Penicillium Antibiotic Effect

       The Penicillium genus of fungi naturally produces the antibiotic penicillin. Students will grow cultures of historically relevant Penicillium rubens and Pencillium crysogenum fungus on both solid and liquid media. Three species of bacteria will be cultured to observe differences between penicillin-resistant and penicillin-sensitive bacteria. Both fungal and bacterial concentrations will be normalized before co-cultivating, and bacteria will be quantified to determine antibiotic effect in liquid culture and on solid media. This lesson will teach students about natural product antibiotics and experimental design and application.

       This experiment allows students to practice several laboratory techniques, which are essential to careers in the biological sciences. They will cultivate and quantify microbiological organisms, practice accurate measurement techniques using several tools, conceptualize experimental design, and connect laboratory experiences to themselves and their understanding of medicine.

       The activity requires one week (five days) of class time. Students should be organized into groups of two to four. Each group will work with one species of bacteria. Groups will collaborate to compile a complete data set for the experiments. A minimum of 3 groups is required to complete the data set.

[Download Student Guide for Word]

 


Penicillium Fungus and Antibiotic Effect Classroom Protocol.

Background

The discovery of penicillin revolutionized medicine. Developing penicillin product and manufacturing process might have been the greatest contribution to medicine of the 20th century. Before the discovery of penicillin and other antibiotics, bacterial infections were common causes of death around the world. In the age before antibiotics, bacterial infections killed more people than heart disease and cancer combined. Today bacterial infections are considered trivial and are often treated with simple, inexpensive pills or ointments.

Penicillin was the first antibiotic discovered in 1928 by Dr. Alexander Fleming. After returning from a vacation, Fleming noticed a greenish mold contaminating one of his bacteria plates. Fleming observed that the bacteria would die within a zone around the mold leaving a clear ring. He isolated the mold in pure culture and at first determined that it was Penicillium. Many scientists would have dismissed this as a ruined experiment but Alexander Fleming was observant. His curiosity drove him to write a short scientific article on his observations.

Fleming’s discovery caught the attention of Dr. Howard Florey, Dr. Norman Heatly, and Dr. Ernst Chain. These researchers discovered that penicillin could be used to cure bacterial infections in a living organism and was suitable for use in mice and ultimately humans.

Although at first researchers worked to mass-produce penicillin from P. rubens, they quickly realized that P. rubens could not produce enough penicillin for hospital demand. These researchers began seeking a species that could produce more of the antibiotic and even asked local citizens for rotten vegetables from their kitchens. They soon discovered just what they needed on a moldy cantaloupe from a grocery in Peoria, Illinois. This cantaloupe was being consumed by Penicillium crysogenum, a species that produced around ten times the penicillin of P. rubens. P. crysogenum was capable of meeting the hospital demand for penicillin until many years later when the process for producing penicillin was improved.

Our experiment

To understand what the early researchers might have witnessed, we can design an experiment to test the antibiotic effect of the two species of Penicillium mentioned above. As a class we will grow the two different fungal species in liquid cultures. Each fungus will be tested using three different bacterial species to see the effect of penicillin. We will be using the common bacteria Staphylococcus epidermidis, Micrococcus luteus, and Enterobacter aerogenes. We will measure the amount of bacteria in each flask after growing overnight with the fungus.

We can measure using optical density (OD600) to determine the amount of bacteria in the liquid. For accuracy we will need to filter the fungus out of the culture, then we will compare the OD600 of the bacteria to compare the antibiotic effect in each case.

Each group will work with a single species of bacteria and we will pool the results from the class for the full experiment.

Experimental Goals:

  • Observe which fungus is more effective at killing bacteria
  • Observe possible bacterial resistance to penicillin
  • Consider experiments designed for accurate results

Materials (recipes below)

  • P. rubens culture plate, grown 7 days on Potato Dextrose Agar (PDA) 
  • P. crysogenum culture plate, grown 7 days on PDA
  • S. epidermidis culture plate, grown overnight at 30-37°C, can be stored at 4°C for 1 month
  • M. luteus culture plate, grown overnight at 25°C, can be stored at 4°C for 1 month
  • E. aerogenes culture plate, grown overnight at 25-37°C, can be stored at 4°C for 1 month
  • 125mL LB (Miller) broth
  • 1x pack of inoculating loops or reusable metal loop
  • 2x LB agar plates
  • 5x sterile cotton tipped applicators
  • 2x sterile LB (1.8ml) in 2ml microcentrifuge tubes
  • 3x sterile LB (25ml) in 125ml Erlenmeyer flasks
  • 1x sterile 250ml Erlenmeyer flasks
  • 2x sterile 5ml culture tubes
  • 1x Sterile 50ml graduated cylinder or falcon tube
  • 3x non-sterile collection tube (15ml Falcon tubes work best)
  • Funnel, ensure spout fits in collection tube
  • 5x non-sterile coffee filters
  • 6x cuvettes
  • Spectrophotometer (or colorimeter)
  • Orbital shaker

Procedure

Penicillium (fungal) spore suspensions

  1. Begin by initializing and blanking your spectrophotometer (spec)

(for Thermo Spectronic 200):

  1. Plug in and turn on. Wait until it shows “Remove cuvette and press ok…” Check that the spec is empty and press the ↵. Select OD600 from the menu options and press ↵ when the next screen appears
  2. Fill a cuvette with 900ml LB. Put it into the holder in the spec. Face the ribbed side towards the front of the spec. Close the lid and press the “0.00” button.  You will need to repeat blanking each cuvette immediately before use.
  3. If using a WPA CO7500 colorimeter: Turn instrument on, place 1ml LB cuvette in the holder in the correct orientation and press “Z”.

 

  1. Label one of the 2ml centrifuge tubes containing LB with an “R”. Dip a sterile cotton swab into the LB tube.
  2. Using sterile technique, collect spores from the P. rubens plate by rubbing the LB soaked cotton swab on the plate. Collect an area roughly the size of your pinky finger. Press firmly while rubbing, but not so firmly to break the agar. These plates can be shared among several groups. Select a fresh area of the culture for harvesting each time.
  3. Put the cotton swab with spores back in the “R” tube and twirl it to mix in the spores. Continue about 10 seconds until the suspension looks dark green and homogenous.
  4. Make a 1/10 dilution in a new cuvette:
    1. Votex spore suspension 5-10 sec.
    2. Pipette 900ml LB into a new cuvette and blank the spec.
    3. Pipette 100ml of your spore suspension into the same cuvette.
    4. Pipette up and down to mix thoroughly.
    5. Make sure you leave all the suspension in the cuvette after mixing.

 

  1. Make sure that the OD600 reading on the spec is above 1.0.  If not, you may need to add more spores from the plate to the 2mL tube
  2. Measure the OD600 with the spec and record the result in Table 1 below
  3. Label the other 2ml tube with LB “C” then repeat steps 2 through 7 with the P. crysogenum spores. Record your results in table 1.
  4. Remember the spec reading is 1/10 the Actual OD of the spores in your tube because you made a dilution. Multiply your Spec Readings by 10 to get the Actual OD.

     Table 1.

Measurement

Spec Reading

Actual OD

P. rubens spore suspension OD600 measurement

 

 

P. crysogenum spore suspension OD600 measurement

 

 

 

  1. In order to normalize the amount of fungal spores you add to each culture, you will need to calculate the dilution of spores needed for a final OD of 0.05 in the final volume of 25mL.

a.  For the dilution use the calculation equation C1 x V1=C2 x V2

b.  This can be re-written:

(Actual OD in your 5ml tube)  x  (Volume you need in ml) = (0.05 final OD)  x  25ml

c.  (Volume you need) = (0.05 x 25ml)/(Actual OD in your 5mL tube)

d.  Multiply answer by (1000ml /1ml) to convert to ml

  1. Mix the “R” rubens spores by inverting the tube 3 times.

Pipet the volume from step d into one of the flasks containing 25ml LB.

Label this flask with the contents, your group initials, and the date using tape.

  1. Repeat step 11 for the “C” crysogenum spores.
  2. Place both flasks on a shaker to be left for 72h or 3 full days at 25°C shaking at 100rpm.

(You will have 1 of your 3 flasks of LB left over. Set it aside and keep it sterile. You will use it as a bacteria control in step 16.)

  1. Finally, we will use some of the remaining spores of each fungus to make a vertical stripe on LB agar plates.
    1. Label one LB agar plate rubens, the date, and your initials, repeat with crysogenum for the other plate.
    2. Dip a new sterile cotton swab into the “R” tube.
    3. Make one swipe across the center of the rubens LB agar plate.
    4. Repeat with the crysogenum plate from the “C” tube.

                                 

Culturing Bacteria (DAY 3)

  1. Collect a patch of your bacteria from the plate using a sterile 200ml pipette tip.
    1. Use a 200ml pipette to hold the tip.
    2. Touch the tip to a bacterial colony.
    3. Eject the tip into a culture tube containing 5ml LB.
    4. Label the tube with the bacteria name, your initials, and the date

 

  1. Incubate your tube with the rest of the class by shaking at 180 rpm overnight at 30°C.

Co-cultivation

On the 4th day of the experiment, the cultures are ready to be co-cultivated. (bacteria added to fungus)

  1. Determine the relative bacteria concentration by measuring OD600 of your bacterial culture.
    1. Blank the spectrophotometer with 900ml LB in a cuvette.
    2. Pipette 900ml LB into a new cuvette.
    3. Make sure your bacteria is well mixed by vortexing one of the 5ml overnight cultures (the other culture is back-up and does not need to be measured yet)
    4. Pipette 100ml of bacteria into the cuvette containing 900ml LB and pipette up and down to mix.
    5. Measure OD600 and record value for Spec Reading and Actual OD (Spec Reading times 10 = Actual OD)
    6. Record your results in table 2.

Table 2

Bacteria

Spec Reading

Actual OD600

 

 

 

  1. Co-cultivating flasks with bacteria:

 

  1. Prior to co-cultivating the bacteria with the fungus, you will normalize the three bacteria cultures so that you are adding relatively the same amount of each culture to the fungus. Each dilution of bacteria will be normalized to OD600 at 0.05, this time you will dilute in 100ml of LB broth (Recall C1V1=C2V2 from step 8.)
  2. Label a 250ml flask containing 100ml LB broth with the name of your bacteria.
  3. Add the normalized concentration volume of bacteria culture you calculated in step 18a to the flask and swirl to mix.
  4. Using a sterile 50ml Falcon tube, add 25ml of your bacteria to each of the three 125ml flasks containing P. rubens, P. crysogenum, & sterile LB (your bacteria only control)

 

  1.  Incubate the mixed cultures at 25°C overnight shaking at 100rpm.
  2. The last step for today is to inoculate bacteria on the vertical stripped Penicillium LB agar plate you made the other day.
    1. Using sterile technique, dip a sterile inoculating loop into your 5ml overnight bacterial culture.
    2. Make two stripes starting at the edge of the plate perpendicular to the fungus moving the loop toward the fungus.

                                              

  1. Label with the bacteria name just under the stripes.
  2. Get together with two other groups who have the other bacteria and repeat steps 20a – 20c using their bacteria. You can use both sides of the fungal stripe.
  3. Incubate at 25°C over night.

Measurement

After 24h the bacteria will have experienced the effect of the antibiotic and is ready for measurement.

  1. Label your 3 collection tubes with the names of your bacteria and fungus as listed in the table above.
  2. The cultures need to be filtered to separate the bacteria from the fungus. Fold a coffee filter into quarters. Pull one fold apart from the others so that it makes a cone and place this into the funnel.

  1. Place the funnel in a collection tube. Swirl the flask to re-suspend the bacteria then pour less that 10mL from the bacteria only flask through the filter. You can use the graduations on the side of a falcon tube collection tube for this measurement as is doesn’t have to be precise, just less than 10mL.
  2. Throw away the coffee filter and shake off the liquid from the funnel into the biohazard waste. Save the filtrate in the collection tube.
  3. Repeat steps 21 – 24 for all 3 flasks.
  4. Measure the OD600 of the filtrate from each tube:
    1. Add 1mL of the filtrate to a cuvette. Measure the OD600
    2. The spectrophotometer is able to read up to 2.50OD. If the spec is flashing 2.50, you need to dilute the filtrate.
    3. Dilute in a cuvette by adding 100mL filtrate from the falcon tube to 900mL LB as in step 5. (Remember to record the ACTUAL OD600.)
  5.  Record your data in table 3 and the class data in table 4.

Table 3

Your group bacterial species

 

 

 

Sample

Spec Reading

Actual OD600

LB ONLY

 

 

P. rubens

 

 

P. crysogenum

 

 

   

 

 

 

 

 

 

 

 

          Table 4

 

P. rubens only

P. crysogenum only

 

 

 

Actual OD600 Data

LB pure culture

P. rubens

P. crysogenum

S. epidermidis

 

 

 

M. luteus

 

 

 

E. aerogenes

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Measuring the vertical stripe plates.

  1. Using a ruler, measure the distance from the vertical stripe of fungus to the first sign of bacteria in the bacterial stripes. Do this for each of the bacteria on your plate.
  2. Record any observations and differences you can see. Focus on the way the bacteria are growing. How are they different from each other? How does the bacterial growth differ on the P. rubens plate vs. the P. crysogenum plate?

Recipes

LB broth (Miller)

1.0% Tryptone (10g/L)

0.5% Yeast extract (5g/L)

1.0% NaCl (10g/L)

Autoclave to sterilize

LB agar (Miller)

1.0% Tryptone (10g/L)

0.5% Yeast extract (5g/L)

1.0% NaCl (10g/L)

1.5% Agar (15g/L)

Autoclave to sterilize and pour plates when warm

Potato Dextrose Agar (PDA)

20.0% Potato (200g/L)

2.0% Dextrose (20g/L)

1.5% Agar (15g/L)

Preparation of PDA

  1. Boil sliced, unpeeled potatoes in 1L distilled water for 30 min.
    1. Use a flask that is twice the volume you intend to make (i.e. 2L flask for 1L media.
  1. Filter through cheesecloth, saving effluent.
  2. Bring to 1L with grad cyl.
  3. Add dextrose while mixing to dissolve.
  4. Add agar.
  5. Autoclave 15 min at 121°C to sterilize.
  6. Pour plates @ ~25mL/plate (don’t measure just pour until media covers bottom).
  7. Allow plates to “dry” 24h before bagging. Store at 4°C as long as is needed.

TEACHER Supplemental Notes

If possible the first day should be completed on a block day.

The fungal incubation can proceed over the weekend from Friday to Monday or Tuesday (3-4 days)

The bacteria must be measured 24hrs after co-cultivation.

This experiment can easily become a great way for the teacher to introduce statistics techniques either in class or in collaboration with the math department.