Staining

Gram Staining Procedure, Principle, and Results

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

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

  • Primary stain: Crystal violet (mixer of Solution A and Solution B. Solution A contain 20 mL 95% ethyl alcohol, 2 g Crystal violet, and solution B contain 100 mL distilled water, 0.8 g ammonium oxalate ). Mix solution A and B, then Keep for 24 hours and filter. Store in an amber-colored bottle.
  • Mordant: Gram’s Iodine (mixer of 100 mL distilled water, 2 g potassium iodide, and 1 g iodine crystals, Mix and Store in an amber colored bottle.)
  • Decolorizer: made of acetone and alcohol (95%).
  • Counterstain/secondary stain: Safranin (mixer of 800 mL distilled water, 4.0 g Safranin O, and 200 mL 95% ethano)

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).

Example of Gram-Positive Bacteria

Lactobacillus, Nocardia, Bacillus, Clostridium, Staphylococcus, Propionibacterium, Actinomyces, Streptomyces, Enterococcus, Listria,Gardnerella, Cornyebacterium, Mycoplasma, Streptococcus etc

Example of Gram-Negative Bacteria

Helicobcater, Escherichia, Shigella, Hemophilus, Neisseria, Pseudomonas, Klebsiella, Enterobacter, Vibrio, Chlamydia, Salmonella, etc.

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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