Procedure
• Do all work in a sterile laminar flow hood or on a countertop.
• Use all standard precautions with the Bunsen burner, (tying back hair, wearing goggles, etc.).
• Dispose of any bacteria-contaminated products in autoclave bags and/or 10% bleach
solution.
37ϒC
1. Obtain a Petri plate of LB agar containing
isolated colonies, a 50-mL sterile, conical
centrifuge tube, and a bottle of sterile LB
broth.
2. Flame-sterilize (as demonstrated by the
supervisor) the top of the LB broth bottle.
3. Using a sterile 10-mL pipet, transfer 10
mL of LB broth from the bottle to the
50-mL tube.
4. Flame-sterilize (as demonstrated by the
supervisor) an inoculating loop, cool it
on a spot of uncontaminated agar, and collect
a colony of E. coli from the streaked plate. If
using a sterile plastic loop, collect a colony of
E. coli from the streaked plate.
5. Holding the tube at a 45° angle, add the
colony to the broth, with a twist of the loop,
to ensure that most of the colony gets into the
broth (see Figure 4.12). Reflame the loop to
remove any remaining bacteria. If using the
plastic loop, discard it into disinfectant.
6. Incubate the broth culture in a shaking hot
water bath or a shaking incubator oven at
37°C for 24 hours (see Figure 4.13).
7. Before leaving the lab, discard any biological
waste in the biohazard bag, disinfect your
workspace, and wash your hands.
Note: After 24-hour incubation, remove 1 mL
of culture and store (-20°C) in a sterile tightly
capped 1.7 mL tube for Lab 4l.
Figure 4.12. Inoculating a Broth
Culture. Hold the tube “sideways” to
decrease the chance of contaminants falling
into the container.
Figure 4.13. In industry, large shaker flask ovens
keep cells moving, aerated, and warm, so that
they grow and divide at a maximum rate.
Photo by author.
Data Analysis/Conclusion
Evaluate your broth culture. Does it look the way you think it should look? Is it cloudy and
obviously full of bacteria cells? How do you know? How can you be sure that there are enough
of the right kinds of cells? Propose a method for che