Gram staining, negative staining, native
preparation
1. practice
Cytology and morphology of bacteria
Fidrich (2018)
Gram staining
o differentiate G+ a G- bacterial cells
• Fix dry microscopic slide by the flame after air dry
• Crystal violet (1 min)
– Rinse with H2O
• Lugol solution (30 s)
– Rinse with H2O
– Wash the preparation by ethanol (10-15 s)
• Safranin (1 min)
– Rinse with H2O
Native preparation
1. Drop of water
2. Transfer small ammount of cells in drop of
water
• Do not smear the drop
3. Cover drop with cover slide
4. Observe
• Bright field
• Phase contrast
Negative staining
o Nigrosin
1. Drop of nigrosin + loop of water + culture
2. spread over the slide with another slide
3. Allowed to air dry
o Kongo red
1. Drop of Kongo red + culture
2. spread over the slide with another slide
3. Allowed to air dry
• 1% HCl on dry microscopic slide
What to observe ?
o Native preparation (2)
• 2x Bacilli
o Gram staining (2)
o 1x pure culture
o 1x mix of cultures (2 or more)
o Negative staining (2)
• 1x with Nigrosin
• 1x with Kongo red
Bacilli:
Bacillus sphaericus
Bacillus cereus
Bacillus megaterium
Paenibacillus polymyxa
Cocci:
„Azotobacter vinelandii“
Leuconostoc mesenteroides
Sporosarcina ureae
Staphylococcus aureus
Micrococcus luteus
Rods:
Serratia marcescens
Escherichia coli
Archaea:
Haloarcula hispanica
Eukaryota:
Saccharomyces cerevisiae
Yarowia lipolytica