Gram Staining: Principle, Procedure, Results

Danish physician Hans Christian Gram developed the Gram staining method in 1884. Gram staining procedure uses four chemicals; crystal violet, iodine, alcohol, and safranin, to stain bacteria

Gram staining is still the cornerstone of bacterial identification and taxonomic division. This differential staining technique separates most bacteria into two groups based on cell wall composition.

  1. Gram-positive bacteria- stains purple
  2. Gram-negative bacteria-stains red/pink

Nearly all clinically important bacteria can be visualized using the Gram staining technique, the only exceptions being those organisms;

  1. That exists almost exclusively within host cells, i.e., intracellular bacteria (e.g., Chlamydia)
  2. Those that lack a cell wall (e.g., Mycoplasma)
  3. Those of insufficient dimensions to be resolved by light microscopy (e.g., spirochetes)

Steps of Gram Staining

Classic Gram staining techniques involve the following steps:  

An easy way to remember the steps of the Gram stain
Come In and Stain! is an easy way to remember the steps of the Gram stain
  1. Fixation of clinical materials to the surface of the microscope slide either by heating or by using methanol. (Methanol fixation is recommended rather than heat fixation. Methanol fixation preserves the morphology of host cells and bacteria. Heating the slide causes cell distortion, could increase cell debris, and may cause erroneous Gram stain results.).
  2. Application of the primary stain (crystal violet). Crystal violet is a dark blue to purple dye. It stains all cells blue/purple.
  3. Application of mordant: The iodine solution (mordant) is added to form a crystal violet-iodine (CV-I) complex; all cells continue to appear blue.
  4. Decolorization step: The decolorization step distinguishes gram-positive from gram-negative cells. The organic solvent such as acetone or ethanol extracts the blue dye complex from the lipid-rich, thin-walled gram-negative bacteria to a greater degree than from the lipid-poor, thick-walled, gram-positive bacteria.  The gram-negative bacteria appear colorless, and gram-positive bacteria remain blue.
  5. Application of counterstain (safranin): The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue.

Find information and process for the Preparation of Gram Staining Regent

Principle of Gram Stain

Bacterial cell wall
Image 2: Cell wall of Gram-positive and Gram-negative Bacteria

The differences in Gram-positive and Gram-negative bacteria cell wall composition account for the Gram staining differences. Gram-positive cell wall contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking, which resists decolorization.

In aqueous solutions, crystal violet dissociates into CV+ and Cl – ions that penetrate through Gram-positive and Gram-negative cell walls. The CV+ interacts with negatively charged components of bacterial cells, staining the cells purple. When added, iodine (I- or I3-) interacts with CV+ to form large crystal violet-iodine (CV-I) complexes within the cytoplasm and outer layers of the cell.

The decolorizing agent (ethanol or an ethanol and acetone solution) interacts with the lipids of both gram-positive and gram-negative bacteria membranes.

  • The outer membrane of the Gram-negative cell is lost from the cell, leaving the thin peptidoglycan layer exposed. With ethanol treatment, gram-negative cell walls become leaky and allow the large CV-I complexes to be washed from the cell.
  • The highly cross-linked and multi-layered peptidoglycan of the gram-positive cell is dehydrated by the addition of ethanol. Thus ethanol treatment traps the large CV-I complexes within the cell.

After decolorization, the gram-positive cell remains purple. In contrast, the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.

Procedure of Gram Staining

 Smear Preparation

Fix material on a slide with methanol or heat. If the slide is heat fixed, allow it to cool to the touch before applying the stain.

Methanol fixation

Place or hold the slide over a paper towel and flood the slide with absolute methanol for two minutes. Alternatively, you may dip the slide into a Coplin jar filled with methanol.

Once two minutes have passed, tilt the slide and drain off the excess methanol and let the slide air dry. Do not wipe or blot the slide, as this can remove cells.

gram-stain-procedure
Image 3: Procedure of Gram Staining; note the color change after each step

If you are struggling to remember the staining reagents used in this procedure and their order you can remember this sentence “Come In And Stain” i.e. the order is Crystal violet, Iodine, Alcohol/Acetone and the final one is Safranin.

Gram Staining Procedure

  1. Flood air-dried, heat-fixed smear of cells for 1 minute with crystal violet staining reagent. Please note that the quality of the smear (too heavy or too light cell concentration) will affect the Gram Stain results.
  2. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
  3. Flood slide with the mordant: Gram’s iodine. Wait 1 minute.
  4. Wash slide in a gentle and indirect stream of tap water for 2 seconds.
  5. Flood slide with decolorizing agent (acetone-alcohol decolorizer). Wait 10-15 seconds or add drop by drop to slide until the decolorizing agent running from the slide is clear.
  6. Flood slide with a counterstain, safranin. Wait 30 seconds to 1 minute.
  7. Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent.
  8. Allow the slide to air dry by tilting it onto a paper towel or over a sink. Alternatively, gently dry the slide by blotting it using a lint-free bibulous paper. Please do not use a wiping motion, as it can remove the smear.
  9. The slide is now ready to view under the microscope. First, focus on the image using the high dry objective lens marked 40x. Then, without removing the slide, switch to the high-power oil immersion objective lens marked 100x. Use immersion oil and observe the staining procedure’s results under oil immersion (100x) using a bright field microscope. This will result in an overall magnification of 1,000x.

After performing a gram stain, the technician should first determine whether the Gram stain is adequate. In an appropriately stained biological specimen, the nuclei of neutrophils are red. If the nuclei are blue, the decolorization is insufficient.

Results

  • Gram-negative bacteria will stain pink/red and
  • Gram-positive bacteria will stain blue/purple.
Gram Staining Procedure

Reporting Gram smears

The report should include the following information:

Staphylococcus in Gram Stain
Staphylococcus in Gram Stain
  • Numbers of bacteria present, whether many, moderate, few, or scanty
  • Gram reaction of the bacteria, whether Gram-positive or Gram-negative
  • Morphology of the bacteria, whether cocci, diplococci, streptococci, rods, or coccobacilli. Also, whether the organisms are intracellular.
  • Presence and number of pus cells
  • Presence of yeast cells and epithelial cells.

Example
A urethral smear report might read: ‘Moderate numbers Gram-negative intracellular diplococci and many pus cells.’

Limitations

The sensitivity of the Gram stain procedure is low. Sometimes, you may fail to see the organism in Gram Stain smear, but the same clinical specimen may yield organisms when cultured. To be visible on a slide, organisms that stain by the Gram method must be present in about 10to 105 organisms per milliliter of centrifuged fluid.

Gram staining technique is not recommended for spirochetes and mycobacteria. Mycobacteria stain weakly with gram stain, and bacteria such as Mycoplasma, Rickettsiae, Chlamydiae do not take up the dyes used in Gram stain or are too small to be seen with light microscopy.

Not all bacteria can be seen in the Gram stain. This is the list of medically important bacteria that can be not been in Gram-stain.

NameReasonAlternative Microscopic Approach
Chlamydiae, including C. trachomatisIntracellular; very smallInclusion bodies in the cytoplasm
Legionella pneumophilaPoor uptake of red counterstainProlong time of counterstain
Mycoplasma pneumophilaPoor uptake of red counterstainNone
Mycobacterium, including M. tuberculosisToo much lipid in cell wall so dye cannot penetrateAcid-fast stain
RickettsiaeIntracellular; very smallGiemsa or other tissue stains
Treponema pallidumToo thin to seeDark-field microscopy or fluorescent antibody

Quality Control

Always check new batches of stain and reagents for correct staining reactions using a smear containing known Gram-positive and Gram-negative organisms.

Variations in Gram Reaction

Various factors influence the results of Gram staining. Sometimes the result might be entirely different than you have anticipated. 

  1. Gram-positive bacteria may lose their ability to retain crystal violet and stain Gram negatively for the following reasons:
    • cell wall damage of bacteria due to antibiotic therapy or excessive heat fixation of the smear.
    • over- decolorization of the smear
    • use of an old Iodine solution that is yellow in color instead of brown (always store in a brown glass or other light opaque containers).
    • preparation of smears from old culture.
  2. A thick smear will require more decoloration than a thin smear. When the smear is too thick, Gram-negative bacteria may not be fully decolorized during decolorization steps and appear as Gram-positive.

Pitfalls in the Interpretation of Gram’s Stains

OrganismClassic PresentationVariant PresentationComments
Streptococccus pneumoniaeGram-positive, lancet-shaped, diplococciElongated cocci, resembling short bacilliMay be misinterpreted as mixed organisms; over-decolorized cells may be mistaken for gram-negative coccobacilli.
Acinetobacter spp.Gram-negative coccobacilliGram-negative cocci; gram-variable staining is commonMay be mistaken for Neisseria spp. and reported as gram-negative cocci; search the smear to find some organisms that demonstrate elongated forms, which are not seen in Neisseria.
Clostridium perfringensBoxcar-shaped gram-positive bacilliGram-positive cocciMay be mistaken for Streptococcus pneumoniae and reported as gram-positive cocci; in addition to a coccal form, cells retain crystal violet tenaciously during decolorization.
Clostridium perfringensBoxcar-shaped gram-positive bacilliGram-variable or Gram-negative bacilliMaybe mistaken for gram-negative bacilli; the boxcar shape is a clue that the organism is gram-positive; other Clostridia and Bacillus spp. May also appear similar.
Yeast, especially Cryptococcus neoformansGram-positive round or oval cells with buddingGram-variable cellsMay be mistaken for artifacts; size and shape distinguish them from bacteria.

References and further reading

Acharya Tankeshwar

Hello, thank you for visiting my blog. I am Tankeshwar Acharya. Blogging is my passion. As an asst. professor, I am teaching microbiology and immunology to medical and nursing students at PAHS, Nepal. I have been working as a microbiologist at Patan hospital for more than 10 years.

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