Grantville Gazette, Volume I Page 36

  "You said once that the czar supports Gustavus Adolphus, didn't you? Or is that his father's doing?"

  "Some of both I think." Vlad leaned forward. "Money. Always a problem, the money. The Poles cleaned out the Muscovy treasury. The time of troubles left roving bands of thieves that travel through Russia, some of them even now, after nearly twenty years of Mikhail's rule. Mikhail is loved by the people but he is not very strong. He is governed by the boyars and Duma men. I respect your system of government, Brandy. I really do. But how much of it can be adapted to Muscovy . . . that is harder to say. I don't know how much we can do. We have Natasha. We have your Berna, even. I will work for change, with all my heart."

  "I'll help." Brandy stood up. "As much as I can."

  NON-FICTION

  Crude Penicillin:

  Potential and Limitations

  By Kim Mackey

  "That which we know frequently impedes us in acquiring new knowledge."

  Claude Bernard

  (1813-1878),

  French physiologist.

  Background and Early History of Penicillin

  The Age of Disinfection began with the work of Pasteur and Lister in the 1860s and 1870s. While this initial work focused on external disinfection, doctors and scientists were soon looking for ways to use substances for "internal disinfection," that is, to rid the human body of disease-causing organisms. Unfortunately, these initial efforts were limited. "Their attack," Iago Goldston wrote, "was too direct, too primitive for the wily ways of nature. They thought to catch sunbeams in a butterfly net." [1, 81].

  "At the Congress of Internal Medicine held in Wiesbaden in 1883, the assembled body of scientists solemnly recorded it as their sober judgment that "inner disinfection is an impossibility." [1, 80]. Influenced by the work of Von Behring and others, scientists turned to anti-toxin therapies to cure human ills rather than chemotherapy. This attitude changed somewhat in the early twentieth century with the work of Paul Ehrlich and the discovery of salvarsan, but between 1915 and 1935 there was little progress and scientists began to doubt that the theories of Ehrlich were correct. It was in this context that Alexander Fleming, a member of the staff of the Inoculation Department at Saint Mary's Hospital in London, and recently appointed Professor of Bacteriology at the University of London, discovered penicillin in September, 1928. Unfortunately, Fleming limited his observations on penicillin's bacteria-killing power to "a few sentences or very short paragraphs in medical journals, most of them with very limited circulation." [2, 1]. Thus proof of the remarkable antibiotic power of penicillin would have to wait for the work of Florey and his colleagues twelve years later, well after the introduction of sulfanilamide's.

  As with any major discovery, the history of penicillin is filled with facts, pseudo-facts, omissions and myths. For example, because of the way in which penicillin attacks bacteria ("it could only act on them during the very short phase in their life history when they were actually dividing" [2, 3]), it is highly unlikely that the mold contaminated the plate after the bacterial colony had been established. This is important, because "if the phenomenon had been produced in the manner Fleming thought it had, it would be a very common occurrence in bacteriological laboratories all over the world, and Fleming could claim credit only for observing something unusual and acting upon it. In doing so he did himself an injustice, because the stringent requirements in terms of time when the mold reached the plate and the temperature to which it was subsequently subjected render its accidental production almost impossible in countries with tropical or continental climates, and very unusual in those with temperate climates. Fleming was a great deal more fortunate than he ever realized." [2, 3-4].

  Another myth that can be dispelled is the source of the mold. In 1945 when a film about penicillin was being produced, Fleming told the producer, for whatever reason, that the mold must have drifted in off the street. This was highly unlikely, however, since the windows were seldom open because they were too difficult to reach. In fact, the plates were most likely contaminated by mold from the mycology laboratory run by Dr. C. J. La Touche on the floor below Fleming's lab, since, as noted in Flemings original paper, one of La Touche's molds, "had exactly the same cultural characteristics as the mold on the original plate, and, although he did not say so, ability to produce the same amount of penicillin." [2,4]

  The dispelling of this myth is important for the 1632 universe. Why? Because knowing that molds and other substances in the soil have antibiotic properties and can be isolated and cultured is a huge percentage of the battle. Once this fact was appreciated up-time, many substances were quickly isolated that were useful in fighting a wide variety of bacterial infections.

  It will be very important for down-time scientists and physicians to appreciate the potential and limitations of antibiotics. While penicillin can treat a wide variety of bacterial infections, there are also a wide variety that it cannot, especially in the crude form. Fortunately, production of crude penicillin, once you have an appropriate mold strain, is fairly simple.

  Alexander Fleming did not start serious experimentation with the penicillium mold until December 1928, and when he did much of the work was done by a research assistant, Dr. Stuart Craddock, who had just qualified in medicine in July 1928. "It was soon found that penicillin could be produced by growing the mold at room temperature in the laboratory's routine broth, which was made in small batches from a tryptic digest of bullock's heart muscle. A pellicle formed on the surface, the fluid below became bright yellow and was usually free of mold particles. Although they could be removed by filtration without loss of penicillin, this was usually omitted." [2, 6]

  The procedure for estimating penicillin content "consisted in making serial dilutions in fresh broth, to each of which were added a few drops of a staphylococcal suspension. Following incubation, the highest dilution in which no growth of organisms had occurred was recorded as the titre. It was soon found that after growth at room temperature for five to seven days the titre was generally 1/100 to 1/300 and very occasionally 1/600. Thereafter, it began to fall so that all but a trace of penicillin had gone after fourteen days." [2, 6]

  In January 1929 attempts were made to concentrate the penicillin and it was fairly quickly discovered that penicillin was readily soluble in both ether and alcohol, but not significantly so in acetone. It is important to note that penicillin's instability was not a major problem for the experimenters. What drew Fleming and his assistants away from testing penicillin for antibiotic purposes were experiments with rabbits which seemed to indicate a quick elimination of penicillin once introduced into the body. Unfortunately, Fleming drew the wrong conclusion from the experiments and felt that the reason was the absorption of the penicillin by tissues and serum, rather than elimination through the urine.

  The earliest successful uses of crude penicillin to cure bacterial infections seem to have been the attempts in the early 1930s by Dr. C. G. Paine in Sheffield [3]. Paine obtained a culture of the mold from Fleming and grew the mold much as Fleming had done. Both gonorrheal infections in babies and an eye infection in a miner containing Pneumococcus were cured. Unfortunately, Paine did not publish any of the results of his experiments with crude penicillin and the next wave of crude penicillin work did not take place until the 1940s.

  With the coming of World War II and the work at Oxford by Florey and his associates, penicillin's antibiotic properties became well-known. Unfortunately, while the Oxford product was very potent, it was not very pure, nor was it initially available in large quantities. "This led a number of workers to re-examine the therapeutic properties of crude penicillin filtrates. Essentially, three methods were developed for the topical application of crude penicillin. These were 1) use of liquid filtrates which were usually applied using lint or other absorbent material; 2) the use of dressings inoculated with P. Notatum often in conjunction with liquid filtrates; and 3) the application of crude penicillin in agar, the so-called pen-agar method. In addition, crude filtrates wer
e also occasionally administered by injection." [4,41]

  Due to the fact that the partially purified product became more widely available after 1945, the large-scale production of crude penicillin covered only a short period of time between 1942 and 1945. However, in this time period, numerous doctors and scientists grew and used crude penicillin to cure serious illnesses involving bacterial infections. From Cairo to Hawaii, Boston to New York, doctors and scientists refused to wait for the limited supplies of pure penicillin and manufactured crude penicillin to effectively treat thousands of infections. [4], [5], [6], [7]

  Crude Penicillin Production in the 1632 Universe

  As I've already noted, one of the biggest hurdles to penicillin production is just the idea that organisms found in the soil can destroy or inhibit the growth of bacteria. Once this idea is accepted, then it becomes necessary to find the organisms. Fortunately for Grantville, it is at least somewhat plausible that in a school the size of Grantville's, a biology teacher would have in stock kits containing Penicillium Notatum. An investigation of one of the premier suppliers of biological materials, Carolina Biologicals, reveals numerous microbiology kits that contain the mold. But culturing the mold is not producing penicillin. Doing that will require active and ongoing cultivation, like any crop. And, like any crop, you can improve your yield by fertilization, trace nutrient supplementation, and better media. In addition, like some crops, you will want a "biocide" to kill off organisms wanting to consume your product.

  Most improvement of penicillin yields will come about through active experimentation. While corn steep liquor was a preferred medium for commercial production in the mid-twentieth century, there are other media just as good, if we assume lower levels of production in a seventeenth-century environment. For example, "an extract of ground dried peas at 10 percent concentration has been reported as a successful penicillin media . . ." [10, 695] Another media from the same source was cottonseed meal. Still a third possible surface culture medium was "wheat bran moistened with an equal weight of water." [8, 262].

  It seems clear given the historical evidence, that an adequate medium for relatively large-scale (beyond small laboratory batches) production of crude penicillin will be found and utilized.

  But obtaining the media for crude penicillin production is just one factor. Another will be obtaining the appropriate containers and manpower to produce the crude penicillin on a regular schedule. Another factor that will be important is some substance or substances that can act as an effective "biocide."

  In terms of an effective biocide, the most useful for penicillin production is borax. In a 1945 study done in Wisconsin, "37 different chemicals were tested for their ability to prevent the growth of contaminants and still allow penicillin production in contaminated shake flask fermentations. Of the chemicals tested, only borax and boric acid could be used at a level high enough to delay the growth of contaminants and still not interfere with penicillin production." [8, 515]. The importance of this to 1632 is that borax was one of some twenty-seven common mineral substances used in medicinal or cosmetic recipes [11, 125]. While the most expensive mineral ingredient (2–3 guilders per pound), borax nevertheless was available throughout much of Europe. The amount necessary to prevent contamination of penicillin cultures is quite small, two-tenths of one percent. Since borax has other important uses however, as in the making of borosilicate glass, it is likely that resources in Tuscany will be developed fairly quickly, helping to drive down the price.

  Uses and Limitations

  From the literature on the use of crude penicillin, it is apparent that it will be most useful for infections in open wounds, and infections involving staph, strep, and gonorrheal bacteria. Syphilitic sores might also respond to treatment, although a complete cure would likely have to wait until large doses of a partially pure product could be injected. Diphtheria is also penicillin sensitive, so application as throat and nasal drops might drastically lower mortality rates. Unfortunately, penicillin will not work with cases of typhoid, cholera, plague and typhus, nor will it be effective against viral diseases. Crude penicillin will also have other limitations. Batches will tend to be highly variable in all but the best laboratories or hospitals. There will have to be a continuous production line because without refrigeration, penicillin loses its potency fairly rapidly over a span of about two weeks. Even with refrigeration the maximum lifespan will be measured in weeks, not months. There will be a constant need to maintain sterile conditions to prevent contamination. Hence either autoclaves or dry heat ovens will be required.

  Crude penicillin production, however, will be much more likely to be attempted and utilized than sulfanilamide and chloramphenicol production outside the USE. Technologically speaking, it is much closer to the typical activities attempted by down-time medical practitioners of all kinds. Crude penicillin production will foster the development of sterile procedures, instrumentation, glassware and microscopes. As technology improves, the work on crude penicillin will set the stage for purification of a pure product that can be used internally through the use of injections or IVs. It will also foster investigation of the soil for the complete cornucopia of organisms that are part of the modern arsenal of antibiotics.

  Now the crucial question, of course, is can a hospital or laboratory produce enough crude penicillin to serve a population of thousands? Evidence from Hawaii in 1945 seems to indicate that this is indeed possible [4, 47]. In Hawaii approximately forty liters of crude penicillin filtrate were being prepared every week. This was enough for 16,000 dressings and this amount in turn was enough to serve 100 physicians around Hawaii as well as any ship or naval depot that required the product. Given the likely per capita ratio of doctors in 1945, this was sufficient to serve at least 100,000 people. Given the necessary incubation period for maximum yield, maintaining this level of production on a weekly basis probably requires somewhere in the vicinity of 500 to 600 liter bottles or containers. This certainly seems doable within the context of 1632, especially given the resources that were expended on hospitals by many urban centers.

  Bibliography

  [1] Behind the Sulfa Drugs: A short History of Chemotherapy. Iago Galdston. 1943.

  [2] New Light on the History of Penicillin. Ronald Hare. Medical History, 1982, 26: 1-24.

  [3] C.G. Paine and the earliest surviving clinical records of penicillin therapy. Milton Wainwright and Harold T. Swan. Medical History, 1986, 30: 42-56.

  [4] The History of the Therapeutic Use of Crude Penicillin. Milton Wainwright. Medical History, 1987, 31: 41-50.

  [5} Robert Pulvertaft's Use of Crude Penicillin in Cairo. H.V. Wyatt. Medical History, 1990, 34:320-326.

  [6] The Use of Gauze Inoculated with Penicillium Notatum or Impregnated with Crude Penicillin In The Treatment of Surface Infections. R.S. Myers, R.H. Aldrich, R.W. Howard, and R.A. Walsh. New England Journal of Medicine. Volume 231 No. 23. December 7, 1944.

  [7] Crude Penicillin: Its Preparation and Clinical Use Externally. Charlotte Dunayer, Lillian Buxbaum, and Hilda Knobloch. Annals of Surgery. Volume 119 No. 5. May 1944.

  [8] The Effect of Medium Constituents on Penicillin Production from Natural Materials. Bhuyan BK, Johnson MJ.Appl Microbiol. 1957 Jul; 5(4): 262-267.

  [9] The Control of Contaminants in Penicillin Fermentations by Antiseptic Chemicals. Knight SG, Frazier WC.J Bacteriol. 1945 Nov; 50(5): 505-516.

  [10] Microbiological Aspects of Penicillin: IX. Cottonseed Meal as a Substitute for Corn Steep Liquor in Penicillin Production. Foster JW, Woodruff HB, Perlman D, McDaniel LE, Wilker BL, Hendlin D.J Bacteriol. 1946 Jun; 51(6): 695-698.

  [11] Midwifery and Medicine in Early Modern France: Louise Bourgeous. Wendy Perkins. 1996. ISBN 0859894711.

  Penicillin: Its Practical Application. Alexander Fleming, editor. 1946.

  Launching The Antibiotic Era: Personal Accounts of the Discovery and Use of the First Antibiotics. Carol L. Moberg and Zanvil A. Cohn, editors. 1990. ISBN 0-874770-047-7.

  The Effect of Cert
ain Mineral Elements on the Production of Penicillin in Shake Flasks. Koffler H, Knight SG, Frazier WC.J Bacteriol. 1947 Jan; 53(1): 115-123.

  Comparative Study of Penicillin Production with Vegetative and Spore Inoculum of Penicillium chrysogenum. Bhuyan BK, Ganguli BN, Ghosh D.Appl Microbiol. 1961 Jan; 9(1): 85-90.

  Chemical Changes in Submerged Penicillin Fermentations. Koffler H, Emerson RL, Perlman D, Burris RH.J Bacteriol. 1945 Nov; 50(5): 517-548.

  Microbiological Aspects of Penicillin: III. Production of Penicillin in Surface Cultures of Penicillium notatum. Foster JW, Woodruff HB, McDaniel LE.J Bacteriol. 1943 Nov; 46(5): 421-433.

  Microbiological Aspects of Penicillin: II. Turbidimetric Studies on Penicillin Inhibition. Foster JW, Wilker BL.J Bacteriol. 1943 Oct; 46(4): 377-389.

  Microbiological Aspects of Penicillin: IV. Production of Penicillin in Submerged Cultures of Penicillium Notatum. Foster JW, Woodruff HB, McDaniel LE.J Bacteriol. 1946 Apr; 51(4): 465-478.

  Mode of Action of Penicillin: I. Bacterial Growth and Penicillin Activity—Staphylococcus aureus FDA. Lee SW, Foley EJ, Epstein JA.J Bacteriol. 1944 Oct; 48(4): 393-399.

  Penicillin. III. The Stability of Penicillin in Aqueous Solution. Benedict RG, Schmidt WH, Coghill RD

  , Oleson AP.J Bacteriol. 1945 Jan; 49(1): 85-95.

  Evaluation of Precursors for Penicillin G. Singh K, Johnson MJ.J Bacteriol. 1948 Sep; 56(3): 339-355.

  Microbiological Aspects of Penicillin: VIII. Penicillin from Different Fungi. Foster JW, Karow EO.J Bacteriol. 1945 Jan; 49(1): 19-29.

  The Relation of Natural Variation in Penicillium notatum to the Yield of Penicillin in Surface Culture. Whiffen AJ, Savage GM.J Bacteriol. 1947 Feb; 53(2): 231-240.