15 Activity 2: Turbidimete
We will swab bacterial cells from your agar plates (BUG Agar: BUG (Biolog Universal Growth) Agar w/ 5% sheep blood), dilute in Biolog IF-A inoculation fluid, and check turbidity with a turbidimeter.
Each student researcher will need:
- IF-A inoculating fluid
- BUG agar plate with bacteria (incubated overnight at 33°C from your TSA plates)
- Biolog GEN3 plate
- One 200/300 μl multichannel micropipette (p200)
- Micropipette tips for p200
- Wipes
- Tip disposal container
Getting Started
1. Calibrate the turbidimeter to a “blank” sample of inoculation fluid in a tube
a. Using the Biolog Turbidimeter, blank the turbidimeter with a clean tube (wiped clean of dirt and fingerprints) containing uninoculated IF-A. Because the tubes used are not optically uniform, they should be blanked individually.
b. Set the 100% transmittance adjustment knob so that the meter reads 100%.
c. Record reading.
2. Use a turbidimeter with bacterial sample in the inoculation fluid in a tube
a. Collect bacteria from your streak plate by touching the Inoculatorz™ swab to the bacteria (be gentle: do not push the swab through the agar media).
b. Open your Inoculating Fluid tube (IF) tube and submerge the swab into the liquid. Swirl the swab around in the fluid for 15 seconds to ensure inoculation. Do not leave the liquid uncapped longer than necessary to prevent contamination. The tubes are glass: work carefully.
c. Cap the tube and gently invert the three (3) times to ensure thorough mixing (no bubbles).
d. The target cell density is 95%T for our protocol.
i. Add more bacteria with the swab if necessary to get as close to this value as possible by continuing to add more bacteria with the swab to the tube.
ii. Work carefully and keep the tube capped. If necessary, use another swab if you think it has touched another surface.
3. Once you’ve reached the target cell density at 95%T, cap the tube and proceed to the next Activity.
