In this lesson students will use the Penicillium chrysogenum fungus, which naturally produces the antibiotic penicillin, to investigate the effect of naturally produced antibiotics on bacteria in laboratory cultures. Students co-culture P. chrysogenum with three species of bacteria to observe differences between penicillin-resistant and penicillin-sensitive bacteria. They will normalize fungal spore suspension and bacterial culture concentrations before inoculating co-cultures. After bacteria have been exposed to the antibiotic, students will quantify culture density to determine antibiotic effect in liquid culture and on solid media. Students will learn about natural product antibiotics as well as experimental design and application.

Introduction

Antibiotics are powerful tools that greatly reduce the risk of serious illness or death from bacterial infections. Before the 1940s, bacterial infections accounted for more deaths than heart disease or cancer, and a small cut could cause a lethal infection. Infections now rarely advance to a serious condition because of the use of antibiotics.

The clinical use of antibiotics began with a discovery by Sir Alexander Fleming (1881–1955), a microbiologist working to understand Staphylococcus bacterial infections. Finding his culture dishes contaminated with mold, Dr. Fleming noticed an interesting phenomenon: the bacteria on the plate were unable to grow near the mold. Fleming termed the observation the “zone of inhibition” and began investigating the cause. He identified the mold as Penicillium, commonly known as green bread mold, and named the antibiotic compound he isolated “penicillin”.

By 1940, Fleming's discovery caught the attention of medical Drs. Howard Florey, Norman Heatly, and Ernst Chain, who began to research penicillin in mice. One of Florey's human patients, Albert Alexander, developed a severe Staphylococcus infection that doctors were certain was fatal. When Dr. Florey began treatment using penicillin, Alexander's fever decreased and he appeared to be recovering. Unfortunately, Alexander required more penicillin than the hospital could produce, and his infection returned, ultimately causing his death.

Alexander did not die in vain. His treatment showed doctors that penicillin could treat bacterial infections in human patients, and researchers focused on finding a sufficient source of penicillin to serve the hospital. They found a candidate fungus on a moldy cantaloupe from a market in Peoria, Illinois. The fungus was identified as Penicillium chrysogenum, a close relative of Fleming's Penicillium rubens. Upon characterization, P. chrysogenum produced several times more penicillin than P. rubens, greatly improving penicillin availability in hospitals by 1945 (ACS & RCC, 1999).

To illustrate these early observations, we designed a set of experiments to test the antibiotic effect of P. chrysogenum. Students practice microbiology techniques, growing fungus both in liquid culture and on agar plates. The fungus is tested for penicillin antibiotic activity against three bacterial species with different levels of penicillin sensitivity: Staphylococcus epidermidis, Micrococcus luteus, and Enterobacter aerogenes. To determine bacterial growth, students measure optical density using a spectrophotometer or colorimeter capable of measuring light at 600 nm (OD600) (Brown, 1966; Morris, 1978). This activity will establish an understanding that living organisms in nature produce antibiotics, and that antibiotic sensitivity differs among bacterial species.

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.bio5.org/biotech_lab_activities/penicillium

Preparation

These steps can be performed by the instructor, teacher's aids, or the students.

  • -

    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 per liter.)

The remaining steps of the protocol are to be performed by the students.

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.

    • 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 mlV1=(0.05×25 ml)/13.6=0.092 ml or 92μl

    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 equaling 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).

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.

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.

Table 1.
Example data for fungal spore suspension.
MeasurementSpec ReadingActual OD600
P. chrysogenum spore suspension 1.36 13.6 
MeasurementSpec ReadingActual 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.

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.

Day 3: Start bacterial cultures and observe fungal growth

  • -

    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 bacterial contamination.

  • -

    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.)

Day 4: Co-cultivate fungus and bacteria in liquid media

  • -

    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 required).

    • 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 mlV1=(0.05×75 ml)/5.28=0.710 ml or 710μl
  • -

    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:

  • -

    Co-cultivate all three bacteria onto solid media with fungal stripe (Figure 2).

    • 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.

Table 2.
Example data for results of bacterial overnight growth.
Bacterial SpeciesSpec ReadingActual OD600
S. epidermidis 0.528 5.28 
M. luteus 0.499 4.99 
E. aerogenes 0.478 4.78 
Bacterial SpeciesSpec ReadingActual OD600
S. epidermidis 0.528 5.28 
M. luteus 0.499 4.99 
E. aerogenes 0.478 4.78 

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.

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.

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.

SampleOD600 of pure bacterial cultureOD600 of fungus/bacteria co-culture
S. epidermidis 5.73 2.57 
M. luteus 1.95 0.045* 
E. aerogenes 4.84 3.39 
SampleOD600 of pure bacterial cultureOD600 of fungus/bacteria co-culture
S. epidermidis 5.73 2.57 
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 experiments.
Bacterial speciesDistance to stripe 1Distance to stripe 2Average distance
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 
Bacterial speciesDistance to stripe 1Distance to stripe 2Average distance
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 

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 measured distance between the fungus and the bacterial growth by calculating the arithmetic mean, (A + B)/2.

Discussion

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 until homogenous.

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).

Conclusion

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.

Extensions

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 fungal ITS region and sequencing the amplified DNA. Students may also identify any unknown bacterial species through a similar method by PCR amplifying and sequencing the 16S ribosomal region of the bacteria. This allows students to use modern DNA technologies and DNA computer databases, bringing modern technologies into the spectrum of their learning and into an experiment that changed the face of medicine.

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