21 Procedure
Each group will need:
- Slides and cover slips
- Bacterial culture/plate for isolates
- BUG agar plate with bacteria (incubated overnight at 33°C from your TSA plates)
- Invitrogen LIVE BacLight kit
- MycoLight™ rapid fluorescence bacterial Gram stain kit.
- Micropipette tips for p200, p20, and p10
- Wipes
- Tip disposal container
Bacterial Staining
- Take bacteria (1 or 2 colonies) from a plate using a sterile swab or loop.
- Mix bacteria into 1 mL PBS (bacterial suspension).
- Combine equal volumes of Component A and Component B in a microfuge tube and mix thoroughly. You were given 5 µL of each component.
- Add 3 µL of the dye mixture per mL of bacterial suspension.
- Mix thoroughly and incubate at room temperature in the dark (drawer) for 15 minutes.
- Trap 20 µL of each stained bacterial suspension between a slide and a 24×50 mm rectangular coverslip.
- Observe in a fluorescence microscope equipped with any of the filter sets listed in Table 1. Live Gram-negative organisms should fluoresce green and Gram-positive bacteria should fluoresce red.
