Gram Staining Flashcards
What is gram staining ?
Gram’s Stain is a widely used method of staining bacteria as an aid to their identification.
Gram’s Stain is devised by…….?
Christian Joachim Gram
What are Gram negative ?
Bacterial species with walls containing small amount of peptidoglycan, and characteristically lipopolysaccharide, are Gram-negative.
What are Gram-positive?
Bacteria with walls containing. Relatively large amounts of peptidoglycan and no lipopolysaccharide are Gram positive.
If an infection has inflammation, which Gram species do we suspect? Why?
It may Gram-negative species.
The lipopolysaccharide acts as an endotoxin. These cause infalmmation in the human body.
Which gram species infections are much difficult to treat? Why?
Gram-negative infections are much more difficult to treat with antibiotics.
Because
- many antibiotics penetrate Gram positive walls much more easily than Gram-negative.
- the gram-negative bacteria are much less prone to the action of lysozyme, an enzyme secreted in tears, sweat and saliva.
Principle of gram staining ?
Gram positive bacteria have more acidic peptidoglycan which may account for their retaining the basic primary dye more strongly than gram negative bacteria. The peptidoglycan of gram positive is thick and thus able to retain the dye iodine complex.
The high lipid content of gram negative bacteria makes them permeable to secondary dye after decolorisation with organic solvent like acetone and alcohol.
Differences between gram positive and gram negative bacteria?
Gram positive bacteria
- peptidoglycan is thick
- theicoic acid present
- less polysaccharide
Gram negative
- peptidoglycan is thin
- theicoic acid absent
- more polysaccharide
Mechanism of gram staining( Crystal violet)- gram positive
- When bacteria is stained with primary stain crystal violet and fixed by mordant, some bacteria are able to retain the primary stain and some are decolourized by alcohol or acetone. The cell wall of gram positive bacteria have a thick layer of protein sugar complexes called peptidoglycan.
- Crystal violet dissociates in aqueous solution into crystal violet(CV+) ion and Cl- ion. This ion penetrates through the cell wall and cell membrane of both gram positive and gram negative cells. CV+ interacts with negatively charged components of bacterial cells and stained the cells purple.
Mechanism of gram staining (Iodine)- gram positive
Iodine interacts with CV+ and form large complexes of crystal violet and iodine within the inner and outer layers of cell. Iodine is often refers. To as a mordant but is a trapping agent that prevent the removal of CV-I complex and therefore coloured the cell.
Mechanism of gram staining (decolourizing agent)- gram positive
Decolourizing the cells causes thick cell wall to dehydrate and shrink which closes the pores in the cell wall and prevent the stain existing the cell so that alcohol or acetone cannot removed the CV-I complex that is bound to the thick layer of peptidoglycan and appears purple/violet in colour.
Mechanism of gram staining- gram negative bacteria
In case of gram negative bacteria, cell wall also take the iodine complex but due to the thin layer of peptidoglycan and thick outer layer which is formed by lipids, CV-I complex gets washed off. A gram negative cell wall loss its outer lipopolysaccharide membrane and the inner peptidoglycan is left exposed. Then the CV-I complexes are washed from gram negative cell along with the outer membrane.
Reagents? 4
- Crystal violet
- Grams Iodine-fixer
- Decolourizing agent- acetone/alcohol
- Safranin- counter stain
Procedure
- Take a clean grease free slide
- Prepare the smear of suspension on the clean slide with a loopful of sample.
- Prefer to pick up colonies with loop and smear on clean glass slab then air dry and heat fixed.
- After that air dry and heat fixed the smear for 1 minute. Then flood air dry with fixed smear of cells for 1 minute with crystal violet.
- Wash the slide in a gentle and indirect stream of tap water for 2 sec.
- Flood the slide with mordant (gram iodine) fro 1 minute
- Wash the slide in a gentle and indirect stream of tap water for 2 sec.
- Flood the slide with decolourizing agent- acetone/alcohol then rinsed the slide with distilled water.
- Pour the counter stain on the slide for 1 minute.
- Wash the slide in a gentle and indirect stream of tap water for 2 sec.
- Read the slide with oil immersion lens under microscope.
Type of gram positive organism
- All cocci except Neisseria and Branhamplla
* yeast
Example of gram positive bacteria
- Staphylococcus
- . streptococcus
- Clostridium
- Bacillus
- Nocardio
Normal flora of skin and nose?
Staphylococcus
