The Gram staining technique is the most important and widely used microbiological differential staining technique. It was developed by Dr. Christian Gram in 1884, and categorizes bacteria according to their Gram character (Gram positive or Gram negative).
In addition this stain also allows determination of cell morphology, size, and arrangement. It is typically the first differential test run on a specimen brought into the laboratory for identification.
In some cases, a rapid, presumptive identification of the organism or elimination of a particular organism is possible.
Principle of Gram staining
The structure of the organism’s cell wall determines whether the organism is gram psitive or negative. When stained with a primary stain and fixed by a mordant, some bacteria are able to retain the primary stain by resisting declorization while others get decolorized by a decolorizer.
Those bacteria which retain the primary stain are called Gram positive and those bacteria which gets decolorized and then get counterstained are called Gram negative.
Crystal violet (CV) dissociates into CV+ and Cl– ions in aqueous solutions. These ions penetrate through the cell wall and cell membrane of both Gram-positive and Gram-negative cells. The CV+ ion interacts with negatively charged components of bacterial cells and stains the cells purple.
Iodine (I), used as mordant interacts with CV+ and forms large complexes of crystal violet and iodine (CV–I) within the inner and outer layers of the cell.
When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. Since Gram negative organism have thin peptidoglycan layer(1-2 layers) and have additional lipopolysaccharide layer which gets dissolved due to the addition of alcohol, so gram negative organism fails to retain the complex and gets decolorized as the complex is washed away.
In contrast, a Gram-positive cell becomes dehydrated from an ethanol treatment. This closes the pores in the cell wall and prevents the stain from exiting the cell. The large CV–I complexes become trapped within the Gram-positive cell also due to the thick and multilayered (40 layers) nature of its peptidoglycan.
After decolorization, the Gram-positive cell remains purple and the Gram-negative cell loses its purple color. Counterstain, which is usually positively-charged safranin or basic fuchsin, is applied last to give decolorized Gram-negative bacteria a pink or red color.
Requirements and preparation of reagents
- Primary Stain : Crystal violet
Solution A :
- Crystal violet = 2 gm
- Ethyl alcohol= 20 ml
Solution B :
- Ammonium oxalate = 0.8 gm
- Distilled water = 80 ml
Mix solution A and B. Keep for 24 hours and filter. Store in an amber colored bottle.
- Mordant : Gram’s Iodine
- Iodine = 1 gm
- Potassium iodide = 2 gm
- Distilled water = to 100 ml
Mix and Store in an amber colored bottle.
- Decolorizer : 95% Ethanol or 1:1 acetone with ethanol
- Acetone = 50 ml
- Ethanol (95%) = 50ml
- Counterstain: safranin
- Safranin O = 0.34 gm
- Absolute alcohol = 10ml
- Distilled water = 90ml
Mix, filter and store in ambered colored bottle.
Procedure of Gram staining
Smear preparation :
- Take a grease free dry slide.
- Sterilize the inoculating loop on a flame of a Bunsen burner.
- Transfer a loopful of culture (or the specimen) by sterile loop and make a smear at the center. Smear should not be very thin or very thick.
- Allow the smeat to dry in the air.
- Fix the dry smear by passing the slide 3-4 times through the flame quickly with the smear side facing up.
Gram Staining :
- Place the slides on the staining rods.
- Cover the smear with crystal violet stain and leave for 1 minute.
- Wash carefully under running tap water.
- Flood the smear with Gram’s iodine solution and leave for 1 minute.
- Drain off the iodine Wash the slide for the again in a gentle stream of tap water.
- Flood the slide with the decolorizing agent then wait for 20-30 seconds. This can also be done by adding a drop by drop to the slide until the decolorizing agent running from the slides runs clear.
- Gently wash the slide under running tap water and drain completely.
- Counterstain with safranin for and and wait for about 30 seconds to 1 minute.
- Wash slide in a gentile and indirect stream of tap water until no color appears in the effluent and then blot dry with absorbent paper.
- Observe under microscope.
Interpretation of Gram staining
- Gram Positive : Dark purple
- Gram Negative : Pale to dark red
- Yeasts : Dark purple
- Epithelial cells : Pale red
Examples of Gram Positive Organisms
Bacillus, Nocardia, Clostridium, Propionibacterium, Actinomyces, Enterococcus, Cornyebacterium, Listria, Lactobacillus, Gardnerella, Mycoplasma, Staphylococcus, Streptomyces, Streptococcus etc
Examples of Gram Negative Organisms
Escherichia, Helicobcater, Hemophilus, Neisseria, Klebsiella, Enterobacter, Chlamydia, Vibrio, Pseudomonas, Salmonella, Shigella
Animation of gram staining