differential stains

gram stain explained

• Gram Stain

• First test done to determine unknown pathogen.  

  • Take advantage of differences in the bacterial cell wall 

  • Gram Positive 

    • Thick Peptidoglycan layer 

  • Gram Negative

    • Thin peptidoglycan layer

    • Outer membrane 

    • Disadvantages 

      • Need to be fixed

gram stain steps

• Slide is flooded with crystal violet for 1min, then rinsed with water

  • Result: All cells are stained purple

  • Sticks to peptidoglycan, so thick will remain with primary stain (violet) 

•  Slide is flooded with iodine for 1 min, then rinsed with water

  • Result: iodine acts as a mordant; all cells remain purple

  • Makes a stable complex and traps it in the cell

• Slide is rinsed with solution of ethanol and acetone for 10-30 sec, then rinsed with water

  • Results: Smear is decolorized; gram-positive cells remain purple, but gram-negative cells are now colorless. 

• Slide is flooded with safranin (counter stain) for 1 min, then rinsed with water and blotted dry. 

  • Results: gram-positive cells remain purple, gram-negative cells are pink. 

• Gram positive is purple, gram negative is pink.

negative staining

• Acidic dyes (anionic) that are repelled by the negative surface charge of a cell

• Bacteria with this capsule add to the virulent factor, prevent immune system from fighting 

• Smear, fix, stain 

• Might not completely kill, contra-indicated for virulent pathogen 

  • Also called a capsule stain

  • Acidic - negatively charged 

  • Cell remains colorless but background is stained to give contrast 

  • Creates a capsule effect. 

acid fast staining

• Stains mycobacterium and norcardia (tuberculosis, leprosy, lung and skin infection)

• For cells that resist decolorization 

• Thick, lipid or waxy layer in cell wall, so water soluble dyes dont work

  • Primary: carbolfuchin red

  • Heat to permeate waxy layer

  • Decolorized with hydroclorich acid and alcohol and removes non-acid fast cells. Cells that resist are acid fast 

  • Counterstain w/ methylene blue 

  • Cell remains colorless but background is stained to give a contrast 

giemsa staining

• Mix of multiple dyes, acidic and basic dyes. Neutral overall charge 

  • giemsa : mix of methylene blue, eosin and azure 

  • Works well on human blood cells, stain nuclei of white blood cells blue/purple and cytoplasm pink

  • Diagnosis of malaria, protozoa, chlymidia, borriela, histoplasma, pneumonia