Methods and results
The activity requires one week (five days) of class time. Students
should be organized into six to eight groups. The complete list of
materials and protocol is available at our website: http://biotech.
These steps can be performed by the instructor, teacher’s aids, or
− Start fungal cultures from stock onto potato dextrose agar
(PDA) two weeks prior to activity. Incubate at 25°C (room
temperature) for one week. Subculture fungus by spreading
spores from primary plates onto secondary PDA plates to create a lawn one week prior to activity and incubate at 25°C for
one week. Two to four groups can share one plate. Each inoculation step should require no more than 5 minutes.
− Prepare bacterial cultures from stocks on LB agar no more
than two weeks and no less than two days before start of
the activity. Incubate S. epidermidis and M. luteus at 30°C
for 48 h and E. aerogenes at 30°C for 24 h. Bacterial culture
plates may be stored at 4°C until activity.
− Sterilize 25 ml LB broth (Miller) pH 7.2 in 125 ml Erlenmeyer flasks (6 per group + 2 per class) and 75 mL 2 × LB
broth (Lennox) pH 7.2 in 250 ml Erlenmeyer flasks (3 per
group). (2 × LB broth consists of twice the amount of all
components required to make LB broth. For 2 × LB broth
(Lennox), mix 20 g Tryptone, 10 g Yeast, and 10 g NaCl
The remaining steps of the protocol are to be performed by the
Day 1: Harvest fungal spores and inoculate fungus
in liquid media, LB broth
− Harvest spores from P. chrysogenum using sterile swabs. Wet a
sterile swab with sterile LB in a 2 ml micro centrifuge tube,
and wipe firmly back and forth in a 5 cm-by-1 cm rectangle
on the PDA plate containing P. chrysogenum until swab is dark
with spores. Suspend spores by twirling swab in approximately 1.8 ml LB broth (Figure 1A and 1B).
− Quantify the concentration of fungal spores in suspension by
measuring OD600 using a 1:10 dilution. Dilution is required
because the suspension is too turbid.
Figure 1. (A) Penicillium lawn on PDA showing the ideal area of light green to cyan colored spores harvested and the ideal density of
spores on a swab. Full-color images can be found at http://biotech.bio5.org/biotech_lab_activities/penicillium (B) Spore suspension
showing ideal density by color of LB/spores in 2 ml microcentrifuge tube. (C) Pure fungal cultures in 50 ml LB showing ideal growth
after four-day incubation. (D) Bacterial culture tubes showing pipette tip inoculation and ideal density after 24 h incubation.