25 or <25 for each, WBC and epithelial cells.
c. If <25 WBC’s/LPF contact the nursing unit or physician and request a new specimen. The
physician may elect to have the specimen cultured regardless of the number of WBC’s seen
on gram stain. Discard plates if the specimen is rejected. Report gram stain results on LIS.
2. Report the number and morphology of organisms present using the guidelines below:
Rare (<1 per oil immersion field)
Small # (1 – 5 per oil immersion field)
Moderate # (5 – 10 per oil immersion field)
Large # (> 10 per oil immersion field)
3. Synovial fluid Gram stains.
a. Report crystals if present. Also comment on their shape, either “needle” or “rhomboid.”
b. Extra care should be taken when staining this type of fluid as this fluid type is prone to stain
precipitate formation. It is recommended that these are hand stained.
4. GHS Core Lab: CSF Gram stains should be resulted as soon as possible, especially those on in-house
patients.
5. GHS Core Lab: Gram stains reported on PM and night shifts will be reviewed by day shift and any
necessary changes will be corrected.
6. GHS Core Lab: Report Cytospin slides when used.
7. Refer to Lab 0130 Critical Call Values, Lab Reporting Protocol for specific microbiology critical results
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