Gram Staining Test Procedure and Results (With Flowchart)

Gram staining test procedure is a differential bacterial staining technique used to differentiate bacteria into Gram Positive and Gram Negative types according to their cell wall composition.

Gram Staining Definition

Gram staining test procedure is a common technique for distinguishing two large groups of bacteria based on differences in cell wall constituents. Gram staining differentiates between Gram positive and Gram negative groups by colouring cells red or violet. Gram-positive bacteria stain violet because their cell walls contain a thick layer of peptidoglycan, which retains the crystal violet with which they are stained. Gram negative bacteria, on the other hand, stain red due to a thinner peptidoglycan wall that does not retain the crystal violet during the decoloring process.

Principle of Gram Staining

Gram staining differences can be attributed to the differences in cell wall composition between Gram-positive and Gram-negative bacteria. The cell wall of Gram-positive bacteria contains a thick layer of peptidoglycan with numerous teichoic acid cross-linking that is resistant to decolorization.

Crystal violet breaks up into CV+ and Cl– ions in water, which can get inside Gram-positive and Gram-negative cell walls. When the CV+ comes into contact with negatively charged parts of a bacterial cell, the cells turn purple. When I- or I3- is added, it reacts with CV+ to form large crystal violet-iodine (CV-I) complexes in the cell’s cytoplasm and outer layers.

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, whereas the gram-negative cell loses the purple color and is only revealed when the counterstain, the positively charged dye safranin, is added.

Steps of Gram Staining In Order

Fixation: Heating or methanol are used to fix clinical materials to the surface of the microscope slide. (Methanol fixation preserves the morphology of host cells and bacteria, which is particularly useful when examining bloody specimen material.)

Primary staining: The primary stain also known as methyl violet 10B or hexamethyl pararosaniline chloride, is a triarylmethane dye is applied crystal violet. All cells are stained blue/purple by crystal violet.

Mordant application: An iodine solution (mordant) is added to form a crystal violet-iodine (CV-I) complex; all cells remain blue.

Decolorization: The decolorization step differentiates between gram-positive and gram-negative cells. The blue dye complex is extracted to a greater extent by organic solvents such as acetone or ethanol from lipid-rich, thin-walled gram-negative bacteria than from lipid-poor, thick-walled gram-positive bacteria. Gram-negative bacteria are colorless, while gram-positive bacteria are blue.

Counterstain: The red dye safranin stains the decolorized gram-negative cells red/pink; the gram-positive bacteria remain blue.

Gram Staining Requirements

  1. Sample bacteria
  2. Gram Staining Kit
  3. Glass slide
  4. Inoculating loop
  5. Bunsen burner
  6. Staining rack
  7. Wash bottle
  8. Microscope

Reagents for Gram Staining

Crystal Violet: Dissolve 2 g crystal violet in 20 ml of 95% ethyl alcohol. Dissolve 0.8 g ammonium oxalate monohydrate in 80 ml deionized water. Mix the crystal violet and ammonium oxalate monohydrate solutions to make the crystal violet stain. Filter the stain if necessary.

Gram’s Iodine: Dissolve 1 gm of Iodine, and 2 gm of potassium iodide in 300 ml distilled water. Mix properly till the iodine dissolve and keep the solution in a dark bottle.

Decolorizing Solution: Mix 50 ml of acetone with 50 ml of 95% ethanol. Mix properly till the iodine dissolve and keep the solution in a bottle.

Safranin Solution: Mix 2.5 gm of safranin-O in 100 ml of 95% ethanol. Mix 10 ml of the above solution with 90 ml of distilled water to prepare a working solution.

Carbol-fuchsin: Dissolve 3 gm of basic fuchsin in 100 ml of 95% ethanol, Mix 5 ml liquid phenol with 95 ml of distilled water to prepare 5% phenol solution. Mix 10 ml of basic fuchsin solution with 100 ml of 5% phenol solution. Let the solution sit for 24 hours at room temperature and Store in a dark bottle.

Procedure of Gram Staining

i) Smear Preparation

Suspensions Culture: Place a loopful of the broth culture on the slide with a sterile cooled loop. Spread the inoculating loop in a circular motion to about one centimeter in diameter. Excessive spreading may cause cellular arrangement to be disrupted. A good smear will allow you to look at the typical cellular arrangement and isolated cells (Figure 1).

Bacterial Plate Cultures: Place a drop of sterile water or saline solution on the slide with a sterile cooled loop. Sterilize and cool the loop once more before picking up a very small sample of a bacterial colony and gently stirring it into the drop of water/saline on the slide to create an emulsion.

Gram Staining Test Procedure and Results

Figure 1: Gram Staining Procedure in Flowchart.

ii) Gram Staining Procedure

  1. The bacteria in the smear are killed by the heat, the smear is firmly attached to the slide, and the sample is more easily stained.
  2. Place the slide with the heat-fixed smear on the staining tray. Flood the smear gently with crystal violet and set aside for 1 minute.
  3. Tilt the slide slightly and use a wash bottle to gently rinse it with tap water or distilled water. Flood the smear gently with Gram’s iodine and set aside for 1 minute.
  4. Tilt the slide slightly and use a wash bottle to gently rinse it with tap water or distilled water. On the slide, the smear will appear as a purple circle.
  5. Decolorize using 95% ethyl alcohol or acetone. Tilt the slide slightly and apply the alcohol drop by drop for 5 to 10 seconds until the alcohol runs almost clear. Be careful not to over-decolorize.
  6.  Immediately rinse with water. Gently flood with safranin to counter-stain and let stand for 45 seconds.
  7.  Tilt the slide slightly and gently rinse with tap water or distilled water using a wash bottle.
  8.  Blot dry the slide with bibulous paper. View the smear using a light-microscope under oil-immersion.

Interpretation of Gram Staining

The bacteria that retain the primary stain, i.e. Crystal violet and appears Violet/Blue are Gram-Positive Bacteria.

The bacteria that retain the Counterstain, i.e. Safranine and appears Pink in color are Gram-Negative Bacteria.

Gram staining

Gram Positive Bacteria: Actinomyces, Bacillus, Clostridium, Corynebacterium, Enterococcus, Gardnerella, Lactobacillus, Listeria, Mycoplasma, Nocardia, Staphylococcus, Streptococcus, Streptomyces ,etc.

Gram Negative Bacteria: Escherichia coli (E. coli), Salmonella, Shigella, and other Enterobacteriaceae, Pseudomonas,Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio, acetic acid bacteria, Legionella etc

Applications of Gram Staining

Gram staining test procedure is a research tool that separates bacteria into two categories: gram-positive and gram-negative. Gram staining test procedure is in medical laboratories for the purpose of identifying the infectious agent

Gram staining test procedure is utilized in hospitals for the purpose of selecting an antibiotic treatment spectrum prior to the complete identification of bacteria. Gram staining test procedure is for the examination of the morphology of bacteria

Limitations of Gram Staining

Decolorization: Two conditions may occur during decolorization, resulting in incorrect results. Over-decolorization of the smear may result in false gram-negative results, whereas under-decolorization of the smear may result in false gram-positive results.

Thickness of smear: Smears that are too thick or viscous may retain too much primary stain, resulting in incorrect results. Furthermore, Gram-negative organisms may not decolorize properly and retain primary stain, causing them to appear under the microscope as Gram-positive or Gram-variable organisms.

Precipitation of Stain: As the stain ages, it may precipitate out in the stain solution, which could lead to inaccurate results. Better results are obtained by using freshly made staining solutions or by filtering the stain solution through gauze to remove extra crystals.

Antibiotic therapy: Gram-stained smears from patients on antibiotics or antimicrobial therapy may have altered Gram stain reactivity because many antibiotics alter the bacterial cell wall and cell membranes, resulting in variable gram staining.

Variations in staining: Some Gram-negative organisms like Campylobacter sp. and Brucella sp., faintly stain by the Gram-staining technique and for such organisms alternative counterstains, e.g. basic fuchsin can be used for better results.

Gram Staining 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.