What is Gram stain?
"Gram staining" is a technique to stain the colorless bacteria so as to observe them under the microscope. This staining technique distinguishes bacteria into two large groups based on their cell wall characteristics. Hans Christian Gram, who developed this method to stain bacteria in 1884 named this technique as Gram staining. Gram observed that bacteria possess either thick or thin cell walls. He used his stain, Gentian Violet, to examine the round bacteria in the lungs of pneumonia patients.
In 1921, G. J. Hucker modified the components of Gram staining, substituting Crystal Violet for Gentian Violet. He prepared the Crystal Violet staining reagent by mixing Crystal Violet with ethanol and ammonium oxalate to enhance its stability.
Purpose of Gram Staining
Gram staining, a differential staining
process, is utilized to differentiate bacteria with thick cell walls from those
with thin cell walls. The cell wall, is made up of peptidoglycan made up of sugar and amino acid molecules, serves
as the outermost protective layer of bacteria.
Gram positive bacteria with thick cell wall (up to 80 nm) has peptidoglycan as 90% of cell wall's dry weight and it also contains teichoic acid. The teichoic acid is present only in gram positive bacteria and provides flexibility to the cell wall. Thick cell walled bacteria after staining with the Crystal Violet reagent retain the violet color and appear blue or violet. The high thickness of the cell wall traps the stain, maintaining the color even after washing with ethanol, hence termed Gram-positive bacteria.
On the other hand, Gram negative bacteria with thin (7 to 8 nm) cell walls has 10% peptidoglycan. Gram-negative bacteria after staining with crystal violet when de-stained with ethanol, becoming colorless. Gram negative bacteria also has an outer membrane enveloping the cell wall.
These colorless Gram-negative bacterial cells can
be stained with a counterstain called Safranin, adding a red or pink color to the cell wall. This staining procedure is not applicable to eukaryotes and
archaea, as they lack a cell wall.
How is Gram staining useful to microbiologists or doctors for treating patients?
Gram staining results can help doctors in selectively choosing antibiotic treatment against specific infection causing agents. Most commonly available antibiotics, such as penicillin and cephalosporins, prevent bacterial cell wall synthesis, leading to bacterial death. Other antibiotics that inhibit protein synthesis can easily enter Gram-positive bacterial cells because they lack the outer membrane and the cell wall is not selectively permeable.
However, Gram-negative bacteria possess an outer membrane. The cell wall is situated between the outer and inner membranes. The outer membrane is selectively permeable, preventing the entry of antibiotics or expelling them out using outer membrane proteins This protects the cell wall from antibiotic action. Therefore, it is crucial for doctors to identify the bacteria causing the infection before initiating treatment. Random trials can adversely impact treatment outcomes and promote antibiotic resistance, which can be dangerous.
Reagents used for Gram staining
1. The primary stain is Crystal Violet reagent,
consisting of Solution A and Solution B:
Solution
A: 2 grams
of Crystal Violet mixed in 20 ml of 95% ethanol.
Solution
B: 8 grams
of Ammonium oxalate mixed in 80 ml of distilled water.
Mix
Solution A and B to prepare the Crystal Violet staining reagent and filter it
through filter paper before use.
2. Gram's Iodine is utilized as the Mordant,
composed of 1 gram of iodine and 2 grams of potassium iodide mixed in 300 ml of
distilled water.
3. Decolorizer (95% Ethanol) is employed for
de-staining bacterial cells. Alternatively, a mixture of ethanol and acetone
(1:1) can be used.
4.
The counterstain (Safranin) is used to stain
the decolorized cells:
Stock solution: 2.5
grams of Safranin mixed in 100 ml of 95% ethanol.
Working solution: Mix 10
ml of stock solution with 90 ml of distilled water.
1. Take a sample containing bacteria and make a smear
on a clean glass slide.
2.
Air dry the smear and heat-fix
the bacterial cells.
3.
Flood the smear with Crystal Violet (CV)
staining reagent for 1 minute.
(The
Crystal Violet reagent dissociates into positively charged CV and negatively
charged Cl. The CV+ interacts with negatively charged components of the cell
wall and cell membrane, staining the cell.)
4. Wash the slide with an indirect stream of tap water
for 5 seconds to remove excess Crystal Violet stain.
5.
Flood the smear with Gram’s Iodine for 1
minute.
(Iodine
combines with Crystal Violet absorbed in the peptidoglycan layers of the cell
wall, to form the Crystal Violet-Iodine “CV-I” complex.)
6.
Decolorize the smear drop-wise with
decolorizer until the violet color stops appearing.
(Decolorizer
dehydrates and shrinks the cell wall, making it difficult for the large CV-I
complex to leach out from the multiple layers of thick-walled Gram-positive
bacteria. However, this complex can easily leach out from the thin peptidoglycan
layers of Gram-negative bacteria, making them colorless.)
7. Flood the smear with positively charged Safranin
(counterstain) for 30 seconds to stain any colorless Gram-negative bacteria
present.
8.
Wash the slide with an indirect stream of tap water
for 5 seconds to remove excess Safranin.
9. Observe the results under the microscope at 40X or
using oil immersion at 100X magnification.
Result:
Gram-positive bacteria
will appear blue or violet.
Gram-negative bacteria
will appear red or pink in color.
How does the age of bacteria change Gram stain results?
Fresh samples or bacterial cultures should be used
for staining because old cultures/samples contain a greater number of dead and
damaged cells, which adversely affect their ability to uptake stains, thereby
altering staining patterns.
Does Gram staining work for all bacteria?
Gram stain is not applicable to microorganisms without
a cell wall, such as Mycoplasma, Ureaplasma, and some smaller bacteria like
Chlamydia and Rickettsia (although Gram-negative), but these bacterial cells
may weakly uptake the counterstain.
Factors affecting Gram staining include:
·
The age of the culture,
· Samples from patients undergoing prolonged
antibiotic treatment (which may impair cell wall synthesis or increase the
number of dead cells, leading to false results),
·
Excessive heat fixation during staining,
·
Excessive decolorization of the smear, and
·
Insufficient use of Iodine.
Can we differentiate between Gram-positive and Gram-negative bacteria without using Gram staining?
In liquid culture media, Gram-positive and
Gram-negative bacteria can be differentiated easily by using sodium
hydroxide (3%). Gram-negative bacteria cells easily brake down in presence of sodium hydroxide due to their thin
peptidoglycan cell wall, releasing a thick, viscous mixture of cytoplasm and
DNA. Conversely, the thick cell wall of Gram-positive bacteria resists the
action of NaOH, preventing lysis.
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