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1. Gram Staining

2. Objectives & Scope

Gram staining technique is used for staining bacteria, yeasts and aerobic actinomycetes.

This Standard Operating Procedure is applicable to all technical staff in the microbiology section that have been
trained in performing this procedure and are competent and authorized to perform this procedure.

3. Abbreviations and definitions

For general abbreviations, definitions and terms refer to quality manual chapter 1 “General”.

 SOP Standard Operating Procedure

 QM Quality Manual

4. Tasks, responsibilities and accountabilities

For general authorizations refer to the Authorization Matrix.

Task Authorized Responsible

Preparation of reagents LA LM
Preparation of slide LT LM
Staining of slide LT LM
Interpretation of results LT LM
Reporting of results DC LM

5. Principle
The composition of the cell wall of a microorganism determines whether it will retain crystal violet dye or be decolorized
and made visible only with the counterstain, safranin. Those organisms that retain the crystal violet dye will stain blue,
those that do not are stained red with safranin. Blue micro-organisms are Gram positive. Red micro-organisms are Gram
negative.

6. Safety and environment

Safety rules must be taken into consideration when working with potentially infectious patient materials. For general
safety rules refer to the biosafety manual.

Aliquot blood cultures and prepare slides from blood cultures in a biosafety cabinets.

7. Sample
Gram stains can be made from blood culture and solid culture. Of both types of samples only a small quantity is
needed:
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Sample type: Minimal quantity Storage conditions

Blood culture: 1 drop 4°C, room A2, refrigerator 1
Solid culture: 1 colony 4°C, room A2, refrigerator 1
After incubation cultures are stored at 4°C in a refrigerator.
Stains are stored at room temperature.

8. Equipment and supplies

Supplies: Storage location:
Glass slides Room A3, cabinet 1
Syringe with needle Room A3, cabinet 1
Pasteur pipettes Room A3, cabinet 1
Loop Room A3, cabinet 1
Staining rack Room A1, safety hood

Equipment (equipment code): Storage location:

Slide warmer (B001) Room A1, safety hood
Light microscope (B005) Room A1, table

9. Reagents and chemicals

Item (SOP for reference) Storage location Storage condition
Crystal Violet (P23) Room A3, safety cabinet 3 Room temperature
Iodine (P23) Room A3, safety cabinet 3 Room temperature
95% ethanol/acetone (P23) Room A3, safety cabinet 3 Room temperature
Safranin (P23) Room A3, safety cabinet 3 Room temperature
Immersion oil Room A1, table

10. Quality Control

Perform the quality control below once per week. The quality control consists of reading prepared slides with Gram
positive and Gram negative organisms. For Gram positive Staphylococcus aureus is used, for Gram negative E. coli is
used.

Interpretation and acceptable limits:

 Gram Positive S. aureus are visible as blue cocci
 Gram negative E. coli are visible as red bacilli

Processing of the results:

 Record the QC results on the Gram stain QC sheet provided in Annex 1.
 Record unacceptable QS results also on a nonconformity form to initiate problem analysis and corrective
action.
 Review the QC results every month.

Actions in case results are outside acceptable limits (performed by the laboratory technologist):
 Stain new control slides
 If still unacceptable, check the quality of the reagent, check if the staining procedure was followed correctly
and check if the correct reference strains were used.
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11. Procedure
11.1 Preparation of smears
From blood culture:
1. Using a Pasteur pipette or a loop, remove a small aliquot and place a drop or loop ful of blood on a glass slide
2. Place on slide warmer to dry and fix slide for approximately 30 minutes

From colonies:
1. Place a drop of sterile distilled water or saline onto a glass slide
2. Using a 1 µl loop, remove a colony and emulsify in the droplet
3. Place on slide warmer to dry and fix slide for approximately 30 minutes

11.2 Stain procedure

1. Flood dried slide with crystal violet and let stand one minute
2. Rinse with tap water and drain off excess water
3. Flood slide with gram’s iodine and let stand for one minute
4. Rinse with tap water and drain off excess water
5. Decolorize with 95% ethyl alcohol/acetone until most of the crystal violet is removed in thin areas (length of
decolorizing time depends on thickness of smear)
6. Rinse with tap water and drain off excess water
7. Counterstain with safranin for 10 seconds
8. Rinse with tap water and drain off excess water
9. Place on slide warmer until dry or blot gently on paper towel

11.3 Examination
1. Place a drop of immersion oil on the slide
2. Examine using oil immersion (100x) objective
3. Focus using coarse and fine adjustment knobs until objects are in focus

11.4 Results
Interpretation of results:
 Blue organisms – Gram Positive
 Red organisms – Gram Negative
 Yeasts stain Gram Positive
 Background material, cell cytoplasm stain red

11.5 Reporting of results

1. Describe organisms by their Gram reaction (Gram Positive - blue, Gram negative – red) and their microscopic
morphology and arrangement (e.g. cocci in pairs, chains, clusters; bacilli, small, large, filamentous, yeasts). For
example: “Gram positive cocci in chains”
2. Record the findings in the Result Register
3. Follow the normal reporting procedure as described in SOP P4v2 “Reporting of Results”

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11.6 Limitations
1. The length of time of the decolorizing step (ethanol/acetate) is critical. Thin smears require less time than thick
smears. Too much decolorizing will render everything on the slide red; not enough decolorizing will render
everything on the slide blue
2. Gram positive organisms, especially bacilli, from cultures that are not fresh (>48 hrs.) may not retain the crystal
violet and stain red
3. Some species of bacteria are described as “Gram variable” and may stain blue or red or show both colors (e.g.
Gardnerella vaginalis)

11.7 Cleanup
1. Used staining liquid should be discarded in the liquid waste chemical containers
2. Gram smears should be discarded in the autoclave bin

12. Related document

 QM1 “General”
 Biosafety Manual, present in every laboratory room and the administration office
 SOP P23 “Preparation of Gram staining reagents
 SOP E4 “Use and maintenance of light microscope”
 SOP P4 “Reporting of results”

13. Related Forms

 P4F1 “Result report”
 Patient register, secretariat

14. References
 Manual of Clinical Microbiology, Chapin, KC, Lauderdale, T., ASM Press, 2003, Washington, DC. Chapter:
Reagents, Stains and Media.
 Patient Safety Monitoring & International Laboratory Evaluation portal. Accessed on February 20, 2013.
Available at: http://resources.psmile.org/resources/process-control/section-specific-
information/microbiology/bacteriology/Pro6.7-A-12%20Gram%20Stain%20%20.doc/view

15. Attachments
 Annex 1 “Quality Control Sheet Gram Stain”

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Annex 1: Quality Control Sheet Gram Stain

Acceptable results:
 Gram positive: blue cocci (Staphylococcus aureas)
 Gram negative: red bacilli (E. coli)
Record all unacceptable quality control results on a nonconformity to start the problem analysis and corrective
action process.

Year: ________

Quality Lot number Lot number Lot number Result Quality control done
control date Crystal violet Iodine Safranin by:
Gram Gram
positive negative

Signature of laboratory manager for review: Date: Signature: