Gram staining- Principle, Procedure and Result

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Overview:  

The Gram staining was developed by Danish physician Christian Gram in 1884; he is the most widely employed staining method in bacteriology. Gram staining is the common and most important differential staining technique for microbiology. Gram’s staining helps to differentiate whether the bacteria are Gram positive or Gram Negative and is used to classify bacteria on the basis of their forms, sizes, cellular morphologies but does not help to identify the exact genus and species

Purpose:

To differentiate the bacteria into Gram positive and Gram negative based on the presents of cell wall composition.

Principle:

The difference in Gram positive and Gram negative bacterial cell wall bacteria is base on cell well composition. Gram positive cell wall contains a thick layer of peptidoglycan with teichoic acid cross linking and low lipid content, which closes the pores in the cell wall and prevents the stain existing the cell, its resists decolorization of crystal violet and iodine complex, that is help to bound to the thick layer of peptidoglycanof Gram positive bacteria and appear purple in color. The Gram negative bacterial cell wall binds with crystal violet and iodine complex bud due to the thin layer of peptidoglycan and thick outer layer which is formed of lipids, crystal violet and iodine complex gets washed off. The decolorizer dissolves the outer lipid layer during the decolorization, the stained with safranin, they take the stain and appears red or pink color.

Sample container:  Sterile containers or sterile swabs

Sample type:

Urine

Sputum

Throat swab

Pus

CSF & other body fluids

Wound swab

Vaginal swab

Urethral discharge

Required equipment and reagents:

Equipments:

        Bunsen burner

        Biological safety cabinet

        Light Microscope - 1000 magnification (oil immersion – lens)

Reagents:

        Sterile normal saline

        Gram’s stain

        Crystal Violet

        Gram’s Iodine

        Decolourizer (Acetone or ethanol)

        Safranin

        Cedar wood oil / Immersion oil

Procedure:

1. Make a thin smear of the specimen on a clean-labelled slide and allow it to air dry. Fix the smear by gentle heat.

2.  Add Crystal violet and kept for 1 minute, Pour off the stain and wash with water

3.    Flood with Gram's Iodine solution for 1 minute and wash with water.

4.  Then pour decolourizer agent (95% Acetone or ethanol) over the smear for about 10 seconds, till the purple color runs off. Wash quickly with tap water. Usually this is sufficient for satisfactory decolourization, though thick smears may require repetition of this step. Shake off the excess water.

5. Flood the smear with Safranin for 1 minute, Pour off the stain and wash with tap water. 

6.   Air dries the water and Observe under oil immersion – lens.

Quality control procedures

Check the reliability of stains with known bacteria / ATCC strains of bacteria whenever new batches of stain are used

Gram positive cocci: Staphylococcus aureus 25923

Gram negative bacilli: Escherichia coli 25922

Results and interpretation:

           Gram Positive bacteria appear Violet or Purple color.

           Gram Negative Bacteria appear Red or Pink color.

 

  

Examples of Gram positive bacteria:

Gram positive cocci: Staphylococci sp., Streptococci sp., Enterococci sp., etc,.

Gram positive bacilli: Bacillus sp., Clostridium sp., Lactobacillus sp., Corynebacterium sp. and etc,.

Examples of Gram positive bacteria:

Gram negative cocci: Neisseria sp., Moraxella sp., Acinetobacter sp., and etc. 

Gram negative bacilli: E. coli, Klebsiella sp., Salmonella sp., Pseudomonas sp., and etc.

Gram Staining Interpretation for Vaginal Swab

Assign scores, add up total score and interpret the results.

Organism Morphotype

Number/oil immersion

Score

Lactobacillus-like (parallel-sided, Gram-positive rods)

>30

5-30

1-4

<1

0

0

1

2

3

4

Mobiluncus –like (curved, Gram negative rods)

>5

<1-4

0

2

1

0

Gardnerella/Bacteriodes-like (tiny, gram variable coccobacilli and rounded, pleomorphic, gram negative rods with vacuoles)

>30

5-30

1-4

<1

0

4

3

2

1

0

 

Score

Interpretation

0-3

Normal

4-6

Intermediate, repeat test later

7-10

Bacterial Vaginosis

 

Gram Staining Interpretation for Sputum samples

The Bartlett score measure the quality of sputum for microbiology testing

Bartletts grading:

No. of neutrophils per 10X low power field

Grade

<10

0

10-25

+1

>25

+2

Presence of mucus

+1

No. of epithelial cells per 10X low power field

Grade

10-25

-1

>25

-2

 A final score of 0 or less indicates lack of active inflammation or contamination with saliva.

References

  1. Practical Medical Microbiology by Mackie & McCartney 14th Edition, Page No-796 – 798.
  2. Koneman’s Color Atlas and textbook of Diagnostic Microbiology, Sixth edition by Washington et al., 2006.


 

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