Gram Stain (Gram stain procedure)
- Gram stain can be done on sputum, pus, tissue, and urine.
- The sample can be obtained from the infected ulcer or wound.
- The CSF may be stained.
Indications for Gram stain
- Gram stain differentiates between gram-positive and gram-negative organisms.
- To diagnose the presence of bacteria in sputum, pus, or any other tissue or fluids.
- To diagnose bacterial meningitis.
- It can stain yeast, and this needs to be reported.
History of Gram stain
- The name comes from its inventor, Hans Christian Gram. He published a gram stain method in 1884.
- He was searching the organism and diagnosed pneumonia.
- This is a special stain for diagnosing the gram-positive or gram-negative organism in various samples like sputum, pus, urine, etc.
- It divides bacteria into two physiologic groups:
- The gram-negative bacterial wall consists of four layers. The following diagram shows the layers of the wall.
The procedure of Gram staining:
It consists of four steps :
- Fix the slide by heat.
- Primary stain: Stain the slide smear with gentian violet or crystal violet. It will penetrate the cell membrane.
- Mordant: wash the violet stain and flood the smear with the Iodine solution. This acts as mordant and forms a complex with Crystal violet.
- Decolorizer: Wash off the smear and flood it with alcohol (95 %) or Acetone-alcohol mixture.
- This will remove the outer cell membrane in gram-negative bacteria, where the complex will also be washed of.
- While in Gram-positive bacteria cell membrane remain intact, and the stain will not be washed off after alcohol treatment.
- Decolorization with acetone or alcohol will lead to:
- Gram-positive bacteria block the dye extraction (still, this step is unclear).
- This step will decolorize gram-negative bacteria and not gram-positive bacteria.
- Secondary stain: Counterstain the smear with safranin O, a red dye.
- In gram-positive bacteria, counterstain cannot enter, so the bacteria are purple.
- While in gram-negative bacteria, safranin can enter and give pink color.
Results of gram stain
- Gram-positive bacteria are blue-purple.
- Gram-negative bacteria are pink magenta.
Morphological classification of gram stain bacteria:
|Type of microorganism||Morphology in gram stain|
|Bacillus||Gram + thick spore-forming bacilli|
|Streptococcus pneumoniae||Gram + lancet-shaped diplococci|
|Staphylococci||Gram + cocci as grape-like clusters|
|Streptococci||Gram + cocci in chain|
|Neisseria||Gram – kidney-shaped diplococci|
|Cronybacteria||Gram + small bacillus in Chinese letter|
|Vibrio||Gram – curved rods|
|Clostridia||Gram + thick spore-forming spores|
|Listeria||Gram + small bacilli|
|Actinomyces||Gram + branching, filamentous, bacilli|
|Enterobacteriaceae||Gram – straight barrel-shaped rods|
Examples of Bacterial Infections:
- Gram-positive cocci infections: Staphylococcal aureus can cause skin infections and toxic shock syndrome.
- Gram-negative cocci infections: N.meningitidis causes meningitis. N.gonorrhoeae causes gonorrhea.
- Gram-negative bacilli infections: E.coli causes urinary tract infection.
- Gram-positive bacilli infections: B.anthracis leads to skin infection and pneumonia. Listeria monocytogenes may cause food-born infection.
Viruses do not stain with gram stain.