Day 4: Co-cultivate fungus and bacteria in liquid
− Normalize bacterial concentrations to 0.05 A in 250 ml flask
containing 75 ml 2 × LB broth (Lennox).
Measure each overnight bacterial culture concentration by
OD600 and record in Table 2 (a 1/10 dilution is again
Calculate volume of each bacterial culture needed for
0.05 A final concentration in 75 ml 2 × LB broth (Lennox).
(Recall C1V1 = C2V2)
• For OD600 = 5.28 A, calculate:
5.28 × V1 ml = 0.05 × 75 ml
V1 = (0.05 × 75 ml)/5.28 = 0.710 ml or 710 μl
− Prepare culture flasks:
All measurements must be made using sterile technique.
Measure 25 ml of diluted bacteria (at OD600 = 0.05 A)
into one flask containing 25 ml LB broth (control) and
one flask containing 25 ml LB broth with P. chrysogenum
(experimental). This will result in two flasks, each with
50 ml final volume.
Repeat for each of the other two bacterial species (in 250 ml
flasks) for a total of six flasks per group.
Shake all cultures at 100 rpm and 25˚C overnight.
− To the two classroom flasks of P. chrysogenum, add 25 ml sterile 2 × LB broth.
For the solid media plates with a vertical stripe of P. chrysogenum:
Dip a sterile 1 μl loop into the overnight bacterial culture tube.
Swipe the end of the loop with bacteria starting at the
edge of the plate, moving perpendicular to the fungal
stripe until the loop touches the fungus.
Swipe again just below the first with the remaining bacteria on the loop.
Day 5: Measure antibiotic effect
− Gravity filter ~5 ml of each pure and co-cultivated growth
through a coffee filter folded into a cone in a funnel (Figure 3).
Collect the filtrate (bacteria) in an appropriately sized tube
(12 ml, 15 ml, or 50 ml).
− One group will also filter the two classroom P. chrysogenum
pure culture control flasks and make OD600 measurements.
This serves as a control to ascertain that the fungus is not
passing through the filter and having no effect on the optical
density after filtration.
− Record and compare results (Table 3).
− To quantify the antibiotic effect on solid media, use a metric
ruler to measure distance in mm from the edge of the growth
of P. chrysogenum to the start of the growth of bacterial colonies (Figure 2). Record results in Table 4.
Some of the bacterial stripes may have colonies within this distance
with non-continuous growth (Figure 2). Record the results in Table 4.
The zone of inhibition on plates should be obvious after 24 h, but
the effect continues to develop over several days. Average the
Table 2. Example data for results of bacterial
Bacterial Species Spec Reading Actual OD600
S. epidermidis 0.528 5.28
M. luteus 0.499 4.99
E. aerogenes 0.478 4.78
Figure 3. Apparatus for filtering co-culture of bacteria and
fungus consisting of coffee filters, conical tubes, a funnel and a
rack capable of holding conical tubes upright. Bacteria will
readily flow through the coffee filter and funnel into the conical
tube. Fungus will remain trapped in the coffee filter. Measure
concentration of filtered bacteria at OD600.
Table 3. Example data for control and experimental
cultures. Of the three bacteria, E. aerogenes is least
sensitive to penicillin, S. epidermidis is moderately
sensitive, and M. luteus is the most sensitive to penicillin.
S. epidermidis 5.73 2.57
OD600 of pure
M. luteus 1.95 0.045*
E. aerogenes 4.84 3.39
*This concentration is less than 0.1 and must be measured neat (undiluted).
Table 4. Example data for zone of inhibition
S. epidermidis 1.4 cm 1.1 cm 1.25 cm
M. luteus 3.1 cm 2.7 cm 2.9 cm
E. aerogenes 0.0 cm 0.0 cm 0.0 cm