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.
- Gram stain differentiates between gram-positive and gram-negative organisms.
- To diagnosis 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.
- The name comes from its inventor, Hans Christian Gram. He published a gram stain method in 1884.
- This is a special stain for the diagnosis of the gram-positive or gram-negative organism in various samples like sputum, pus, and 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.
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 form 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.
- Secondary stain: Counterstain the smear with safranin O Which is 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.
- Gram-positive bacteria are blue-purple.
- Gram-negative bacteria are pink, magenta color.
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.