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Week9_Penicillin2

Week9_Penicillin2 - THE LANCET FURTHER OBSERVATIONS ON...

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Unformatted text preview: THE LANCET] FURTHER OBSERVATIONS ON PENICILLIN E. P. ABRAHAM,* D. PHIL. OXFD E. CHAIN,* PH.D. CAMB. C. M. FLETCHER; LLB. CAMB., M.R.C.P. A. D. GARDNER, DJI. OXFD, F.R.C.S. N. G. HEATLEY,T PILD. CAME. M. A. JENNINGs,* 13.31. oxrn H. \V. FLOREY, M.B. ADELAIDE, F.R.S. (The Sir William Dunn School of Pathology and the Radclifie Infirmary, Oxford ) THE work on penicillin briefly reported by Chain and others (1940) is here presented in greater detail, and its further development to the stage of human therapy is described. Growth of Penicillin-producing Mould The mould will grow and produce penicillin on a variety of different media, but that used by Clutterbuck, Lovell and 'Raistrick (1.932) is easy to prepare and gives horse-muscle digests, &c. This modified Czapek-Dox medium consists of: NaNO3 3 g., KHZPO4 1 g., KCl 0-5 g., MgSO4.7H20 0-5 g., FeSO4.7H20 001 g., glucose 40 g., with water to 1 litre. Oxford tap-water has proved as good as distilled water for this purpose. Yeast-extract has usually been added to speed up the growth of the mould (details later). The medium, sterilised by autoclaving, is sown with a. spore suspension made by shaking up sterile water in a . The growth becomes more voluminous during the next day and on the 3rd day, if the liquid layer is not more than 1 cm. thick, this reaches the surface of the medium and throws up dry white mycelium, isolated foci, ' pact,'often corrugated, dark greenish-blue felt whose upper surface cannot be wetted by water. myce um becomes more faded and grey . ,The 6 es in the appearance of the mould are accompanied ‘ ' 3rd day~usually as the dry mycelium is fo It tAltelthis , reaching 5 as t e greenish-blue colour appears . f I 1 medium an traces 0’ penicillin can be detected. The PH’coutinues to rise and the titre of penicillin and the: averages both increase rapidly. As the myceliuni . , H rises more and more slowly; Macmexeeeds 8-8. Penicillin subduction is _ at about 1111 7, an " L' “at? days 61' my fan f'th stgie ofth _, j ; 1most‘useful31: ,0 >6, W from an assay for mm Medical Ream 06 an. r RMRsssarch s‘i‘r‘hhshr- deGlNAleTRTlCLES [Avon 16, 194l of development may be greater or less than that described, depending largely on the depth of the medium. A systematic study of the factors influencing penicillin- production was begun, but it could not owing to the very variables, and to the fact that the assay-method then in use could only detect large differences of titre. following conclusions, however, could be drawn : l. Penicillin production seems wide range of oxygen tension. anaerobically.) 2. The mould grows satisfactorily at 24° C. At lower temperatures growth is delayed and as harvesting of the medium is carried out in the incubator higher temperature." have not been studied, 24° C. being about the upper limit of comfort. Fleming (1929) in his original description stated that the mould would not grow at 37° C. and this has been confirmed. 3. Crude attempts to change the pH of the medium or to maintain it at a constant val to take place ,over a (The mould will not grow If deeper than 2 cm. diffusion is visibly inadequate, for two distinct layers can be seen in it, the upper being yellow, the lower colourless. 5. When the medium is fit to be harvested it can be drawn penicillin will form in about half the time required for the initial production. The medium can be changed several times in this way; with one batch it was changed 14 times. 6. The mould must be grown and the medium harvested replaced under strictly sterile conditions since penicillin is destroyed by certain bacteria (Abraham and Chain 1940). 7. The addition of yeast-extract (Gladstone and Fildes 1940) accelerates the growth of the mould but does not affect the yield of penicillin. In large-scale growth we have always used yeast-extract ; in starting a batch the medium is made up to contain 10% of it, but the medium used for replacement contains only 2%%. The accelerating effect of the yeast is not impaired by prolonged autoclaving at a. high temperature. Mannite or malt extract have no efl'ect on the rate of growth or on the yield of penicillin. 8. The yield is not greatly afi'ected halving the strength of the medium.‘ either by doubling or by i i 9. If the sodium nitrate is replaced by ammonium lactate Very little penicillin is produced. ' ' i' * i " 10. The sodium nitrate can be wholly or partly replaced by peptone or by peptic and tryptic digests of muscle, .and‘ used in place of glucose, without materially altering the yield of penicillin. ' ‘ * ' mould. To test this, pure-line cultures were made from single cells of two batches, one of which had given a. yield ofpenicillin . and the other none. Five pure lines of the former andnl'na of the latter were inoculated in triplicate into bottles of medium and the penicillin titre was follOwed.‘ ‘No difl' ' could be detected be lines, , o. a special rack which Wtherlid of the Petri dish half an inch'jabove the . “apart. When dry the seeded plates'are removed Mthe incubator and can be kept in the refi-igelrzttfirs- of glass tubing, of which will be seen from the inset of fig. 1, are placed on the agar. The lower edge of the cylinder is care- fully ground level and has an internal bevel so that the thin edge tends to sink into the agar and make a water- and bacteria-tight '_ seal. Vitreous porcelain cylinders of the same size and shape, coloured at the non-bevelled end to facilitate their orientation, ~ have also proved satisfactory. The cylinders are filled with the fluid to be tested, and the plates, resting on a block of wood, are incubated for 12—16 hours at 37° C. (If placed directly on the warm incubator shelves moisture may condense on the lids of the Petri dishes and drop on the agar, thus obscuring the results.) By the end of incuba- tion most of the fluid in the cylinders has disappeared and each cylinder is surrounded by a circular zone .Where no bacterial growth has occurred. The diameter of the zone depends on the concentration of the penicillin, the type of relation being shown in fig. 1. The possibilities and limitations of this method of assay have not yet been fully worked out but the following points may be noted. ' l. The diameter in mm. of the zone of inhibition (which we have called the “ assay value ”) is only slightly smaller (1—2 mm.) when the cylinder is half filled than when it is fully filled. 2. The assay value is unaffected by the pH of the fluid being tested, provided it is not strongly buffered and lies within the range pH 5—8-5. , 3. No inhibition is produced by a saturated aqueous solution of ether or by water containing free droplets of chloroform. 4. Difi‘usion of penicillin seems to be practically complete in 2—3 hours, and assay values after 14 hours of incubation are only very slightly smaller (0-5—1 mm.) than after a further 8 hours at 37° C. 5. Provided the plate is not jarred the fluid cannot escape from the cylinders, and even if the fluid is not sterile the con- taminating bacteria are confined to the inside of the cylinder. 6. The assay value is not affected by the thickness of the agar provided it is between the limits of 3 to 5 mm. 7. The assay value varies slightly with different batches of plates and with the density of bacterial population at the beginning of incubation. For this reason uneven seeding of the plates must be avoided. 8. Sometimes the clear zone of inhibition is surrounded by a halo of partial inhibition, which varies from a faint ghost to almost complete inhibition. So far no explanation for, or means of controlling, this phenomenon has been discoveredj 9. When the antibacterial activity of blood is to be assayed plasma or serum must be used, since red cells tend to form a layer immediately on top of the agar which seriously impedes the diffusion of penicillin and leads to low values. \\\\\\\\\\\\\ \\\\\\\\\\\\\~ _- r .\ I , 2 , 4 Conc. of penicillin (units per c.cm.). ~, FIG. l—Relation between assay value and ‘ concentration of penicillin insulation being tested. Inset—Median vertical section throughassay cylinder; dimensionsinmm. High accuracy cannot be claimed for this method of assay, but if it is done in triplicate (preferably on three different plates), and if the unknown solution is diluted so as to give an assay value of not more than 25 mm. (before the curve has flattened out), the error is probably not greater than : 25% and. may be considerably less. We have no evidence that under suitable conditions this the dimensions ' " I y, , .7 upoth ’syhichallthe assay values may be related. We have been using as such a' standard a partially urified solution of purely arbitrary . It is made up in dilute phosphate- bufier, is saturated with ether and is kept in the ice-chest. It gives an av may value of24 mm.'and as far as can be seen its activity has not altered during the three months in which it has been in use. We have adopted as the arbitrary unit (until penicillin is obtained chemic- ally pure) that amount of penicillin which when dissolved in 1 c.cm. of water gives the name mm as this standard. The material used in the human therapeutic tests usually contained 40—50 of these units per mg. Large-scale Production of Penicillin Culture vessels and sowing.—After many types of con- tainers had been tried a. satisfactory ceramic vessel was eventually desi ed,1 the shape and. dimensions of which can be seen in g. 2. - The vessels are glazed only on the inside ; this both reduces the cost and renders them easier to handle and less liable to slide when stacked one on top of the other. The inset of fig. 2 shows a con- venient way in which they can be stacked for autoclaving, sowing and so on ; each plug iswell separated from the other but no bench space is lost, and should the medium boil in the autoclave the plugs are unlikely to be wetted. One litre of medium fills the vessels to a depth of about 1'7 cm. When a batch of vessels is first set up the medium (containing 10% of yeast-extract) is sterilised in the vessels, which are then inoculated with a few drops of a spore-suspension ’ and incubated at 24° 0. Apart from an occasional test the vessels are not touched until the medium is ready to be harvested. Arrangements for withdrawing and re- placing medium.— The penicillin-obn- taining fluid is withdrawn from the vessels and replaced with fresh culture-medium. To carry out these two processes without introducing contamination is far from easy and needs thorough bacteriological precautions. In order to hasten the production of penicillin we attempted to reduce these precautions to a minimum, and at first obtained satisfactory results; but lately contamination has become so frequent that substantial modifications have had to be introduced, some of which are still on trial and therefore cannot usefully be described. The Withdrawal of culture—medium is done by suction, the medium being replaced with sterile air. In the original method the refilling was done by simple pouring, but safer methods (not yet finally determined) have now been introduced, and precautions at all stages are being;r tightened up. Dimensions m u nttmcl res amt 37.. Lnl’uul dune!" FIG. 2—Eerthenware culture vessel. Abm.——Vessels stacked for autoclaving. The operations can conveniently be carried out on the “ changing~trolley ” (fig. 3), made by replacing the top of a small all~metal table by a wooden plank A, to which are hinged the four wooden slabs B on which the vessels are placed. The raised rim 0 is prolonged into a handle D, by which the slab (and the vessel on it) can be tilted forward. The slab is not hinged square with the edge of A, but at a slight angle to it, so that on depressing the handle D the fluid in the vessel drains not merely to the front edge but to the corner nearest the spout. The medium is drawn off by suction with the help of a special pistol-shaped holder E, carrying two detachable sterilised tubes F and G. F is plugged with cotton-wool before sterilising, and through it is blown into the vessel a current of (sterile) air at a somewhat faster rate than 1. Made for us by J. Macintyrc and 00., who also made the'porCelaEn a assay cylinders. .. The strains of penicillium used in this work have been obtained from Prof. Alexander Fleming of St. Mary’s Hospital, London. THE LAxcrr] the fluid is drawn out by tube G. Suction and pressure are ‘ ' rolled by the single trigger H of the “ pistol."7 A fresh t... G is used for every batch of four vessels and F is changed after every two batches. Both tubes are sterilised wrapped in - paper and only one end of the tube is unwrapped when being SUCTION M“ FIG. 3~Changing trolley. fitted into the pistol ; the tubes are unsheathed from the re. maining paper wrappings immediately before use. From the pistol the harvested medium passes to the inverted bottle K, in which it is filtered through a silk bag. The filtered liquid than passes to the collecting bottle L which is changed after every batch of four vessels. When not in use the pistol is hung on the pin M. For refilling the vessels are tilted at about 45’. A fitting on each wooden slab enables the vessel to be wedged in this position (as shown by vessel N in fig. 3), so that both hands are free to manipulate plug and bottle of medium. EXTRACTION FROM CULTURE MEDIUM Principle—Penicillin can be extracted by ether, amyl acetate and certain other organic solvents from an aqueous solution whose pH has been ‘adjusted to 2. From the organic solvent the penicillin may be re- extracted by shaking with phosphate-bufi'er or with water the pH of which is kept at 6—7. Penicillin is quickly destroyed at pH 2 at room temperature, so the first extraction must be carried out rapidly or at a low temperature. Once it has been extracted into solvent the penicillin is stable for some days. Laboratory large-scale extraction.———It was planned to produce a weekly output of about 500 litres of the crude medium containing penicillin, the working up of which by band would obviously have been out of the question. A continuous countercurrent extraction apparatus was devised, an adequate description of which cannot be included in this paper. The crude penicillin, having been filtered and acidified, is passed through special jets which break it up into droplets of uniform size. These are allowed to fall through a column of amyl acetate, to which the penicillin is given up. The crude solution is acidified immediately before entering the jets, so that the aqueous solution is at a. pH at which penicillin is unstable for only a few seconds before it has been extracted. As the crude solution is passed through a cooling c011 surrounded by circulating tap-water probably very little destruction of penicillin takes place. Phosphoric acid is used for the acidification as the pK of the first stage of dissociation is 31:1)ther 2 ; it will therefore not as its own buffer and inaccurate correlation of the rates of flow of the acid and of the crude solution will not cause big. fluctuations in [thermal pH. Fresh solventis con- tinuousl fed in to the bottom of the column, from the topxof $0]: an equal~ amount of penicillin-rifli‘solvcnt is collected for furthelworkingnm This solvent has [mi-Ifb‘bf-the volume of the crude solution from which * the: ' ha: been extracted, and ,' impurities,- ’ ‘ ' n eliminated. lyfilose forming emulsions, have PROFESSOR FLOREY AND OTHERS: FURTHEOB‘ OBSERVATIONS ON PENICILLIN ~z'ghich contahs perhaps 80% of the .5 e. , .. p Y glasSwarehaving been‘rinsod Wi a: tune charactcfisficsmcll and amen-m [AUGH 16. 1941 179 The preparation used in the mouse-experiments previously reported (Chain et a1 1910) was purified only to the stage of recovering the sodium salt from a first ether extract (ether was at that time used instead of amyl acetate). Though strongly active this product probably had less than 1’10 of the activity of our present " thera- peutic penicillin.” A batch intended for injection into man was first tested on mice. A mouse was little affected by the intravenous injection of 10 mg. in 0-3 c.cm. of water but became very ill after receiving 20 mg., though it recovered in about 11} hours. When given intravenously to an afebrile human subject 100 mg. of the same material caused a shivering attack with a rise of temperature in about an hour. Trial on other subjects gave the same result and left no doubt that a pyrogenic substance was present in the preparation. The pyrogen can be assayed by the method used by Lees and Levvy (1940) in which five rabbits of uniform weight (a little over 2 kg.) are kept at a constant temperature (67° F.). On the day of the test they are deprived of food and the rectal tempera- ture is taken hourly. Twenty milligrammcs of the substance used in the mouse experiments dissolved in l c.cm. of distilled water, injected intravenously, gave a rise of about 2° F. in each rabbit, but “middle fraction ” from the adsorption column (see below) had no pyrogenic effect. The further purification here described secures a pyrogen-free product suitable for intravenous use. Further purification—Batches of 3 litres each of the penicillin-containing solvent as delivered from the extraction-apparatus are extracted with five successive amounts of 300 ml. each of water, using baryta to adjust the pH to 6-5—7. The five watery solutions have already been used for former extractions and are used in order of decreasing concentration of penicillin. The first extract, which is the strongest, is set aside for further working up, but a sixth extract is made with fresh water so that the number of watery solutions remains the same. The amyl acetate, which may still contain a. small amount of penicillin, is recirculated in the extraction apparatus. In the course of time pigment accumulates in the solvent and is periodically removed by extraction with 1% sodium hydroxide followed by thorough washing. The strongest aqueous extract is partially decolorised by shaking with about 8% of animal charcoal and filtering. The charcoal residue is washed twice, the washings being put through the extraction apparatus or used in other ways. The partially decolorised solution is cooled, acidified and extracted into successive amounts of ether ; the strongest of the ether extracts is then passed through an adsorption column of Brockmann alumina. The spent ether contains solid matter but no penicillin, which remains behind in the column. Though the chromatograms vary, four main zones can be seen, their boundaries overlapping to some extent. These are, starting from the top : . 1. A dark brownish-orange layer whose depth is inversely proportional to the amount of charcoal used for the decolor- isation and which may be absent altogether. This layer con~ tains some penicillin. ' * 2. A light yellow layer containing most of the penicillin but none of the pyrogen. \. 3. An orange layer which contains some penicillin and some or all of the pyrogen. 4. A brownish or reddish-violet layer which contains practically no penicillin. The violet pigment disappears on exposure to light. * . The column is washed by passing successive small amounts of ether through it and is then divided into the four fractions, of which the fourth is discarded. - others are eluted with successive amounts of Milli phosphate buffer (pH 7-2), from the strongest of which the'penicillin is again extracted mto ether, while-tho more dilute solutions are used for the elutions of next batch. Finally the penicillin is extracted water using» sodium hydroxide 'to adfimttho the soluti "not buffered the greatest ‘ taken in ding the alkali, for penicm destroyed in’alkalinp solution.) . , I The “non—WT”. or “1.11me en—freo ants, . the latter-.9 It,. a d reddishorange Quid, yellow in dilutemtiou,with .13 extracted into 1) ' prawn actually erg: 3W in a form suitable for intravenous Theme are many stages in the preparation at which the occurandaefaraspossiblethesehavebeen checked individually but owing to overlapping of d...
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