Gram +/- Bacteria

Gram +/- Bacteria

1. Positive

   • Bacillus Subtilus
   • Purple
   • Thick Peptidoglycan
   • no Lipopolysaccharide
   • Sensitive to Penicillin
     • interferes with interpeptide links of peptidogylcan
       • weak and collapse/disintegrate when divide
         • human cells = no cell wall = no harm
   • Peptidoglycan binds to crystal violet
     • Alcohol closes cell wall pores
       • Can't wash off dye = Purple

2. Test

   • Loop-full bacteria onto sterile drop of water
     • Spread on glass slide
       • Air-dry and heat-fix - pass over flame
         • Add Crystal violet (30 seconds) - drain + wash
           • Add Lugol's Iodine (1 minute) - drain + wash
             • Add Ethanol (30-45 seconds)
               • Add Safranin (30 seconds) - drain + wash

3. Negative

   • Red/Pink
   • E.coli
   • Thin Peptiodoglycan
   • Has lipopolysaccharide layer
   • less sensitive to penicillin
     • not affected by antibacterial enzyme lysozyme
       • supplemented with lipop layer
     • require antibiotics
       • interfere with ability to make proteins
         • different to humans so not harmful to us
   • Cell wall = complex = outer layer of lipopolysaccharide
     • Ethanol dissolves this layer
       • Crystal violet washed away
         • Cell wall stained by Safranin = Red

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