Add 900 μl LB broth to a cuvette, blank the instrument
with that cuvette, then add 100 μl of spore suspension
and mix by pipetting.
After measurement, multiply the value reported on the
instrument by 10 to determine the actual OD600 of the
suspension. Record in Table 1.
− Normalize fungal spores to an optimal concentration of
OD600 (0.05 Absorbance Units (A)) in 25 ml LB broth.
Introduce the C1V1 = C2V2 equation.
In this case, C1 = actual OD600 reading, V1 = ml of spore
suspension to be added to the flask, C2 = optimal OD600
(0.05 A), and V2 = final volume 25 ml in the flask.
For example: OD600 = 13.6 A, calculate:
13.6 × V1 ml = 0.05 × 25 ml
V1 = (0.05 × 25 ml)/13.6 = 0.092 ml or 92 μl
In this example, the addition of 92 μl of the spore suspension
to 25 ml of LB broth will result in a culture with OD600 equ-
aling 0.05 A.
− One group per class will inoculate two flasks with spores that
will be not be co-cultivated. These two flasks will serve as a
classroom control on the last day of experiments.
− Inoculate three flasks of 25 ml of LB with fungus and shake at
100 rpm and 25˚C for three days.
− Harvest fungal spores and inoculate fungus on solid media, LB agar.
− Using the same 2 ml tube from the liquid inoculation, dip a
sterile swab in the remaining spore suspension (Figure 2)
and swab a line down the middle of a LB agar plate. This is
the solid media companion to the liquid media antibiotic
effect study (adaptation of From Mold to Medicine, FSU, 1992).
Day 3: Start bacterial cultures and observe fungal
− Make observations of fungal culture growth. Compare to
Figure 1C; P. chrysogenum will appear as small specks,
and the broth should otherwise be clear and free of cloudy
− Preparing bacterial cultures for testing:
Inoculate 5 ml LB broth in a culture tube with each bacterial species. Be sure to label tubes.
• Aseptically attach a sterile 20-200 ul pipette tip to a
p20 or p200 micropipette. Alternatively, sterile
toothpicks can be used to inoculate tubes.
• Collect ~1 mm3 or 1–2 colonies of bacteria from
a plate at the end of the pipette tip or sterile toothpick.
• Eject the tip or drop toothpick directly into a tube
containing 5 ml LB broth.
− Shake tubes upright in a rack at 30˚C and ~180 rpm overnight
(Figure 1D). (See discussion for an option if a shaking incubator is unavailable.)
Table 1. Example data for fungal spore suspension.
Measurement Spec Reading Actual OD600
P. chrysogenum spore suspension 1.36 13.6
Figure 2. (A) P. chrysogenum vertical stripe plated from spore suspension grown 4 days on LB agar. (B) Result of vertical stripe
co-cultivation showing placement of bacterial stripes perpendicular to fungal stripe on LB agar. The bacterial cultures show
differential sensitivity to the penicillin diffused into the agar by the fungus.