Friday, May 04, 2007

How To Make Penicillin: Cheap and Tasty Version

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Equipment:
  • Viable spores or a live culture of a strain of Penicillium chrysogenum suitable for submerged (vat) culture of penicillin
  • Tanks for holding the culture broth that are capable of being sterilized
  • A means for aerating the broth in vats with sterile air
  • Purified water
  • Lactose (20 parts per 1000) and corn steep solids (20 parts per 1000) (or corn steep liquor) for the fermentation tank, along with trace amounts of substances such as sodium nitrate (3 parts), dipotassium phosphate (0.05 parts), magnesium phosphate (0.125 parts), calcium carbonate (1.8 parts), and phenyl acetic acid (0.5 parts). All these items must be completely sterile.
  • Filtering material, such as parachute silk
  • A weak acid and a weak base
  • Amyl acetate or ether (for removing the penicillin from the broth)
  • Aluminum oxide powder or asbestos (to filter microorganisms and "pyrogens" - fever-causing impurities - from the penicillin)
  • Freeze drying equipment such as a rotary freeze dryer (for removing the water from the penicillin to make a storable crystalline compound)
  • Microscopes and slides (for testing the activity of the penicillin)
Thoughtful people might add other items likely to be necessary to this list, such as electricity, laboratory glassware, and agar agar. For simplicity, I am leaving such background items of indirect necessity off the list - for now.

Procedure:
  1. Sterilize the tanks and aeration equipment.
  2. Dissolve the sugar, corn steep liquor, and other substances in the water in the tanks.
  3. Introduce the mold to the culture medium.
  4. When the mold is reproducing, begin aeration with sterile air. Ideally, maintain the temperature at approximately 24 degrees Celsius. Using aseptic methods, test the broth regularly for penicillin concentration and antibacterial activity. (See note.)
  5. When the broth has reached a high level of penicillin concentration, filter the mold juice through a physical filter, such as parachute silk.
  6. Acidify the mold juice to a pH of 2-3 using the weak acid (such as citric acid).
  7. Thoroughly shake the mold juice with the solvent by hand or using an apparatus.
  8. Allow the mold juice and penicillin-containing solvent to sit until they reseparate.
  9. Drain off the dirty water.
  10. Filter the penicillin-containing solvent through the aluminum oxide powder (alumina salts). The top brownish-orange band contains little penicillin; the pale yellow band contains the majority of the penicillin and no pyrogens; the bottom brownish or reddish-violet purple band is full of impurities. (The solvent may be re-used.)
  11. Carefully separate only the yellow band in the aluminum oxide powder; wash it in a buffer to clear off the alumina. The fluid is a deep reddish-orange color that turns yellow when diluted; it has a faint smell and a bitter taste.
  12. Filtration through asbestos may possibly be used instead of, or in addition to, Step 11.
  13. Freeze dry the solution to obtain crystalline penicillin.
Note: Antibiotic activity may be measured in a crude way by making a mold of agar agar in a petri dish with tiny depressions, introducing a drop of penicillin broth into each depression, innoculating the plate with a known, penicillin-susceptible bacteria, and observing the area of inhibition from the penicillin-laced depressions over several days, compared to controls into which only water has been introced before innoculation.

Sources:

Chatwal, Gurdeep R. Synthetic Drugs. Mumbai: Himalaya Publishing House, 2006.
Herrell, Wallace E. Penicillin and Other Antibiotic Agents. Philadelphia: W. B. Saunders Company, 1945.
Lax, Eric. The Mold in Dr. Florey's Coat. New York: Henry Hold and Company, 2004.
Ligget, R.W. and H. Koffler. Corn steep liquor in microbiology. Bacteriol Rev. 1948 December; 12(4): 297–311.
Pathak, S.G., and R.P. Elander. Biochemical properties of haploid and diploid strains of Penicillium chrysogenum. Appl. Microbiol. 22:366-371.
US Army Special Forces Medical Handbook. United States Army Institute for Military Assistance, 1982.
Werner, David. Where There Is No Doctor. London: Macmillan Education Ltd., 1993.

9 Comments:

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