Making Penicillin, Part 3: Verification of Penicillin Production

Introduction

If you've isolated a fungus that is on the spectrum of gray-green with a white edge, you've got something promising. If you flip your PDA plate, look through the bottom and see that the fungal colony is yellow, even better ("Fun With Microbiology" has a great catalog with helpful pictures). However, the colony morphology of many Penicillium species and Aspergillus species overlap, and it can be difficult to distinguish them without a microscope. Furthermore, some species from both genera produce penicillin, while others do not.^1,2

It's helpful to verify that your isolate produces penicillin before going through the trouble of fermenting it and attempting to purify the ferment. You don't need a microscope to do this. You simply need to grow colonies of your isolate on an agar plate in the presence of Gram positive bacteria (more on that later). For now, it is important to note that Gram positive bacteria are more susceptible to Penicillin G than Gram negative bacteria. Thus, if you coculture your isolate with Gram positive bacteria and see "clearing" around the fungal colonies (i. e., there's a gap between the edge of the fungal colony and the bacterial "lawn" where nothing grows) - you've got a penicillin-producing isolate.

You can find a lot of Gram positive bacteria (and few Gram negatives) easily: on your skin.

If you don't see any clearing around your isolate, you may want to culture isolates over different parts of your home. Alternatively, you could pull an Alexander Fleming - ask everyone you know to give you their mold samples. There is the option to purchase a pure isolate from Carolina.

Materials

potato dextrose agar
fungal isolate
autoclaved water or autoclave liquid media
Bunsen burner
inoculation loop

Procedure

1. Set up. Make sure your work space is clean (follow the same procedure as if you were pouring plates) and your Bunsen burner turned on.

2. Add liquid media to the surface of your agar. Take your container of autoclaved water/liquid media. Remove the cover and pass the lip of the container through the flame (as if you were pouring plates). Lift the lid of the agar plate just enough to allow you to pour a small amount of sterile liquid (maybe a puddle the size of a quarter). Replace the lid on the plate. Flame the lip of the container and the cover before replacing the cover on the container.

3. Inoculate the isolate. Flame your inoculation loop until it's red hot, stick it into agar that has no agar (your plate with the puddle is fine) until it hisses, then use the loop to scoop up some of your isolate. Lift the lid of your "puddle plate" just enough to accommodate the inoculate loop, and swirl the loop in the puddle. Remove the loop, replace the lid, and flame the loop.

4. Inoculate the bacteria. Use your naked hand to spread the puddle all over the surface of the agar. When it's spread, the liquid should dry quickly. Wash your hands and allow the fungus and bacteria a few days to grow.

References

1. Brakhage, A.; Browne, P.; Turner, G. 1992. "Regulation of Aspergillus nidulans penicillin biosynthesis and penicillin biosynthesis genes acvA and ipnA by glucose." J Bacteriol. 174(11):3789-99

2. Laich, F.; Fierro, F.; Martin, J. 2000. "Production of penicillin fungi grow on food products: identificaiton of a complete penicillin gene cluster in Penicillium griseofulvum and a truncated cluster in Penicillium verrucosum." Appl Environ Microbiol. 68(3):1211-9