Gram stain protocols provide essential techniques for microbiology students to differentiate between gram-positive and gram-negative bacteria. This guide outlines the historical background of the Gram stain, detailing its development by Hans Christian Gram in 1884. It includes a comprehensive step-by-step protocol for performing the Gram stain, emphasizing the importance of each reagent and the chemical mechanisms involved. Ideal for laboratory courses, this resource aids students in understanding bacterial cell wall structures and their implications in medical microbiology. The document also discusses common pitfalls and variations in Gram staining results, making it a valuable tool for accurate bacterial identification.

Key Points

  • Explains the historical significance of the Gram stain developed by Hans Christian Gram in 1884.
  • Details the four essential steps of the Gram staining procedure, including primary staining, mordant application, decolorization, and counterstaining.
  • Describes the chemical mechanisms that differentiate gram-positive and gram-negative bacteria based on cell wall structure.
  • Highlights common errors in Gram staining and their impact on bacterial identification accuracy.
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9 pages
Microbiology
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Microbiology
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Gram Stain Protocols
| |
Created: Friday, 30 September 2005
Author
• Ann C. Smith
• Marise A. Hussey
Information
History
The Gram stain was first used in 1884 by Hans Christian Gram
(Gram,1884). Gram was searching for a method that would allow
visualization of cocci in tissue sections of lungs of those who had died of
pneumonia. Already available was a staining method designed by Robert
Koch for visualizing turbercle bacilli. Gram devised his method that used
Crystal Violet (Gentian Violet) as the primary stain, an iodine solution as
a mordant followed by treatment with ethanol as a decolorizer. This
staining procedure left the nuclei of eukaryotic cells in tissue samples
unstained while the cocci found in the lungs of those who had succumbed
to pneumonia were stained blue/violet. Gram found that his stain worked
for visualizing a series of bacteria associated with disease such as the
“cocci of suppurative arthritis following scarlet fever”. He found however
that Typhoid bacilli were easily decolorized after the treatment with
crystal violet and iodine, when ethanol was added. We now know that
those organisms that stained blue/violet with Gram’s stain are gram-
positive bacteria and include Streptococcus pneumoniae (found in the
lungs of those with pneumonia) and Streptococcus pyogenes (from
patients with Scarlet fever) while those that were decolorized are gram-
negativebacteria such as the Salmonella Typhi that is associated with
Typhoid fever.
Purpose
The Gram stain is fundamental to the phenotypic characterization of
bacteria. The staining procedure differentiates organisms of the domain
Bacteria according to cell wall structure. Gram-positive cells have a thick
peptidoglycan layer and stain blue to purple. Gram-negative cells have a
thin peptidoglycan layer and stain red to pink.
Theory
The Gram stain, the most widely used staining procedure in bacteriology,
is a complex and differential staining procedure. Through a series of
staining and decolorization steps, organisms in the Domain Bacteria are
differentiated according to cell wall composition. Gram-positive bacteria
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have cell walls that contain thick layers of peptidoglycan (90% of cell
wall). These stain purple. Gram-negative bacteria have walls with thin
layers of peptidoglycan (10% of wall), and high lipid content. These stain
pink. This staining procedure is not used for Archeae or Eukaryotes as
both lack peptidoglycan. The performance of the Gram Stain on any
sample requires four basic steps that include applying a primary stain
(crystal violet) to a heat-fixed smear, followed by the addition of a
mordant (Gram’s Iodine), rapid decolorization with alcohol, acetone, or a
mixture of alcohol and acetone and lastly, counterstaining with safranin.
Details of the chemical mechanism of the Gram stain were determined in
1983 (Davies et al.,1983 and Beveridge and Davies, 1983). In aqueous
solutions crystal violet dissociates into CV
+
and Cl
–
ions that penetrate
through the wall and membrane of both gram-positive and gram-
negative cells. The CV
+
interacts with negatively charged components of
bacterial cells, staining the cells purple. When added, iodine (I
-
or I
3
-
)
interacts with CV
+
to form large CVI 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 the membranes of both
gram-positive and gram-negative Bacteria. The outer membrane of the
gram-negative cell is lost from the cell, leaving the peptidoglycan layer
exposed. Gram-negative cells have thin layers of peptidoglycan, one to
three layers deep with a slightly different structure than the
peptidoglycan of gram-positive cells (Dmitriev, 2004).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. The multi-layered nature of the peptidoglycan
along with the dehydration from the ethanol treatment traps the large
CV-I complexes within the cell. After decolorization, the gram-positive
cell remains purple in color, whereas the gram-negative cell loses the
purple color and is only revealed when the counterstain, the positively
charged dye safranin, is added. At the completion of the Gram stain the
gram-positive cell is purple and the gram-negative cell is pink to red.
Some bacteria, after staining with the Gram Stain yeild a pattern called
gram-variable where a mix of pink and purple cells are seen. The
genera Actinomyces, Arthrobacter, Corynebacterium,
Mycobacterium, and Propionibacterium have cell walls particularly
sensitive to breakage during cell division, resulting in gram-negative
staining of these gram-positive cells. In cultures of Bacillus,
Butyrivibrio, and Clostridium a decrease in peptidoglycan thickness
during growth coincides with an in increasing number cells that stain
gram-negative (Beveridge, 1990). In addition, in all bacteria stained
using the Gram stain, the age of the culture may influence the results of
the stain.
Some bacteria do not stain as expected with the Gram stain. For
example, members of the genusAcinetobacter are gram-negative cocci
that are resistant to the decolorization step of the Gram
stain.Acinetobacter spp. often appear gram-positive after a well prepared
Gram stain (Visca et al. 2001). For Mycobacterium
spp., the waxy nature
of the coat renders the bacteria not readily stainable with dyes used in
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American Society for Microbiology © 2016 3
the Gram stain, though the bacteria are considered to be gram positive
(Saviola and Bishai, 2000). Gardnella has an unusual gram-positive cell
wall structure that causes bacteria of this genus to stain gram-negative
or gram-variable (Sadhu et al 1989).
Misinterpretation of the Gram stain has led to misdiagnosis or delayed
diagnosis of infectious disease (Visca et al., 2001, Noviello et al., 2004 )
RECIPE
(Gephardt et al., 1981)This is Hucker’s modification of the Gram Stain
method. Gram originally used Gentian Violet as the primary stain in the
Gram stain. Crystal violet is generally used today. In Hucker’s method
ammonium oxalate is added to prevent precipitation of the dye
(McClelland, 2001) and uses an alcoholic solution of the counterstain.
Burke’s modification of the Gram Stain adds sodium bicarbonate to the
crystal violet solution. Sodium bicarbonate prevents the acidification of
the solution as iodine oxidizes (McClelland, 2001) and uses an aqueous
solution of Safranin for the counterstain (Gephardt et al., 1981).
The reagents listed below can be made or purchased commercially from
biological supply houses
1. Primary Stain: Crystal Violet Staining Reagent.
Solution A for crystal violet staining reagent
Crystal violet (certified 90% dye content), 2g
Ethanol, 95% (vol/vol), 20 ml
Solution B for crystal violet staining reagent
Ammonium oxalate, 0.8 g
Distilled water, 80 ml
Mix A and B to obtain crystal violet staining reagent. Store for 24 h
and filter through paper prior to use.
2. Mordant: Gram's Iodine
Iodine, 1.0 g
Potassium iodide, 2.0 g
Distilled water, 300 ml
Grind the iodine and potassium iodide in a mortar and add water slowly
with continuous grinding until the iodine is dissolved. Store in amber
bottles.
3. Decolorizing Agent
Ethanol, 95% (vol/vol)
*Alternate Decolorizing Agent
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FAQs

What are the main steps involved in the Gram staining process?
The Gram staining process involves four main steps: applying a primary stain (crystal violet), adding a mordant (Gram's iodine), decolorizing with ethanol or acetone, and finally counterstaining with safranin. Each step is crucial for differentiating between gram-positive and gram-negative bacteria. The primary stain colors all cells purple, while the mordant helps fix the dye. The decolorization step is critical, as it determines whether the cells retain the purple color or become colorless, allowing the counterstain to reveal the gram-negative cells in pink.
What factors can affect the accuracy of Gram staining results?
Several factors can influence the accuracy of Gram staining results, including the thickness of the bacterial smear and the age of the culture. A thick smear may lead to uneven staining, while older cultures can exhibit gram-variable results due to weakened cell walls. Additionally, over-decolorization can cause gram-positive bacteria to appear gram-negative, while under-decolorization may result in false positives. Using fresh reagents and ensuring a proper technique during the staining process are essential for reliable results.
What is the significance of differentiating between gram-positive and gram-negative bacteria?
Differentiating between gram-positive and gram-negative bacteria is crucial in microbiology and clinical diagnostics. Gram-positive bacteria have thick peptidoglycan layers, which retain the crystal violet stain, while gram-negative bacteria possess a thin peptidoglycan layer and an outer membrane that allows them to be decolorized. This distinction not only aids in bacterial identification but also informs treatment decisions, as gram-negative bacteria are often more resistant to antibiotics. Understanding these differences is vital for effective infection control and management.
What are some common mistakes made during the Gram staining procedure?
Common mistakes during the Gram staining procedure include improper timing during the decolorization step and using outdated reagents. Over-decolorization can lead to gram-positive bacteria appearing gram-negative, while under-decolorization may result in gram-negative bacteria appearing gram-positive. Additionally, not preparing a thin smear can cause uneven staining and misinterpretation of results. It is essential for students to practice proper techniques and use controls to ensure accurate Gram staining outcomes.

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