Staining

Gram Staining Procedure, Principle, and Results

Gram-staining used to differentiate between Gram-Positive and Gram-Negative Bacteria.

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This article writter by MN Editors on March 28, 2021

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Gram Staining Procedure, Principle, and Results
Gram Staining Procedure, Principle, and Results

Overview of Gram Stain

  • The importance of the Gram stain in the history of microbiology cannot be overstated.
  • The Gram stain reaction was for many years one of the critical pieces of information used by bacterial taxonomists to construct taxa, and it is still useful in identifying bacteria in clinical settings.
  • The initial researches are done to distinguish bacteria that stained Gram-positive from those that stain Gram-negative were done using model organisms such as Bacillus subtilis (Gram-positive) and Escherichia coli (Gram-negative).
  • At the time, it was believed that all other bacteria would have similar cell wall structures.
  • After Christian Gram revealed the Gram stain in 1884, it soon became visible that most bacteria could be classified into two main groups based on their response to the Gram-staining method.
  • Gram-positive bacteria colored purple, whereas Gram-negative bacteria were pink or red.
  • The difference between typical Gram-positive bacteria and typical Gram-negative bacteria is due to the physical nature of their cell walls.
  • If the cell wall is removed, typical Gram-positive bacteria stain Gram negative. Furthermore, bacteria that never make cell walls, such as mycoplasmas, also stain Gram negative.

Objective of Gram Staining

In Microbiology Gram-staining is the most important differential staining technique. It used to differentiate between Gram-Positive and Gram-Negative Bacteria, which assists in the analysis and differentiation of different microorganisms. In 1884, a Danish Bacteriologist Hans Christian Gram first introduced this stain, that’s why it’s called gram staining.

During gram-staining, the thick cell walls of gram-positive bacteria retain the purple color of primary dye Crystal viole, while the thin cell wall of gram-negative bacteria retain the red/pink color of counterstain Safranin.

Gram staining Principle or Mechanism

  • In gram staining, the bacteria were first treated with the primary stain known as crystal violet.
  • Crystal violet is a positively charged dye that attracted to the bacterial cell’s net negative charge. 
  • In an aqueous solution, Crystal violet (CV) dissociates into CV+ and Cl– ions. Both of these ions can penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells.
  • The negatively charged components of bacterial cell interacts with the CV+ ions and stains the cells purple.
  • In the second step it treated with mordant (mordant is a substance that helps bind the dye tightly to the cell wall ), the Gram’s iodine, it interacts with the CV+ of crystal violet, Making an insoluble complex(CV-I) and thus increasing dye retention.
  • During decolorization step, when the bacteria treated with ethanol, the pores in peptidoglycan layer of gram-positive bacteria started to shrink, as a result, the peptidoglycan prevents the loss of crystal violet from the cell wall.
  • Therefore, the dye-iodine complex is held during the decolorization step and the bacteria remain purple, indeed after the addition of a second dye.
  • On the other hand, the gram-negative bacteria contain a very thin peptidoglycan layer, not highly crossed linked and the pores of peptidoglycan are larger as compared to the gram-positive bacteria. 
  • Hence when t treated with alcohol may extract enough lipid from the outer membrane to increase the cell wall’s porosity further.
  • As a result the alcohol more readily removes the crystal violet–iodine complex and decolorizing the cells.
  • When the cells are treated with negative charge counterstain safranin or secondary stain, It easily stains the decolorized gram-negative cells, as a result of that, they appear red or pink.

Reagents Required for Gram Staining

The Gram Stain technique has been modified by Hucker. Initially, Gentian Violet was utilised as the principal stain in the Gram stain. Today, crystal violet is commonly utilised. In Hucker’s approach, ammonium oxalate is added to prevent dye precipitation (McClelland, 2001), and the counterstain is dissolved in alcohol. Burke modifies the Gram Stain by including sodium bicarbonate into the crystal violet solution. As iodine oxidises, sodium bicarbonate prevents the solution from becoming acidic (McClelland, 2001), and an aqueous solution of Safranin is used as a counterstain.

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 oxalate0.8 g
Distilled water80 ml

Crystal Violet Staining Reagent Preparation

  1. Mix A and B to obtain crystal violet staining reagent.
  2. Store for 24 h and filter through paper prior to use.

2. Mordant: Gram’s Iodine

Iodine1.0 g
Potassium iodide2.0 g
Distilled water300 ml

Gram’s Iodine Preparation

  1. Grind the iodine and potassium iodide in a mortar and add water slowly with continuous grinding until the iodine is dissolved.
  2. Store in amber bottles.

3. Decolorizing Agent

Ethanol, 95% (vol/vol)

*Alternate Decolorizing Agent

Some specialists prefer an acetone decolorizer, but others prefer a 1:1 mixture of acetone and ethanol. A number of combinations are available commercially, with the majority combining 25 to 50 percent acetone with ethanol. A few formulations contain a trace amount of isopropyl alcohol and/or methanol.

Acetone50 ml
Ethanol (95%)50 ml

4. Counterstain: Safranin

Stock solution:

Safranin O2.5 g
95% Ethanol100 ml

Working Solution:

Stock Solution10 ml
Distilled water90 ml

Gram Staining Procedure

Smear preparation

  1. First, make slides grease-free slides by washing them with detergent (rube both sides of the slide with cotton and detergent) and dry it.
  2. Use Bunsen burner to sterilize the inoculating loop, by holding it on flame.
  3. After that, use to sterile loop to transfer a loopful of culture or the specimen on the grease-free slide. Then make a smear at the center (Smear should not be very thin or very thick.).
  4. Now, dry the smear in air.
  5. To fix the dry smear, pass it 3-4 times through the flame quickly with the smear side facing up.
Smear preparation
Smear preparation

Gram Staining

  1. Cover the specimen slide with primary stain, the crystal violet, and leave for 1 min.
  2. Now, gently wash off the stain by running tap water.
  3. Flood the slide with mordant, the Gram’s iodine and leave for 1 minute.
  4. Drain off the iodine, by washing the slide again in a gentle stream of tap water.
  5. Flood the slide with decolorizing agent, the acid-alcohol, and wait for 20-30 seconds. 
  6. Lightly rinse the slide under flowing tap water and drain completely.
  7. Now, cover the slide with counterstain or secondary stain, the safranin, and wait for about 30 seconds to 1 minute.
  8. Wash slide in a gentile and indirect stream of tap water until no color arises in the effluent and then absorb with absorbent paper.
  9. Now, the slide is ready to observe under microscope.
Gram staining procedure
gram staining procedure in flowchart

Gram Staining Result and Interpretation

After the staining the cell will appear in these following colors;

  • Gram-Positive:  The gram-positive bacteria will appear in Dark purple color which is the color of Crystal violet.
  • Gram-negative: The gram-negative bacteria will appear in Pale to dark red color which is the color of Safranin O.
Gram Staining Result
Gram Staining Result

Other Cells,

  • Yeasts: Yeast cells will appear in Dark purple color.
  • Epithelial cells: Epithelial cells will appear in Pale red color.
Colour changes that occurs in bacterial cells due to various Gram stain reagents at each step during the staining Procedure
Colour changes that occurs in bacterial cells due to various Gram stain reagents at each step during the staining Procedure

Limitation of Gram Staining

  1. False gram-negative results can appear, if the smear is Over-decolorized, while under-decolorization can result in the identification of false gram-positive results.
  2. Thick or viscous smear can retain too much primary stain, it can make difficulties in identification.
  3. Living and dead cells can be found in Cultures older than 16 to 18 hours, the dead cells will be deteriorating and will not retain the stain properly.
  4. Filter the old stain before use, it will help to remove excess crystals. Aging of stain can cause precipitate formation. 
  5. Irregularly, pneumococci recognized in the lower respiratory tract on a direct smear will not develop in culture. Some strains are obligate anaerobes.
  6. Faintly staining Gram-negative organisms, i.e. Campylobacter and Brucella, may be imagined by using an alternative counterstain (e.g., basic fuchsin).

Risk of errors

False positive

  • spread out too much (poor discoloration of bacteria in depth),
  • There is a deposit of dye in the gentian violet bottle. To fix this, filter the dye.
  • iodine solution that doesn’t drain well,
  • Bleach didn’t have enough time,
  • Use of fuchsin with certain germs: Neisseria and Acinetobacter, in particular, are “greedy” for fuchsin and concentrate it to a dark red that is hard to tell from violet.

False negative

  • There are Gram- bacteria in every group of Gram+ bacteria, and sometimes there are a lot of them. These Gram- bacteria are dead Gram+ bacteria.
  • low-quality or too-watered-down dyes,
  • Lugol’s plan was put off for too long,
  • bleach left for too long or not rinsed well enough.

Quality control

Spread a thin layer of stool on a slide, and then colour it. There should be a lot of bacteria that are both Gram positive and Gram negative.

Note: One or more tens of slides that have been dried, fixed, and individually wrapped in aluminium foil should be done with the same preparation of diluted stools. The temperature is +4°C. This gets rid of the smear thickness parameter and puts the focus on the quality of the reagents and the times for staining and removing the stain.

Comments and Tips

  • The staining will be different depending on how thick the smear is. The bleaching step is the most important part of the staining process.
  • Too much bleaching can make Gram positive smears look pink or red, which is a sign of a Gram negative result. Not enough bleaching can make Gram negative smears look blue to purple, which is a sign of a Gram positive result.
  • The thickness of the smear determines how much colour change is needed.
  • The ASM group says that cells should be prepared with a thin smear that doesn’t have any clumps or uneven spots. Thin smears should be stained for a short amount of time.
  • Some people pour decolorizing agent over the slide for 15 seconds or less, while others say to add it drop by drop for 5 to 15 seconds, or until the colour of the decolorizing agent coming from the slide no longer shows any colour.
  • It is best to use crops that are young and growing quickly. For an accurate to happen, the cell wall must be whole. Older cultures may have holes in the cell walls and often give different Gram results when a mix of cells is tested.
  • Use brightfield microscopy to look at slides and adjust the brightness so that the colour of the sample can be seen.
  • The KOH test can be used to confirm a Gram stain. If DNA forms a chain in 3% KOH, it means that the isolate is a Gram-negative organism. This is because 3% KOH breaks down the cell walls of Gram-negative organisms, which releases viscous chromosomal material that makes the suspension thick and stringy.
    1. a drop of 3% KOH on a slide for a microscope.
    2. Add a lot of bacteria that has been grown for 24–48 hours to the drop of KOH. Carefully stir
    3. In 30 seconds, the solution of Gram-negative bacteria will be thick and form a chain of mucus.

Gram-positive bacteria and Gram-negative bacteria

Gram positive cocci

Description of the MorphotypeMost Common Organisms
PairsStaphylococcus, Streptococcus, Enterococcus spp.
tetradsMicrococcus, Staphylococcus, Peptostreptococcus spp
GroupsStaphylococcus, Peptostreptococcus, Stomatococcus spp.
ChainsStreptococcus, Peptostreptococcus spp.
Clusters, intracellularStreptococcus spp. Microaerophilic, viridans streptococci, Staphylococcus spp.
EncapsulatedStreptococcus pneumoniae, Streptococcus pyogenes (rarely), Stomatococcus mucilaginosus
In the form of an ancestorStreptococcus pneumoniae

Gram-negative cocci

Neisseria spp., Moraxella catarrhalis.

Gram-positive bacilli

Description of the MorphotypeMost Common Organisms
LittleListeria monocytogenes, Corynebacterium spp.
MediumLactobacillus, anaerobic bacilli
BigClostridium, Bacillus spp.
DiphtheroidCorynebacterium, Propionibacterium, Rothia spp.
Pleomorphic, Gram variablesGardnerella vaginalis
PearlMycobacteria, lactobacilli affected by antibiotics and corynebacteria
FilamentousAnaerobic morphotypes, cells affected by antibiotics
Filamentous, beaded, branchedActinomycetes, Nocardia, Nocardiopsis, Streptomyces, Rothia spp.
Bifid or V shapesBifidobacterium spp., brevibacteria
Gram-negative coccobacilliBordetella, Haemophilus spp. (pleomorph)
MassesVeillonella spp.
ChainsPrevotella, Veillonella spp.

Gram-negative bacilli

Description of the MorphotypeMost Common Organisms
LittleHaemophilus, Legionella (thin with filaments), Actinobacillus, Bordetella, Brucella, Francisella, Pasteurella, Capnocytophaga, Prevotella, Eikenella spp.
BipolarKlebsiella pneumoniae, Pasteurella spp., Bacteroides spp.
MediumEnterics, pseudomonads
BigClostridia or devitalized bacilli
CurvedVibro, Campylobacter spp.
SpiralCampylobacter, Helicobacter, Gastrobacillum, Borrelia, Leptospira, Treponema spp
FusiformFusobacterium nucleatum
FilamentsFusobacterium necrophorum (pleomorph)

gram staining procedure pdf

Why is gram staining important?

  1. An essential test for the rapid presumptive diagnosis of infectious agent 
  2. Gram staining is utilized to distinguish the bacteria as a Gram positive or Gram negative 
  3. Used To examine the morphology of bacteria 
  4. Used To examine the arrangement of bacteria 
  5. Used To find out the evidence of capsule 
  6. Used To find out the evidence of spore 
  7. Used To find out the evidence of pus cells 
  8. Used To find out the evidence of epithelial cells 
  9. Used To find out the evidence of Yeast cells
  10. Used to control initial therapy until definitive identification of microorganism concerned.
  11. Morphology of stained bacteria can sometimes be diagnostic. For example Gram ve- intracellular diplococci in urethral pus provides a presumptive diagnosis of Gonorrhea.
  12. Sometimes specimens may show organisms under a microscope but appear sterile in culture media. In these cases, Gram stain is the only clue to the nature , variety and relative proportion of infecting organism .
  13. Aids in interpretation of culture reports.

Reference

  1. http://www.med-chem.com/pages/lab_procedures/pdf/gram_stain.pdf
  2. https://www.diffen.com/difference/Gram-negative_Bacteria_vs_Gram-positive_Bacteria
  3. https://www.austincc.edu/microbugz/handouts/Stain%20protocols.pdf
  4. https://en.wikipedia.org/wiki/Gram_staining
  5. https://basicmedicalkey.com/role-of-microscopy/
  6. https://microbiologyinfo.com/gram-staining-principle-procedure-interpretation-examples-and-animation/
  7. https://laboratoryinfo.com/gram-staining-principle-procedure-interpretation-and-animation/
  8. https://www.labce.com/spg327545_gram_stain_principle.aspx
  9. https://microbeonline.com/gram-staining-principle-procedure-results/
  10. https://gpatindia.com/staining-microbiology-notes-of-gram-staining-and-acid-fast-staining/
  11. https://www.slideshare.net/manojmahato9638/gram-stain-by-manoj-76506297
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