Making Penicillin, Part 5: Extraction

Introduction

Penicillin-producing fungus will excrete penicillin into the fermentation medium. This means that you don't have to lyse (bust open) the cells in order to recover your product. If you wish, you can siphon off some of the supernatant (the liquid portion of the fermentation) to extract penicillin, and add fresh media to the remainder of the fermentation to continue the culture.

If you're very lucky, you'll recover 1g/L of supernatant. According to Rxlist.com, it takes up to 24 million units (about 15g) per day (divided over 4-6 doses) to treat septicemia, pneumonia, endocarditis, meningitis and syphilis. For treatments lasting 14 days (as is often the case with syphilis), that's 210g of penicillin phosphate - 200 liters' worth of fermentation for the treatment of one person. It is difficult enough to produce that volume of antibiotic, but it is even more difficult given penicillin's finite shelf-life. Pharmaceutical-grade penicillin has an expiry of a year; even in a post-apocalypse scenario, I wouldn't hold onto homemade penicillin any longer than that.

Materials

Penicillium fermentation
coffee filter
hydrogen chloride (aka HCl or hydrochloric acid)
ethyl acetate
pH strips
separatory funnel
ring stand
potassium acetate

• The link above is to potassium acetate salt, which you can use to make a 1% solution. To do so, add 0.5g potassium acetate salt to 50mL of distilled water and mix until the salt completely dissolves.

Procedure

1. Separate solids from liquid. You'll notice growth forming solid structures within the fermentation medium. The first step is to separate the liquid (in which the penicillin is secreted) from the solids. One way to do this is to place the separatory funnel in the ring stand, remove the stopper, curl a coffee filter into a cone shape and fit the apex of the filter-cone into the neck of the separatory funnel. Make sure the stop cock at the bottom of the separatory funnel is in the closed position, and pour the fermentation through the coffee filter.

2. Refrigerate. Once the fermentation has been filtered, remove the solids, stopper the separatory funnel and place it in the fridge. While the liquid is cooling, put the ethyl acetate in the freezer (it won't freeze), and put the hydrochloric acid in a bucket of ice to chill. Leave the fermentation liquid in the fridge for a couple of hours.

3. Adjust the pH. Add the hydrochloric acid solution in a few drops at a time, mix and test the pH. The fermentation should start at a pH of around 5; the more acid you add, the lower the pH will go. You'll probably have to repeat adding acid and testing several times to get a solution to a pH of 2.2, especially since the citric acid in your solution acts as a buffer. This means that as you at acid (protons), citric acid will soak them up (to a point), and your solution's pH won't appear to change much. Try not to get impatient and pour acid into your mix; there comes a point where the citric acid has soaks up all the protons it can, and suddenly the pH will change dramatically with a small addition of acid.

4. Take collection beaker. Take a clean glass vessel (such as a mason jar), measure the mass with your analytic balance. Write the mass in permanent marker on the outside of the jar (you may even want to put a piece of clear tape over your writing to make certain it's legible later). When you've collected your penicillin, you can subtract the weight of the glass vessel from the total weight to calculate the amount of penicillin you produced.

5. Add cold ethyl acetate. On the molecular level, ethyl acetate looks a lot more like penicillin than acidic water - i. e., penicillin is more soluble in ethyl acetate. Add cold ethyl acetate, put the stopper back in the neck of the separatory funnel and shake the whole thing vigorously for at least 30 seconds. This will allow the penicillin to move from the acid water to the ethyl acetate. Ethyl acetate is denser than water, so it will settle on the bottom of the separatory funnel. Put a beaker or container under the separatory funnel, open the stop cock slowly and pour off the ethyl acetate (ideally in a glass container that has been chilled in the freezer). To make sure you've extracted as much penicillin as you can add more ethyl acetate to the remaining acid water solution, shake, and pour off the ethyl acetate layer into the collection beaker.

6. Add potassium acetate to your ethyl acetate solution. A good way to keep your ethyl acetate solution cold is to seat the glass vessel (e. g., mason jar) within a dish of crushed ice. With a 1% solution of potassium acetate, you will have 1g of potassium acetate per 100mL of the solution. At best, you will only get about 1g of penicillin per liter (0.1g in your entire 100mL fermentation), so you won't need a lot of potassium acetate; 100mL should be plenty.¹

7. Evaporate the ethyl acetate. Put your solution in a a well-ventilated area (ideally away from living areas, such as in a garage with a fan - you don't want to breathe the fumes). This will allow the water and ethyl acetate to evaporate, leaving only potassium acetate salt (which will act as a penicillin preservative) and potassium penicillin G salt as the precipitates. It's important that all the liquid has evaporated before you weigh the vessel; any remaining liquid will add weight to the vessel and falsely inflate the calculated amount of penicillin you produced.

8. Weigh and calculate. Weigh the vessel with the penicillin salt in it, then subtract the tared mass of the empty vessel (from step 4). You will also need to subtract the amount of potassium acetate from the total mass to get the final weight of penicillin:

Total mass - empty vessel mass - (1g potassium acetate/100mL solution X mL of solution used) = mass of penicillin

9. Refrigerate. Even with the potassium acetate acting as a preservative, penicillin G has a limited shelf-life (you probably shouldn't use it after a year).

References

1. Sajjad-Ur-Rahman; Rasool, M.; Rafi, M. 2012. "Penicillin production by wild isolates of Penicillium chrysogenum in Pakistan." Braz J Microbiol. 43(2): 476-481

Making Penicillin, Part 4: Fermentation

Introduction
Most bioproducts (products produced biologically, often by microorganisms) are produced by fermentation. Microbes are grown in conditions that make them divide rapidly until the culture is dense. When the easily "digestible" nutrients are spent, the culture reaches maximum density and the microbes "switch metabolic gears." This is usually when they start producing secondary metabolites - the product you want. This is fermentation.

Glucose is a simple sugar that acts as a quick carbon source, jumpstarting the rapid growth of your culture. The idea is to increase the density of your Penicillium so that you have a lot of cells producing penicillin. Yeast extract is a bunch of yeast cells that have broken themselves down (a process called “autolysis”) into component carbohydrates and amino acids. This provides a lot of nutrients and longer-term energy once the glucose is depleted (which happens pretty quickly). Citric acid acts as a buffer, to prevent the pH of your medium from fluctuating and stressing your cells. Lactose is a simple sugar (glucose bonded to galactose) that is often used because unlike glucose, lactose doesn’t inhibit penicillin production. It’s important to use sea salt rather than regular table salt for two reasons: 1) table salt uses iodine as an anti-caking agent, which is great for people (we have thyroid problems if we don’t get enough iodine), but can inhibit microbial growth; and 2) aside from the iodine, table salt is pure sodium chloride, and your cultures need the other minerals and micronutrients found in sea salt.

Materials
Erlenmeyer flask/glass jar
graduated cylinder
100mL distilled water
2g glucose (aka dextrose)
1g yeast extract
2g citric acid
10g powdered nonfat milk

• While this concentration provides enough lactose (a simple sugar) to promote a high concentration of penicillin, this will make the medium thick, and some may not even dissolve. At this concentration, you'll see the growth, but you can dial back the powdered milk if you'd like a clearer view

1g sea salt
forceps/inoculation

Procedure
1. Prepare the medium. Take a clean, dry Erlenmeyer flask or glass jar. Lab scale fermentation is typically performed in shake flasks. As a rule of thumb, whatever the volume of your Erlenmeyer flask is, you never want it more than 20% full with medium. Thus, if for example you have a 500mL flask, only put 100mL of medium in it. Keeping the liquid volume low increases the surface area of the liquid, allowing for faster diffusion of oxygen from the air to the medium, and Penicillium definitely needs oxygen to make penicillin.¹

If you’re shooting for 100mL of inoculated medium, put 2g glucose (aka dextrose), 1g yeast extract, 2g citric acid, 10g powdered nonfat milk and 1g sea salt to your graduated cylinder. Fill the graduated cylinder with distilled water until it reaches the 100mL level. Pour the whole thing into your flask and mix until everything’s dissolved. Cover the top of the flask with a folded pieced of aluminum (it keeps errant microbes out but lets oxygen in), and autoclave.

2. Inoculate with your isolate. Once the medium has cooled to room temperature, aseptically add spores of your Penicillium culture with sterile forceps or inoculation loop. Once you have replaced your sterile aluminum foil cover (after flaming both), you can let the culture sit at room temperature (give it an occasional swirl) for at least a week (but not longer than two weeks).

References
1. Rolison, G. N. 1952. "Respiration of Penicillium chrysogenum in penicillin fermentations." J. gen. Microbiol. 6: 336-343

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

Aseptic Technique: Streaking to Single Colonies

Introduction

Aseptic technique is essential to anyone working with microbes. It ensures that everything that touches your culture is sterile, so you avoid introducing contaminants into your culture. With pure cultures, contaminants introduce an incalculable number of variables, making it impossible to know if your results are attributable to the conditions you set for the experiment or the contaminant. It makes all your hard work, in a word, useless.

Streaking to single colonies can help you obtain a pure culture. Each individual colony - circle of growth - is the result of a single cell dividing into millions of clones. Thus, each cell within that colony is as genetically identical as you're ever going to get.

Materials

sterile agar plate
inoculation loop

• You can make your own by twisting a piece of metal wire (platinum is best, but aluminum is doable).

Bunsen burner
culture

• If you want to use the Penicillium culture featured in the video above, check here. 

Procedure

1. Turn on the Bunsen burner. The flame will heat the air above your work space, lifting the air and any contaminants that might have been disturbed.

2. Sterilize your inoculation loop. Hold the loop in the flame until it becomes red-hot. Then press it to the sterile surface of your agar. You will hear a hiss, indicating that that the loop is cool enough to touch your culture without singing it. Make sure the agar plate is uncovered no longer than necessary; this minimizes the risk of contamination.

3. Inoculate. Touch the inoculation loop to your culture, then touch the loop to the surface of the agar plate to make three parallel lines (again, make sure your agar plate is uncovered only as long as necessary). Sterilize your loop, touch it to an uninoculated part of the agar until you hear a hiss, then drag your loop through the end of the lines you just made. Make two more lines parallel to the last line, but not intersecting your previous three lines (this helps you "dilute" your inoculum). Sterilize your loop again, cool it with the sterile agar, then drag a line through the ending edge of your second set of three lines. If you do this until you have four sets of three lines, you will have single colonies.

Making Penicillin, Part 2: Reculturing

Introduction

Once you've isolated a strain of Penicillium, you'll probably want to subculture it so you can save it for future use. Penicillium grows well on potato dextrose agar (PDA) plates. If you're pouring your own plates, see the section an autoclaving and plate pouring. Once the plate are ready, you'll want to transfer some of your original culture to the plates (see my section on aseptic streaking to single colonies).

Materials

potato dextrose agar

• poured plates
• prepared media + sterile plates
• powdered media
• agar
• dextrose (aka glucose)

water filter (optional)
inoculation loop
cheesecloth (optional)
freeze-dried P. chrysogenum (optional)
filter paper (optional)
sterile petri dish (optional)
glassine envelope 

Procedure

Figure 1: Boiling sliced potato in distilled water.

References

1. "A Simple Way to Preserve Fungal Cultures." Web log post. Cornell Mushroom Blog. Ed. Kathie T. Hodge. Cornell University, 10 Jan. 2008. Web. 18 June 2014.

2. Rafi, M., and Rahman, S. U. 2002. Isolation and identification of indigenous Penicillium chrysogenum series. Int. J. Agr. Biol. 4(4): 553-558

Making Penicillin, Part I: Isolation

Introduction

Sir Alexander Fleming first isolated the species of fungus and the anti-bacterial it produced in the 1920's and 30's, but the genus Penicillium and human health have a long history. People would press the moldy side of a slice of bread to a laceration, or throw it into boiling water to make a tea or broth. Live Penicillium fungus can be safely ingested; if you eat unpasteurized camembert or bleu cheese, you’re digesting P. camamberti and P. roqueforti, respectively. However, naturally-produced penicillin (Penicillin G) is unstable in the highly acidic environment of the stomach, and is destroyed before it can be absorbed by the body. Thus, a Penicillium tea is unlikely to have any antibiotic effect. Penicillium G is injected intravenously or intramuscularly to treat syphilis, meningitis, endocarditis, pneumonia and septicemia.¹ As for an open-air wound, you’re probably better served by pouring sugar or honey onto the wound.

Materials

Penicillium chrysogenum culture (optional)
bread/lemon/cantaloupe (optional)
container to hold moisture (optional)

Procedure

References

1. Rossi, S, ed. 2013. Australian Medicines Handbook (2013 ed.). Adelaide: The Australian Medicines Handbook Unit Trust.

2. Laich, F., Fierro, F., and Martin, J. F. 2002. Production of penicillin by fungi growing on food products: identification of complete penicillin gene cluster in Penicillium griseofulvum and a truncated cluster in Penicillium verrucosum. Appl. Environ. Microbiol. 68(3): 1211-1219

Alexander Fleming and Deserved Inspiration

Imagine being diagnosed with venereal disease. It’s a difficult diagnosis for anyone, but imagine being told that your only possible cure is to be infected with malaria. The brain-damaging high fever that results from malaria would either cure the venereal disease or it would kill you quickly; either way, it was better than the slow, painful death due to VD. Less than a century ago, those were your only options.

Today people with VD – along with hundreds of millions more – owe their lives and their health in large part to one man: Sir Alexander Fleming.

Alexander Fleming was born August 6, 1881 in Scotland. Though he boasted a diverse range of talents – glass blower and rifleman among them – at the age of sixteen he began working in a shipping office. He spent four years in this unlikely occupation until he inherited sufficient money to enroll in medical school.

Throughout World War I, Fleming served as a captain in the Royal Army Medical Corps. It was here that he brought his diverse talents to bear. Despite the liberal use of antiseptics, Fleming saw countless soldiers die from deep wounds. Fleming used his glass blowing skill to make a test tube with conical spikes drawn from the bottom. He filled the test tube with serum, then inoculated it with fecal matter to simulate an infected wound. Repeated washing of the test tube with antiseptics didn’t sterilize the test tube any more than they sterilized the crevices of an actual wound.

After watching so many people lose their lives to sepsis, Fleming began his search for antibacterial agents. His first serendipitous discover came when he was examining one of his plates of bacteria. He had a bit of a head cold, and his runny nose dripped onto the culture. Fleming later noticed that the colonies had cleared around the drip; he had proof that there were antibacterial substances that would not harm the human body. It’s worth pausing on this discovery. It seems so obvious to us now – we live in a world where you can purchase soap with antibiotics – that it’s hard to imagine a world in which the existence of human-friendly antimicrobials was an open question. It’s harder still to realize that such a world existed less than a century ago.

A second serendipity is well-known: Fleming left his lab a dirty mess when he departed for a holiday in 1928. When he returned, he found a fungus contaminating one of his petri dishes, and the bacteria colonies refused to go anywhere near it. The fungus was of course a species of the genus Penicillium. Fleming researched the purification and clinical utility of penicillin for the next decade and a half. Shortly after he dropped the pursuit, Howard Florey and Ernst Boris Chain picked it up. By D-Day (1944), enough penicillin was produced to treat all wounded Allies.

According to ScienceHeroes.com, the discovery of penicillin has saved (and continues to save) over 80 million lives. That doesn’t include the dozens of other antibiotics discovered as a result of Fleming’s ground-breaking work. Over the course of human history, syphilis, typhoid fever and tuberculosis have each killed millions. It is estimated that bubonic plague (the Black Death) killed between 75 and 200 million people over the centuries. Now these diseases are dwindling from the human condition, thanks to Sir Alexander.

A lot of people like to point to Fleming as proof that scientific discoveries are the result of serendipity – as if the only way to improve the human condition is to sit around and wait for a gift from the universe. I regard that as an insult to a man who has save millions. Such remarks overlook the diverse interests Fleming nurtured that made him a better scientist. They ignore the compassion he felt towards his fellow soldiers, compassion that made him a keen observer and propelled his decades-long investigation. He was diligent with his work and careful with his observation, and when inspiration came, Sir Alexander Fleming was ready for it.