In the Laboratory
Production, Extraction, and Qualitative Testing of Penicillin A Biochemistry Experiment for Health Science Chemistry Courses Richard E. Stevens and Kara C. Billingsley Department of Chemistry, Whitworth College, Spokane, WA 99251
Intended Audience This procedure was designed for a biochemistry course taken by nursing and physical therapy students. This course is generally not taken by Chemistry majors. The goal was to produce an experiment that was interesting and pertinent to medical professionals, did not require any advanced techniques, and produced minimal waste. This procedure requires two laboratory periods separated by a week, and a 10-minute viewing period 24 hours after the second laboratory period. Introduction The antibacterial nature of penicillin was first noticed by Alexander Fleming in 1928 (1). Fleming was unable to isolate or stabilize the chemical, delaying its use in medicine for 10 years. In 1938 Ernst Chain and Howard Florey devised a method to purify and extract penicillin (2). The most efficient method of extracting the drug proved to be this solvent method, which involves manipulating the pH of a solution to allow penicillin to pass between water and amyl acetate. The penicillin can then be extracted by using a separatory funnel (3). During wartime, many methods of cultivating and extracting penicillin were researched. An alternative to the solvent method was adsorption onto activated carbon and extraction with acetone (4 ). While this process is not as efficient or cost effective as the solvent method, it is much simpler and produces minimal waste, and thus is the method of choice for this laboratory procedure. Growth of the Penicillin Culture Penicillium chrysogenum is a mutant variety of Penicillium noted for high production of penicillin and is the recommended culture for this lab. It is inexpensively available from scientific supply companies.1 The culture takes approximately 1 week to mature and produce a maximum quantity of penicillin. Under optimum and constantly controlled conditions, the nutrient broth used will produce 200,000 units of penicillin, which is approximately a human dose. The procedure described here will produce a lower yield, yet still demonstrates the process of production and extraction to the undergraduate student. To prepare the nutrient for the Penicillium culture, mix the contents listed in Table 1. Add deionized water to bring the final volume to 100 mL in a 300-mL Erlenmeyer flask. The pH will need to be adjusted up to 5.6 with 1 M NaOH. Place aluminum foil over the mouth of the Erlenmeyer flask and autoclave.2 Add approximately 1.0 g of sterile calcium carbonate to the nutrient broth. This can be accomplished by using an aqueous slurry in a syringe equipped with a disposable syringe filter.3 A flame-sterilized glass rod should 1264
be used to test the pH of the solution. If it is still below 5.6, add more calcium carbonate. Inoculate the broth from a culture tube using sterile technique. Detailed instructions for inoculation procedures are available from the supplier of the culture if needed (5). Replace the foil cover (do not use a stopper) and place the flask in an agitator bath set at 25 °C and oscillating at approximately 30 cycles per minute. The agitation is critical to oxygenate the broth for submerged culture growth. Commercial production facilities add sterile oxygen, but for this laboratory, the substantial airspace above the culture seems to provide enough oxygen. The culture will need to incubate for 1 week until it is ready for extraction. Agitation must continue throughout the incubation period. Extraction of the Penicillin The mycelium must be removed from the broth. This is easily accomplished by pouring the contents of the flask through a very fast qualitative filter or a sieve. Add 3 g of activated carbon to the filtered solution (use granular, such as 12 × 40 mesh, rather than powder to speed up subsequent filtration). Swirl the solution once a minute for 5 minutes or place on an agitator to allow the penicillin to adsorb onto the carbon. Filter through a new fast qualitative filter paper to remove the carbon from solution. Allow a few minutes for the carbon to dry as much as possible. Add 20 mL of 90:10 v/v acetone/H2O to a 50-mL beaker. The activated carbon needs to soak in the acetone solution for 15 minutes. A simple method of soaking is to take the filter paper out of the filter and fold it into a “tea bag”, which is placed in the beaker. Alternately, the carbon can be scraped off the filter paper and into the acetone solution and subsequently separated by filtration.
Table 1. Composition of Nutrient Broth Component
Amount/g
Corn-steep liquor a
4.0
Lactose monohydrate b, c
3.75
Glucose anhydrous b, c
0.27
Sodium nitrate
c
0.3
Magnesium sulfate heptahydrate c
0.025
Potassium dihydrogen phosphate c
0.05
Zinc sulfate heptahydrate c
0.0044
aCorn-steep liquor was obtained from Sigma (800/325-3010). It is a corn by-product used to provide trace minerals and precursors containing the benzyl group, necessary for the production of penicillin. bLactose and glucose provide the energy source for the Penicillium. cPurity and number of waters of hydration are not critical as long as the amount used is adjusted accordingly.
Journal of Chemical Education • Vol. 75 No. 10 October 1998 • JChemEd.chem.wisc.edu
In the Laboratory
Figure 1. A simple flash evaporator constructed from a condenser.
After 15 minutes, the acetone solution will have extracted the majority of penicillin. The final step is to remove the acetone from solution. Since penicillin is heat sensitive, distillation is not an option. Flash evaporation works well, as the time spent in contact with the heat is brief enough to preserve the penicillin. Figure 1 shows a simple flash evaporator, which can be built from a condenser with steam rather than chilled water circulating in the jacket. Place the condenser at approximately a 45° angle so that any liquid flowing down the tube makes good contact with the wall. There need to be two openings at the top of the condenser. In one opening, place a small funnel in a stopper. In the other, run a vacuum line (a water aspirator works well). Pour the acetone into the funnel dropwise. The acetone will evaporate upon contact with the steam jacket, being drawn up through the top of the apparatus by suction. The water containing the aqueous penicillin flows down through the evaporator into a collection Erlenmeyer flask at the bottom. Qualitative Testing of the Penicillin In this procedure, the penicillin solution is tested on a bacterial culture susceptible to the drug. We used Staphylococcus aureus,4 which is widely available and commonly used in microbiology labs. It is used to set the efficacy standard for penicillin and thus has some historical background. Staphylococcus aureus is pathogenic and precautions should be taken when working with it. A safer but more expensive alternative is Staphylococcus chromogenes.5 To conduct the test, a teaching assistant prepares and autoclaves petri dishes filled with agar. After autoclaving, a core of agar 1 cm in diameter is removed from the center of the plate with a sterile instrument. The students inoculate the petri dish with Staphylococcus bacteria and spread the inoculum thoroughly using a sterile bent glass rod. They then fill the hole in the center with their penicillin solutions. Control plates are similarly prepared. We have tried three control solutions, none of which provides any inhibition of the Staphylococcus culture: (i) sterile nutrient solution passed through the adsorption, elution, and flash evaporation process, (ii) 90:10 v/v acetone/H2O passed through the flash evaporator; and (iii) deionized water. Commercially prepared salts of penicillin6 are widely available and provide a good standard comparison of inhibition.
The Staphylococcus cultures are best viewed 24–48 hours after inoculation. A careful extraction will yield almost no bacterial growth in the penicillin-treated agar in 24 hours. After this time, growth should appear at the edges and work its way inward. After 48 hours, mold contamination may show up from Penicillium spores that survived the flash evaporation. The control dish should show verdant Staphylococcus growth after 24 hours. Quantitative analysis of the solutions is beyond the scope of this course and is not well suited to this method owing to the inefficiency of the carbon process. However, there are several methods of analysis, such as immobilized enzyme electrode (6 ) or IR absorption at 1770 cm᎑1 (attributed to the carbonyl group), should one wish to investigate further. We strongly recommend use of the solvent extraction process rather than carbon adsorption for quantitative analysis. Waste Disposal All cultures, filter papers, nutrient solutions, and activated carbon used in the lab should be autoclaved and sent to a landfill in accordance with federal, state, and local laws. The acetone from the flash evaporator may be trapped and disposed of appropriately. There should be no other wastes generated with this laboratory. Acknowledgments We wish to gratefully acknowledge the assistance of T. Mega and K. Stevens in the preparation of this manuscript. Notes 1. Penicillium cultures were obtained from Carolina Science and Math (800/334-5551). 2. The entire autoclaving process will last approximately 1 hour, during which time the instructor may wish to have another exercise for the students to work on. 3. Syringe filters (0.22 µm) were obtained from Carolina Science and Math (800/334-5551). The autoclaving process affects pH, so the final adjustment is necessary. 4. Staphylococcus aureus was obtained from Carolina Science and Math (800/334-5551). 5. Staphylococcus chromogenes (ATCC 43764) may be obtained from the American Type Culture Collection (800/638-6597). 6. Penicillin-G (benzylpenicillin) was obtained in the form of its sodium salt from Sigma (800/325-3010).
Literature Cited 1. Fleming, A. Br. J. Exp. Pathol. 1929, 10, 226–236. 2. Chain, E.; Florey, H. W.; Gardner, A. D.; Heatley, N. G.; Jennings, M. A.; Orr-Ewing, J.; Sanders, A. G. Lancet 1940, 239, 226–229. 3. Abraham, E. P.; Chain, E; Fletcher, C. M.; Gardner, A. D.; Heatley, N. G.; Jennings, M. A.; Florey, H. W. Lancet 1941, 241, 177–189. 4. Whitmore, F. C.; Wagner, R. B.; Noll, C. I.; Bassler, G. C.; Fleming, G. H.; Carnahan, F. L.; Weisgerber, C. A.; Oakwood, T. S.; Herr, C. H.; Patterson, H. T.; Haggard, H. H.; Mraz, R. G.; Hoover, T. B.; DiGiorgio, P. A.; Weisel, C. A.; Lovell, H. L.; Walter, R. N.; Ropp, W. S. Ind. Eng. Chem. 1946, 38, 942–948. 5. Hauser, J. T. Techniques for Studying Bacteria and Fungi; Carolina Biological Supply Company: Burlington, NC, 1986. 6. Mifflin, T. E.; Andriano, K. M.; Robbins, W. B. J. Chem. Educ. 1984, 61, 638–639.
JChemEd.chem.wisc.edu • Vol. 75 No. 10 October 1998 • Journal of Chemical Education
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