measured distance between the fungus and the bacterial growth by
calculating the arithmetic mean, (A + B)/2.
This activity establishes an understanding that antibiotic effect is
dependent on the characteristics of the bacteria. Sensitivity of a
species of bacteria to one antibiotic type is not a general rule for
sensitivity of that species to all antibiotics. For example, E. aerogenes exhibits resistance to penicillin but is more sensitive to other
antibiotics like Tigecycline (Fraser & Sinave, 2017). Penicillin
works by inhibiting formation of the cell wall during division.
Penicillin is a bacteriostatic antibiotic that inhibits growth, resulting in equal cell generation and death rates. Other antibiotics act
with mechanisms, such as forming pores in the cell membrane to
kill bacteria (bactericidal). Either type has a minimum inhibitory
concentration (MIC) and a minimum bactericidal concentration
(MBC). The difference between these concentrations is a characteristic of a bacterial species and an antibiotic. The effect is seen
on the vertical stripe assays. The penicillin produced by the fungal
stripe is diffused throughout the plate in a gradient, strongest near
the fungus and weakest at the plate edge. In Figure 2B: E. aerogenes cultures grow consistently from the edge of the plate to
the fungal stripe indicating resistance; S. epidermidis growth is
inhibited as the culture approaches the fungal stripe, indicating
an interaction with the antibiotic between the MIC and MBC;
and M. luteus exhibits an abrupt zone of inhibition approaching
the fungus, indicating a narrow window between the MIC and
MBC. This effect is present, albeit less noticeable, in the liquid
cultures and manifests as the difference in co-culture concentration of the bacteria.
Growing organisms with different optimal growth conditions
poses some difficulties. We considered conditions and equipment
available in the high school classroom and tested the protocol to
establish a range of growth characteristics. P. chrysogenum and all
three bacteria will grow in LB media. Classrooms without shaking
incubators may place a shaker into an incubator, or use a space
heater to warm the area to 30°C. A confined area would be preferable to maintain consistent optimal temperature.
Liquid cultures of P. chrysogenum may look slightly different from
the pictures included in this article (Figure 1C), appearing as many
small spheres, fewer large spheres, or small “flakey” growths. Consistent growth is better achieved from ideal spore harvesting of four- to
five-day cultures of light green color. Significant difference in accumulated fungal mass may affect the results of the co-cultivation, and
students who observe this should discuss their results in the context
of this differential growth.
Bacterial cultures may form biofilms, stringy mucus-like clumps,
that can affect results and occurs more often in LB (Lennox) than in
LB (Miller). Biofilms can be disassociated with agitation or pipetting
The 2 × LB broth (Lennox) is used to restore nutrients to the
co-cultivation flasks. Nutrient deficiency can affect the growth of
the bacteria and may affect results.
The activity is optimized to conserve the penicillin produced by
the fungus. Penicillin is more stable at 25°C than at higher temperatures (Kheirolomoom et al., 1999).
This lesson allows students to practice laboratory techniques essential to careers in the biological sciences. They will cultivate and
quantify microbiological organisms, practice accurate measurement
techniques, conceptualize experimental design, and connect laboratory experiences to their understanding of medicine.
Using antibiotics to treat human infections comes with inherent
risks. Antibiotic resistances develop naturally in bacteria by the evolutionary force of selective pressure. In the presence of antibiotics,
bacteria that develop mutations to resist the effect of that antibiotic
are more likely to survive and pass on that genetic information.
The risk of resistance development is highest between the bacteriostatic and bactericidal concentrations of an effective antibiotic
because the selective pressure for resistance development is strong.
Resistance can also be passed between bacterial species on DNA
plasmids. Though this is a relatively slow evolutionary process,
increased exposure to antibiotics in the human environment has
led to more rapid adaptations in bacteria. Careless use of antibiotics
creates a risk that resistance will outpace our development of effective antibiotics. This activity is an introduction to exploring the current challenge of antibiotic resistance development. Students may
also be interested in looking for new microbes whose antibacterial
effects have not yet been discovered.
Medicine has extended our lifespans significantly, and successful
treatment of rudimentary infections laid the foundation for medical
development in the 20th and 21st centuries. Antibiotic treatments
for bacterial infections may seem trivial today because antibiotics
are still effective, but the risks of antibiotic resistance development
are currently high. Improper use of antibiotics will significantly
affect our risk of hard-to-treat infections. Maintaining antibiotic
effectiveness against the evolution of antibiotic resistance will require
a two-pronged strategy. Researchers and policy makers must collaborate to outpace antibiotic resistance by developing new antibiotics
and incentivizing research efforts respectively. Work must also be
done to limit the development of resistance through thoughtful
everyday habits, and to inform the community about the risks vs.
benefits of antibiotics. The classroom offers a perfect opportunity
to introduce these concepts early.
If the teacher has experience with statistical analysis, this experiment
can serve to introduce statistical comparisons. Within a class or across
multiple classes conducting this experiment, the results may be compared using tests to determine the significance of the data. A student
t-test may be used to compare data from all groups to determine
whether the optical density of a single co-culture condition is significantly different in the presence vs. absence of P. chrysogenum. To compare the penicillin sensitivity among all three bacteria in one test, a
Two-Way ANOVA may be used. Students can use software for statistics to perform these analyses on their data.
This experiment lends well to further work with antibiotic-producing fungus from the environment. Students can “bait” for
fungus on bread or fruit. Applying newly acquired lab skills and techniques to samples collected by students may increase student buy in
and engagement. Students can determine the species of any fungus
cultured in the baiting experiment using PCR amplification of the