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I. Principle: | II. Clinical | III. Specimen: | IV. Materials: | V. Reagents: | VI. Standardization: | VII. Procedure: | VIII. Limitations: | IX. Results | X. Expected | XI. Quality | XII. References:


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Gram Stain

I. Principle:

The Gram Stain is used to numerate cells and numerate and identify the microbiological flora present. Although the exact mechanism of the Gram Stain is unknown, it is thought that the bacterial cell wall composition (specifically the amount of lipid present) is responsible for the characteristic Gram Stain reactions. Crystal violet is the primary stain that all the bacteria take up and iodine acts as a mordant that fixes the crystal violet to the bacteria. When the decolorizing agent (acetone) is applied, the cells with a high lipid content in their cell wall lose this lipid and become more permeable, allowing the purple crystal violet-iodine complex to escape. The bacteria without the high lipid content is dehydrated by the decolorizing agent, become less permeable, and do not lose the dye complex. These dehydrated cells cannot pick up the Safranin counter-stain and will remain purple. These bacteria are referred to as “Gram positive”. The bacteria that become permeable as a result of the decolorizing agent will now stain with Safranin and the resulting color is red. These are “Gram negative” bacteria.

II. Clinical Significance: N.A.

III. Specimen:

Preparation:

1. Write the specimen accession number on the frosted end of the slide with a lead pencil. Do not use a pen or felt tip pen because these inks will wash off during the decolorization step.

2. Specimens submitted on swabs require no sample preparation before making the Gram Stain slide. Make the Gram Stain slide before inoculating bacteriological media. Gently roll the swab over the inscribed circle on the slide taking care to spread thinly. Rolling prevents the disruption of the cellular components in the specimen and the creation of aerosols when working with pathologic material.

3. Mix urine specimens by swirling for 15 seconds. Change the direction of swirling 3 times before placing an aliquot of the urine within the inscribed circle on the slide.
Note: Do not centrifuge urine specimens before Gram Stain preparation or inoculation of media.

Use a 4 mm loop to obtain the necessary aliquot. Smear one loopful of urine over the entire area of the inscribed circle.

4. Centrifuge body fluids (cerebral spinal fluids (CSF), synovial fluid, peritoneal, etc.) at 1000 g for 15 minutes. Cytospin CSF and non-turbid miscellaneous fluids; refer to “Cytospin Preparation of Miscellaneous Body Fluids” within the Miscellaneous Fluids procedure. Observe the following guidelines:

A. Centrifuge in a sealed sterile tube.

B. Do not centrifuge purulent (grossly turbid) material. Prepare Gram Stains directly from the uncentrifuged material.

C. Centrifuge cloudy or slightly turbid fluids and examine the sediment by Gram Stain.

D. Volume guidelines: 15 ml or less of fluid--the entire specimen is centrifuged to obtain the working sediment.
Greater than 15 ml of fluid--the entire specimen is mixed (20 rapid inversions) and 10-15 ml are withdrawn and centrifuged.

E. Decant the supernatant into a sterile tube. Save the supernatant and send it to Microbiology - Cathedral Square. Mix the sediment with the small amount of fluid that drains back from the sides of the tube. Forcefully aspirate the sediment up and down in a sterile pipette several times to adequately disperse the organisms and cells that remain adhered to the bottom of the tube after centrifugation.
Note: Mixing of the sediment is critical. Use a microbiological loop or sterile Pasteur pipette to prepare the slide and inoculate media.

IV. Materials:

1. Clean slides with a frosted end inscribed with a circle by a diamond point pen and sterilized. (Surgipath #299)

2. Staining rack or area

3. Forceps or tweezers

4. Microscope
Note: Our defacto standard for microscope field # is based on a field size defined by a fixed ocular diaphragm of 18 mm diameter. In order to obviate the need for scope-specific procedures, the scopes with larger field of view have been corrected to the standard by means of a custom reticle that has been installed in the right-side oculars. The reticle superimposes a thin circle over the visible field. When counting formed elements, count only those target elements, which fall within the circle.

V. Reagents:

1. Crystal Violet (Fisher #R40073)

2. Gram’s Iodine (Fisher #R40077)

3. Acetone (CardinalHealth #C4300)

4. Safranin (Fisher #R40079)

5. Methanol (CardinalHealth #3041-1)

VI. Standardization: N.A.

VII. Procedure:

1. Allow the slide to air dry at room temperature.
Note: Do not apply heat to speed up the drying process because this causes distortion of cells and may cause aberrant Gram Staining.

2. Fix the slide after drying using either the heat-fix or methanol-fix method:

A. Heat-Fix Method:
Slowly pass the slide through the flame of a Bunsen burner (2 seconds) three times. (The slide should be hot enough to cause discomfort when placed on the back of the hand but it should not burn the hand.) Allow the slide to cool.
OR
Place the slide for one minute on top of a Bacti-Incinerator that has been preheated for 15 minutes. Allow the slide to cool.

Note: The one minute time period is an approximation-a time much shorter than this may result in improper fixing and loss of the specimen during staining. A time much longer than this may result in disintegration of cellular morphology and aberrant Gram Stain results.

B. Methanol-Fix Method:
Place the slide on a staining rack and flood the slide with methanol. Let the slide fix for 1 minute. Drain the alcohol from the slide and allow the methanol to evaporate prior to Gram staining.
OR
Place the slide in a Copeland jar filled with methanol for 1 minute. Allow the methanol to evaporate off the slide before Gram staining.

3. Flood the smear with crystal violet solution and let stand for one minute.
Note: The crystal violet stage of staining is critical. If the stain is allowed to partially dry and precipitate, artifacts resembling Gram positive cocci may occur.

4. Pick the slide up with a forceps or tweezers. Hold the slide parallel to a stream of gently flowing tap water or Type I water and rinse for 5-10 seconds. Drain off any excess water. Place the slide back on the staining rack after rinsing.

5. Flood the slide with iodine and let stand for one minute. Wash with tap water or Type I water as described in step 4.
Note: This stage of the Gram stain is least critical in terms of timing. However, the Gram’s iodine cannot be allowed to completely evaporate from the slide because of artifact formation. If this occurs, discard the slide.

6. Hold the slide with a tweezers or forceps. Allow acetone to run over and off the entire slide until no further color flows from the slide. This usually takes 5-10 seconds, depending on the thickness of the smear. Rinse the slide with tap water or Type I water as described in step 4. Place the slide back on the rack after rinsing.
Note: The decolorization process is the most critical step in the Gram Stain procedure. Over or under decolorization may result in misinterpretation, improper patient therapy, and personal embarrassment.

7. Flood the slide with Safranin and let it stand for 10 seconds in order to counterstain the smear. Rinse the slide with tap water or Type I water as previously described.
Note: Safranin counterstaining is critical because the precipitate may form red rod-like crystals that resemble Gram negative rods.

8. Allow the slide to air dry before examination. If examination is needed before air drying is complete, use the following procedure to blot the slide dry:

A. Lay a paper towel on the countertop.

B. Lay the slide on the paper towel with the stained side up.

C. Gently fold the towel over the slide and lightly press down on the slide to blot any excess moisture from the slide. Do not rub the towel on the front of the slide as this may result in loss of stained specimen.

D. Blotting slides is less desirable than air drying because of the risk of specimen loss and artifact introduction.

9. Examine the stained dry smear under oil immersion.

A. Gram positive organisms are purple.

B. Gram negative organisms are red.

C. Cellular components (epithelial cells and white blood cells) stain red.

D. White blood cells will be best differentiated in thin portions of the smear. Do not try to “guess” the type of white blood cells in thick areas.

10. Reporting of Gram Stain results:
Note: If a Gram Stain is performed on a specimen that has been prepared using the Cytospin, add the comment “Gram Stain performed on a concentrated specimen.” to the Report Comment field of the LIS.

A. Wound, body fluid, other:
Specimen:  Numeration and differentiation of the white blood cells present, numeration and listing of microbiological flora present (presumptive identification when possible). Technologist/site
 
Example:
Left Foot Wound:  Many polys, moderate Gram negative rods. es/UCL

Note: If unable to differentiate the white blood cells present on the Gram Stain, report as: (Numeration, i.e. few) white blood cells (unable to differentiate).

B. Blood Culture:
Specimen: Critical value (canned comment). Gram Stain results are entered using canned comment with the statement “Microbiology workup to follow” (automatically inserted). Tech/Site
 
Example:
Blood Culture: Critical value. Gram Stain: Gram Negative Rods. Microbiology workup to follow. initials/ucl site.

C. Refer to the “Gram Stain; Sputum and Q-Score” procedure.

11. A Gram Stain report is sent to the physician on the same day the specimen is received.

VIII. Limitations: N.A.

IX. Results Derivation:

1. Some infectious agents can be readily identified on a Gram Stain. These agents are presumptively identified and reported to the physician. The bacterial agents commonly seen in infections and the presumptive reports are listed in Table 1.

2. The technologist must be aware of specimens, which contain normal bacterial flora (sputum, vagina) in contrast to those specimens that are normally sterile. This knowledge is used to determine if the bacteria present on a Gram Stain are pathogens or normal endogenous flora. If a specimen contains normal microbiological flora and no obvious predominant bacterial pathogen is seen on the Gram Stain, a report, “mixed bacterial flora” is issued. Table 2 contains a list of specimen sources and pathogenic organisms most likely to be seen on a Gram Stain.

3. Differentiation of white blood cells found on the Gram Stain provides useful information for the clinician. The presence of polymorphonuclear cells may indicate a bacterial infection while the presence of mononuclear cells may indicate a viral, tuberculous, or fungal agent. The CSF glucose is usually depressed in cases of acute bacterial or tuberculous meningitis and normal in the other meningitides. CSF protein is usually elevated in meningitis. Correlate the results of the cell count and chemistries with the Gram Stain results before reporting the Gram Stain results.

4. Use the following guidelines to numerate polymorphonuclear cells, mononuclear cells, and microbiological flora found on a Gram Stain:

Numeration of Cellular Elements and Microbiological Flora

Number / Oil Field

Report as:

0

No

1

Rare

2 - 4

Few

5 - 10

Moderate

≥ 11

Many

X. Expected Result(s) and/or Critical Values: N.A.

Positive Gram Stains of normally sterile fluids are critical values; refer to the Critical Value Chart.

Table 1: Bacterial Agents Commonly Seen in Infections

Organism:

Microscopic Morphology:

Report As:

Staphylococcus

Gram positive cocci in clumps.

Gram positive cocci (suggestive of Staphylococcus).

Streptococcus

Gram positive cocci in chains.

Gram positive cocci (suggestive of Streptococcus).

Streptococcus pneumoniae

Gram positive lancet-shaped cocci in pairs and short chains,

Gram positive cocci (suggestive of Streptococcus pneumoniae).

Haemophilus

Tiny Gram negative rods

Tiny Gram negative rods (suggestive of Haemophilus).

Coliforms

Large Gram negative rods

Gram negative rods (suggestive of coliforms).

Neisseria sp.

Neisseria gonorrhoeae (from genital specimens)

Gram negative intracellular diplococci. (See note 3)

For males:
“Gram negative intracellular diplococci (suggestive of Neisseria gonorrhoeae)”

For females:
“Gram negative intracellular diplococci (suggestive of Neisseria gonorrhoeae) Culture confirmation is necessary.”
(See note 3)

Gram negative extracellular diplococci (See note 3)

For males:
“Gram negative extracellular diplococci (suggestive of Neisseria gonorrhoeae) Culture confirmation is necessary.”

For females:
(If rare, and associated with other bacteria, usually Gram positive rods, report only as):

“mixed bacterial flora.” (See note 3)

Neisseria meningitidis

Gram negative diplococci

Gram negative diplococci (suggestive of Neisseria sp.).

Candida albicans

Yeast with pseudomycelium

Yeast (suggestive of Candida sp.)

Cryptococcus

Encapsulated yeast (See note 2)

Positive for encapsulated yeast (suggestive of Cryptococcus sp.). (See note 2)

Clostridium perfringens

Gram positive rods (See note 1)

Gram positive rods (See note 1) (suggestive of Clostridium perfringens).

Gardnerella vaginalis

Presence of clue cells; vaginal epithelial cells covered by small gram negative rods.

Small gram negative rods (suggestive of Gardnerella vaginalis).

Note(1): In clinical material, spores are rarely seen with C. perfringens. If spores are found, report out Clostridium sp. Clostridium is only reported in specimens from areas that are normally sterile, report as mixed bacterial flora and quantitate.
Note(2): Encapsulated yeast is seen with an India Ink preparation. Spinal fluids are the most common specimen in which encapsulated yeast are seen.
Note(3): A statement regarding the absence or possible presence of Neisseria gonorrhoeae is reported on all genital specimen Gram Stains except placentas. Because of the difficulty of interpretation (eg. overdecolorized Staphylococcus in males and non-Neisseria Gram negative cocci in females) and the social ramifications, a pathologist must be consulted about any Gram Stain report suggestive of Neisseria gonorrhoeae before reporting. If extracellular but no intracellular Gram negative diplococci are present in a specimen from a male patient, the diagnosis should be confirmed by culture. Refer to
Table 1 item F for examples of reporting. For smears of male urethral exudates, positive correlation with culture of N. gonorrhoeae is greater than 95% for smears containing intracellular Gram negative diplococci. In females, however, smears of endocervical secretions detect only 50-70% of culture positive specimens. Thus, both Gram Stained smears and cultures of endocervical secretions should be done.

Table 2: Specimen Sources and Pathogens Most Often Seen as Determined by Gram Stain

Specimens:

Pathogens:

Lower Respiratory See note 1 and note 2

Streptococcus pneumoniae
Haemophilus
Coliforms
Yeast
Staphylococcus

Urinary System

Coliforms
Streptococcus

Genital System See note 1

Candida albicans
Neisseria gonorrhoeae
Gardnerella vaginalis

Gastrointestinal See note 1

Yeast
Staphylococcus

Abscesses (pelvic, culdocentesis, brain, etc…)

Anaerobes
Streptococcus
Staphylococcus

Wounds

   Deep

 

   Superficial

 

Anaerobes

 

Staphylococcus
Streptococcus

Cerebral Spinal Fluid See note 3 and note 4

Haemophilus
Streptococcus pneumoniae
Streptococcus
Cryptococcus neoformans
Neisseria meningitidis
Listeria monocytogenes

Note(1): Normal flora, which consists of many different mixed bacterial types, is seen in the majority of the specimens.
Note(2): Refer to “
Gram Stain; Sputum & Q-Score” procedure.
Note(3): Pathogens most commonly found in spinal fluids are related to the age of the patient.
Note(4): A pathologist must be consulted before any CSF Gram Stain or Methylene Blue Stain containing organisms is reported. If there are >5 polys/mm3 and no organisms are seen, report the results without calling a pathologist and have a pathologist review the slide as soon as possible during regularly scheduled hours.

XI. Quality Control:

1. Stain a control slide each time a new lot of reagent is put into use and weekly thereafter.

2. Prepare control slides in the following manner:

A. From subcultures:

a. Use subcultures (no older than 48 hours) of S. aureus and E. coli or P. aeruginosa to prepare quality control smears.

b. Place two small drops of water onto each of two inscribed circles on a slide labeled + and -.

c. Use a wooden applicator stick to place a portion of the S. aureus subculture into the + circle and E. coli or P. aeruginosa in the - circle. Spread within the respective circles.

d. Allow the slide to dry and stain according to the Gram Stain procedure.

B. From suspension in water:

a. Prepare a heavy suspension of Staphylococcus aureus and E. coli or Pseudomonas aeruginosa in water.

b. Place a drop of the suspensions onto each of two inscribed circles on a slide labeled + and - in the respective circles.

c. Allow the slide to dry and stain according to the Gram Stain procedure.

3. Acceptable results:

A. S. aureus will stain as purple cocci, which are referred to as Gram positive.

B. E. coli or P. aeruginosa will stain as red rods and are referred to as Gram negative.

C. Record the results on the Quality Control card.

4. Corrective action:

A. If the slide does not stain as expected, the following course of action is followed:

a. Do not report out patient results.

b. Prepare and stain a repeat control slide paying particular attention to the decolorization process.

c. Carefully check the reagents to make sure they are acceptable.

d. Follow the “Incidence Recording” procedure.

5. Save Gram Stained smears for a minimum of 30 days. Place the smears, by accession number, in a slide box. Date the outside of the box once it is full and place in the appropriate drawer. Discard after 30 days have elapsed.

XII. References:

1. Balows, A., Manual of Clinical Microbiology, Fifth edition, American Society of Microbiology, Washington, D.C., 1985, pg. 20-21, 1306-1307

2. Use of the Gram Stain in Clinical Infectious Disease, 1978, Schering Corporation

3. Baron E. J., Diagnostic Microbiology, 1990, Mosby, pg. 216-217

 

i. 5-4-79 J. Schaefer

ii. 3-13-86 C. Harms (Revised: I., X.)

iii. January 1991C. Sullivan (Revised: V., X.)

iv. July 1991 E. Steiner (Revised: I., IV.4.E., VII., XI.)

v. August 1992 E. Steiner (Revised: Table 1 & 2, Table 2.note added)

vi. April 1994 C. Harms (Revised: VII.4., Table 1. Note 1 deleted, Note 3 added, Table 2 revised)

vii. August 2005 J.A. Brennan, M.D. (Revised: Table 1: N. gonorrhoeae (genital spec); Note 3)

viii. December 2007 J. Wedig (Revised: VII.2.B. methanol fixation added)

 

Comprehensive Review:

Pathologist:

Technical Director:

 

Interim Review:

September 2009 S. Hosch (no changes)
September 2010 C. Pregler (Revised: III.4.; V.; VII.10.; X.critical value statement)
October 2011 D. Smothers (Revised: V.p/ns; Table 2-note 1;)

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