a. Classification
size: 0.4-700um
shape – due to rigid cell wall: 3D peptidoglycan/murein mesh
- Spherical: coccus (cocci)
- Rod: bacillus (bacilli)
- Spiral: spirillum (spirilla)
cell wall:
gram positive: thick peptidoglycan/murein layer
retains crystal violet / iodine complex = purple
susceptible to:
- penicillin antibiotic: prevent peptidoglycan cross-links – weaken cell wall for osmotic lysis
- lysozyme enzyme: hydrolyses peptidoglycan intermolecular bonds
gram negative: outer lipopolysaccharide layer + thin inner peptidoglycan/murein layer
alcohol: lose outer with stain – expose inner = red with counterstain safranin
Lipopolysaccharide: resistance to penicillin antibiotic & lysozyme enzyme
metabolic features
antigenic features
genetic features
Gram stain technique
- inoculating loop: transfer small bacteria sample to glass microscope slide
o pass slide through Bunsen flame: fix & kill bacteria
- add crystal violet stain (30 seconds)
o rinse excess with water
- gram’s iodine (1 minute): fix stain
purple = gram positive
- alcohol (30 seconds): decolourise – remove unbound stain & lipopolysaccharide layer (dissolve lipids)
- counterstain safranin
red = gram negative
size: 0.4-700um
shape – due to rigid cell wall: 3D peptidoglycan/murein mesh
- Spherical: coccus (cocci)
- Rod: bacillus (bacilli)
- Spiral: spirillum (spirilla)
cell wall:
gram positive: thick peptidoglycan/murein layer
retains crystal violet / iodine complex = purple
susceptible to:
- penicillin antibiotic: prevent peptidoglycan cross-links – weaken cell wall for osmotic lysis
- lysozyme enzyme: hydrolyses peptidoglycan intermolecular bonds
gram negative: outer lipopolysaccharide layer + thin inner peptidoglycan/murein layer
alcohol: lose outer with stain – expose inner = red with counterstain safranin
Lipopolysaccharide: resistance to penicillin antibiotic & lysozyme enzyme
metabolic features
antigenic features
genetic features
Gram stain technique
- inoculating loop: transfer small bacteria sample to glass microscope slide
o pass slide through Bunsen flame: fix & kill bacteria
- add crystal violet stain (30 seconds)
o rinse excess with water
- gram’s iodine (1 minute): fix stain
purple = gram positive
- alcohol (30 seconds): decolourise – remove unbound stain & lipopolysaccharide layer (dissolve lipids)
- counterstain safranin
red = gram negative
