penicillin · ‘penicillin is an extremely labile substance, very readily inactivated by...

Antibiotics – Exhibition 2016

Penicillin—

The discovery, the development and the production,from Alexander Fleming to the Oxford Research Group at the

“Sir William Dunn School of Pathology”at the University of Oxford with the mastermind of

Lord Howard W. Florey.

—

An article by

Carl Schwedes

—

in collaboration with

The Museum of the History of Science – University of Oxford

and

Hochschule für Technik und Wirtschaft - Dresden

August 30, 2015

“The more complex the world becomes, the more difficult it is tocomplete something without the cooperation with others.”

Sir Alexander Fleming – (1881-1955)

Penicillin - Development and Production

ContentsThe Miracle Drug - Historical Introduction 4

Penicillin - Chemical Structure 10

Development of Penicillin:Howard Florey’s Surface-Culture Method 131. Upstream Processing . . . . . . . . . . . . . . . . . . . . . . . . 13

1.1 Culture Medium . . . . . . . . . . . . . . . . . . . . . . . 131.2 Sterilization (Autoclaving) . . . . . . . . . . . . . . . . . . 211.3 Penicillium Spores . . . . . . . . . . . . . . . . . . . . . . 22

2. Downstream Processing . . . . . . . . . . . . . . . . . . . . . . . 242.1 Harvest the Culture Medium . . . . . . . . . . . . . . . . 242.2 Separate the Culture Medium . . . . . . . . . . . . . . . . 28

(a) Counter-Current Apparatus - Primary Separation 28(b) Separating Funnels - Secondary Separation . . . . 36

2.3 Freeze-Drying, Crystallization . . . . . . . . . . . . . . . . 382.4 Storage, Penicillin Sodium Salt . . . . . . . . . . . . . . . 39

Financial Facts 41

Bibliography 43

by C. Schwedes 3


The Miracle Drug - Historical Introduction

“One sometimes finds what one is not looking for.”

Sir Alexander Fleming

The discovery and development of Penicillin from just a simple substrateright up to a real drug is truly a remarkable story in the history of science.

It happened unexpectedly that Alexander Fleming discovered the effect ofthe Penicillin substance in his laboratory in London in 1928. Penicillin wasmentioned as a miracle- and wonder-drug and, because of its curative prop-erties it was possible to save millions of lives. No-one other than AlexanderFleming was in the position to discover a drug such as the Penicillin oneinside of an ordinary petri-dish which he has used for experiments with“staphylococcus aureus” bacteria. Fleming was already well known as a bac-teriologist in 1928. He discovered for example the enzyme “Lysozyme” in1923 which has a very strong antibacterial effect as well as Penicillin.1“staphyle” and “kokkos” comes from the Greek and means “staphyle” –“grape” and “kokkos” – “granule”. The staphylococcus bacteria seen underthe microscope appear as clusters (cocci) of small round “kokkus”.2 Theseparticular type of bacteria are a normal part of our skin and body and theycan have a disease-causing effect. A dangerous and even deadly influence tothe human body if we produce them in excessive amounts. A reason of thishigher concentration of staphylococcus bacteria can be an infection woundsuch as a cut or bruise in connection with external microorganisms.During Flemings’ work with staphylococcus bacteria at St. Mary’s Hospi-tal, London in 1928, he noticed that some of the culture plates which hadbeen exposed to the air became contaminated with several types of micro-organisms. The culture plates at the laboratory bench were necessarilyexposed to the air during the time of the examination. These contaminatedculture plates (petri-dishes) are normally not useful for the work of bacte-riologists and another scientist would have thrown it in the bin, but notAlexander Fleming. Furthermore he noticed that around a large colony ofcontaminated mould the staphylococcus colonies became transparent andhad partly disappeared. [2;1] It seemed that these mould-colonies couldproduce a particular substance which is able to destroy this special sort ofbacteria, “staphylococcus aureus”.Fleming discovered with a series of different experiments that an antibacte-rial property of the ‘crude mould broth filtrate’ was that the active agent in-hibited the growth of certain gram-positive pathogenic organisms and gram-

1A. Fleming, https://en.wikipedia.org/wiki/AlexanderF leming2staphylococcus aureus, https://en.wikipedia.org/wiki/Staphylococcus

by C. Schwedes 4


negative cocci, but not gram-negative bacilli. The “staphylococcus aureus”bacteria type for example is a gram-positive stem of bacteria.The type of fungus was finally identified as Penicillium notatum by anothergroup of scientists in the end of 1932 and Fleming called this substance fromthe mould “Penicillin” a little time later. Penicillium notatum is a particu-lar type of fungi and grow especially on food. It was already mentioned forthe first time by Johann Heinrich Friedrich Link in his work “Observationesin ordines plantarum naturales” in 1809.3Fleming showed that the activity of the filtrate was massively reduced ifyou boil the broth at about 80◦C as well as that the loss of activity wasmuch more if the broth was alkaline instead of neutral or slightly acid. ThePenicillin is in its nature an acid and most stable if the broth is acidified.In 1929, one year after his discovery of the Penicillin substance, he foundout that if the broth were evaporated at a low temperature to a solid mass4

the active agent could be completely extracted by absolute alcohol, but notwith ether or chloroform. After a series of experiments dealing with therate of killing of bacteria, he even considered that the Penicillin substancebelonged to the group of ‘slow-acting antiseptics’.Penicillin was used for the administration on a number of indolent septicwounds and appeared as a superior chemical for that purpose. Fleming wasalso able to show with his different series of experiments that the filtratedPenicillin broth was more toxic to bacteria than to other cells. The filtratedcrude broth to this point of time (1932) had an exceptionally low activityand it was unfortunately insufficient to show that Penicillin was lacking intoxicity or that the substance retained its activity very well in the body andcould be injected with bacteria to the blood stream.A couple of years later, after a series of experiments, a combination of prop-erties was discovered which made the Penicillin substance into an idealchemotherapeutic agent. It is of some interest to inquire why Fleming’swork on his discovery ceased, despite his appreciation of the fact that Peni-cillin broth differed so greatly from other antiseptics.

In 1940 he said:‘We have been using it in the laboratory for over 10 years as a method ofdifferential culture. It was used in a few cases as a local antiseptic, butalthough it gave reasonably good results the trouble of making it seemednot worthwhile.’ (Fleming, 1940 e.)

Prof. H. Raistrick and some colleagues from the London School of Hygieneand Tropical Medicine had been working for some years with Penicillin andother substances produced by Fleming’s fungus, which was at that time

3Johann Heinrich Friedrich Link, https://en.wikipedia.org/wiki/Penicillium4see section 2.4 Freeze-Drying, Crystallization

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identified as Penicillium notatum. They hoped to extract the Penicillinsubstance from the fungus with ether at a pH-value of 2 with the helpof a synthetic culture medium for the growth process. (Clutterbuck,Lovell, and Raistrick 1932.)

They stated in their summary:‘Penicillin is an extremely labile substance, very readily inactivated byoxidation, evaporation in vacuo at 40◦C-45◦C in both acid (pH 2 and3.5) and alkaline (pH 8) solutions, but is moderately stable at pH 5-6.’(Clutterbuck et al., 1932.)

That investigation work was the first step towards a successful extractionof the Penicillin substance. No-one at that time had any real idea that theproperties and the purpose of Penicillin were so remarkable. It seems thatonly a little bit of interest was given to Raistrick and Clutterbuck throughthe work from Fleming, otherwise they would not have finished their workso early to extract it. Just one worker form the Joseph Hospital, Sheffieldnamed C.G. Paine5 was sufficiently stimulated to try to make use out of thePenicillin substance. His investigations and notes were never published butat least he used the crude broth filtrate for several cases and was even ableto heal a conjunctiva with it. He could remove the injected foreign bodyfrom the eye and recovered 6/6th of the vision of the eye of the administratedpatient.6

To the end of the work of 1932 we could summarize that we have founda fungus which lysed staphylococci and the cultivated broth medium was apotent inhibitor of many pathogenic organisms. Attempts to extract the ac-tive substance from the mould failed and no real experiments had been doneto show the real efficacy of Penicillin. The rest of this historical descriptionduring the work of the 1930’s was described as ‘various ups and downs’.About 11 years after Fleming’s observations, great effort were made to pro-duce Penicillin for therapeutical purpose. All these previous events led tothe work on antibiotics in the “Sir William Dunn School of Pathology inOxford”. Goldsworthy and Florey started to work with lysozyme in 1930which was an antibacterial enzyme which was widely distributed in ani-

5Cecil George Paine, *London in 1905, he worked for a part of his career at St. Mary’sHospital Medical School in London where Fleming was lecturer in bacteriology; NationalCentre for Biotechnology Information, C.G. PAINE AND THE EARLIEST SURVIVINGCLINICAL RECORDS OF PENICILLIN THERAPY, Medical History, 1986, 30: 42-56

6Milton Wainwright, Harold T. Swan; National Centre for BiotechnologyInformation, C.G. PAINE AND THE EARLIEST SURVIVING CLINICALRECORDS OF PENICILLIN THERAPY, Medical History, 1986, 30: 42-56, p.48;https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1139580/pdf/medhist00072-0046.pdf,pdf-document p.8

by C. Schwedes 6


mal tissues.7 Latter investigations in old literature about antibacterial sub-stances produced by micro-organisms such as different types of fungi weremade, and these contained some excellent descriptions about different typesof antibacterial agents. With the discussions about lysozyme and the re-search about antibacterial agents, Chain and Florey decided to undertakesome systematic investigations about antibacterial substances produced bymicro-organisms and sent an application for financial help to the NaturalScience Division of the Rockefeller Foundation in November 1939. It waswell known by this time that if a mixture of micro-organisms is grown to-gether, certain bacteria or moulds will inhibit the growth of other bacteriaor moulds. This behaviour is known as ‘bacterial antagonism’ (Bakterielle-Feindseeligkeit). This inhibiting effect is held by the production of bacterio-static and bactericidal substances into the culture medium. That means thatthe micro-organism starts to produce a substance which is in the position todamage and to kill a particular type of bacteria. The behaviour of bacterialantagonism is observed with the production of substances which are actingagainst cholera, anthrax and diphtheria organisms as well as with organismsproducing substances against pneumococci, streptococci and typhoid. Specialmention should be made of the ‘Penicillium notatum’ organism which pro-duce a very strong bactericidal substance to staphylococci (staphylococcusaureus type), streptococci and pneumococci. After this investigation workand especially the demonstration of the chemotherapeutic effect of Peni-cillin on several forms of staphylococci and streptococci infections (Abrahamet al., 1941; Florey Florey, 1943), it was clear that the effect of the Penicillinsubstance could play a very important role in the successful treatment ofinfection and war wounds.8The first observations on war wounds were made by Lt.-Col. R.J.V. Pul-vertaft in Cairo with material which was sent from Oxford in July 1942.Two further batches of Penicillin were send to him in November 1942 andMarch 1943 and that led to an attempt to manufacture Penicillin in Cairo.Pulvertaft published his observation work with the Oxford material andcame to the conclusion that the Penicillin substance is the most effectiveand remarkable one for the successful cleaning up of infection wounds. [4]Because of the situation of war it was impossible to produce any substantialamount of Penicillin in England and it seemed that absolutely no companywould undertake to try to prepare an industrial large-scale production of thePenicillin drug. Fortunately H.W. Florey and N.G. Heatley had the luck tofind some attention in the United States after a successful visit there in June1941. It was Dr. Weaver, of the Natural Sciences Division of the Rocke-feller Foundation who suggested that an attempt of production of Penicillin

7see discovery of Lysozyme by Fleming 1923, footnote 1 page 38report by H. W. Florey with special references about the Penicillin administration in

war zones in Egypt in North Africa

by C. Schwedes 7


should be made in the United States, which was not at war to this time.After a friendly and successful negotiation it was finally decided to movethe large-scale production to the Northern Regional Research Laboratory ofthe Department of Agriculture in Peoria, Illinois. At the very first meetingon 14th July 1941 with the director of the Research Laboratory Dr. Mayand the Director of the Fermentation Division, Dr. Coghill. Coghill hadalready mentioned that the production of gluconic acid through deep-tankfermentation had been tried and he suggested that this might be the keyof a successful commercial production of Penicillin as well. [2;5]

The huge efforts of the scientific investigations to produce Penicillin in largeamounts were finally used in the United States to produce it to industrialquantities in just two years. Tens of millions of mega-units of Penicillin witha value of more than $100,000,000 was the final outcome.

Finally, it is interesting to note that the effect of the wonder drug hadbeen well known throughout the ages. Folk-lore give us the earliest evidenceof Penicillin administrations from different parts of the world:

Martynowski 1944 stated:

‘. . . when I was there [in the Ukraine] (in 1921-22) I still rememberhow pleasants have cured themselves of their septic wounds and of somediseases of the infectious kind by the appliance of a growth, caused bydampness on the bread in a moderately wet cellar. They take off thisgrowth with a knife and put it carefully on their wounds. It is believedthat mould on the bread possesses better curative properties than chem-ical drugs.’ [1;1]

A more circumstantial account is given by Cliffe (Brunel, 1944):

‘It was during a visit through Central Europe in 1908 that I came acrossthe fact that almost every farm house followed the practice of keepinga moldy rye loaf on one of the beams in the kitchen. When I asked thereason for this I was told that this was an old custom and that whenany member of the family received an injury such as a cut or bruise, athin slice from the outside of the loaf was cut off, mixed into a pastewith water and applied to the wound with a bandage. I was assured thatno infection would then result from such a cut.’ [1;1]

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Sokoloff (1945) spoke of personally encountering in Yugoslavia theuse of mouldy bread for dressing wounds and was told that it wasin use among the peasants of Greece and the Ukraine. Possiblythe following from a letter of B. R. Townend (1943) is an exampleof the same widespread folk-lore:

‘During my undergraduate days at Cambridge, within the period 1911-13, I called at the Botany Laboratory to do a little extra work. We werestudying fungi and the activities of the class were centred at that timeon Penicillium glaucum. It was the custom in the class to be providedwith a growth of the fungus which had been previously grown on oldpieces of shoe leather. Only a portion of the growth was used and on theoccasion of my calling the old laboratory attendant was collecting thestuff left on the students’ benches. I was somewhat curious and askedwhy he was so carefully scraping off the fungus into a jar. He told methat he used it for a salve which had been used in his family for a verylong time. It was used for what he called gatherings. I presume by thishe meant septic wounds.’ [1;2]

’San Jose News’, 7 February 1944, reports an old man of over 80years saying that:

‘His grandmother used to apply mold to infected wounds and sores whenhe was a child. She always kept a culture of it growing, just for emer-gencies.’ [1;3]

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Penicillin - Chemical StructureThe work and investigation about the chemical structure of Penicillin werecarried out by Ernst B. Chain and E. P. Abraham from the Sir WilliamDunn School of Pathology in Oxford and by R. Robinson and W. Bakerand many more later. During this work there was a close collaboration withthe Crystallography Department in Oxford and the research teams in Oxfordand the U.S.A. The research team at Oxford in 1943 showed with evidencethat the Penicillin structure could be broken down into three main parts:

1. Amino acid – penicillamine – β − thiovaline (I)(I)

CH3

C

SH

H3CCH

NH2

COOH

2. Aldehyde – penilloaldehyde – β − γ − hexenoylaminoacetaldehyde (II)(II)

CH3CH2CH = CHCH2CONHCH2CHO

3. Carbon dioxide:

O C O

After a more extensive collaboration with the British and the Americanteam they found a combination (III) of the formulas (I) and (II) what wasthe favoured chemical equation of the Oxford research team in October in1943.

(III)

S

CH3C

H3C

CH

N

CH COOH

CH

CO

NHCOC5H9

[2;2]

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During the work in Oxford, other American companies such as Merck Co.Inc., E.R. Squibb Sons, and Charles Pfizer Co. Inc. made huge progressin solving the production problems and the purification of Penicillin. Thefirst Crystalline Sodium Salt of Penicillin was produced by the Americanpharmaceutical company E.R. Squibb Sons in the summer of 1943.

There were two different types of Penicillin: the English Penicillin which wascalled Penicillin I (F) and the American Penicillin which was called Peni-cillin II (G). The English and the American type of Penicillin were verysimilar but not identical. The two teams in Oxford and America improvedthe activity and the quality of the Penicillin Sodium Salt in an unendingmarathon. The exchange of information showed that the English team hadalways the more impure material but the activity of the solution per ml. wasmuch higher than the American material which was purer but with a loweractivity. The main reason for these two different types of Penicillin and latereven 4 types was the production method. The English team in Oxford usedthe Surface-Culture Method which was developed from Howard W. Floreyfor laboratory-scale production. The fungus could grow on the surface ofshallow fluid layers in culture vessels. The American team in Peoria usedthe Submerged-Culture Method for large industrial-scale production. Thefungus grows in a culture medium in large steel containers called fermenterswhich were constantly agitated and aerated. The fungus grows not onlyon the surface under these conditions but inside of the medium. [2;4] Thetwo different types of Penicillin were later named 42-pentenyl-Penicillin (F)and benzyl-Penicillin (G). The common and general structure of Penicillinis divided into the 6 aminopenicillanic acid with the β − lactam− ring andthe thiazolidine − ring and the acylresidue as variable side chain R. [5](see Fig. 1)

Figure 1: General Structure of Penicillins

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The 6 aminopenicillanic acid group (6-APA) is the result of the early chem-ical equation (I) (see page 8, amino acid – penicillamine – β − thiovaline(I))) and the carbon dioxide part is responsible for the binding of the vari-able side chain R (acyl residue) and the 6-APA.

The general structure is always equivalent (IV), to differ the specific Peni-cillin from others we can use the help of a variable side chain R. (see tablebelow, 1945 - General Penicillin Structure)

(Penicillin IV)

S

CH3C

H3C

CH

N

CH COOH

CH

CO

NHCOR

R = CH3CH2CH = CHCH2– in Penicillin F (I)

R = CH2

– in Penicillin G (II)

R = HO CH2

– in Penicillin X (III)

R = CH3CH2CH2CH2CH2CH2CH2– in Penicillin K

1945 - General Penicillin Structure: Penicillin (IV) [2;3]

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Development of Penicillin:Howard Florey’s Surface-Culture Method

1. Upstream Processing

1.1 Culture Medium

The preparation of the culture medium, such as a nutrient broth, is thebasic and the most important step of a biochemical fermentation process.The final result of those processes should be the production of a particularsubstance in large amounts. A fungus grows on the surface of a nutrientbroth which can produce our particular substance. The remarkable pointis that every type of fungus grows in a specific way in a different type ofculture medium (nutrient broth). This point could be problematical if wetry to find an acceptable medium which is in the position to produce ourdesired product in huge amounts. The whole phase of development aboutthe growth and extraction of Penicillin is a good example to explain howimportant the choice of the culture medium is.I want to describe especially the surface-culture method which was inventedand developed by Howard W. Florey in the early years of the work onPenicillin in Oxford. The surface culture method was mainly used forsmall experiments and large laboratory-scale productions of Penicillin. Thesubmerged-culture method which was used for the industrial large-scale pro-duction in the United States with deep-tank fermentation and stirring tech-nology is surely a very interesting one too but is not a main point of thisarticle, therefore I will explain in a short excerpt, what the main differencesbetween these two methods are. In the surface-culture method, rectangularporcelain culture vessels were mostly used. [3;2] These vessels have a largesurface on which the fungus grows very well, protected from the influence ofair, to produce a suitable amount of Penicillin spores. The main differencein the submerged-culture method which was used for deep-tank fermentationin the United States is that the fungus could grow not only on the surfacebut also inside of the liquid medium with the use of corn-steep liquor, aby-product of the production of animal food and corn-starch9 [7], and bythe use of a stirring technology it was possible to produce huge amounts ofPenicillin spores.Howard W. Florey has put a lot of effort into the surface-culture methodduring the very early time of the development of the production of Penicillinin Oxford. No-one to this point of time had any real idea how they couldproduce large amounts of this substance or which special combination ofchemicals should be used for the culture medium to get the best yields andresults. What followed was a long series of experiments with different chem-

9Corn Steep Liquor, used by Andrew J. Moyer during research work with penicillin,https://en.wikipedia.org/wiki/Corn_steep_liquor

by C. Schwedes 13


icals and culture vessels to find out what would be the best medium andthe most suitable vessel for the growth of the Penicillium notatum fungusto get high active Penicillin material.The result of this investigation work was that they found an acceptableculture medium called Czapek-Dox Broth which is a modification of theCzapek (1902-1903) and Dox (1910, U.S. Dept. Agr.) formula prepared byDr. Charles Thom and Dr. Margarete B. Church (Czapek, 1902-1903) fromthe Northern Regional Research Laboratory in Peoria and by Dr. Thomand Dr. Kenneth B. Raper (Dox, 1910) from the Northern Research Labo-ratory.10 The Czapek-Dox broth was prepared only with inorganic sourcesof nitrogen and several differing sources of carbon. The broth was very use-ful for microbiological procedures and it could produce moderate growth oftypes of fungi such as saprophytic aspergilli, mycelia and conidia.11 [9]

Culture medium – modified Czapek-Dox medium: (used by Clutterbuck,Lovell and Raistrick (1932)) 2 units/ml (Chain/Florey 1940) 12

Contains:Glucose 40gNaNO3 3gKH2PO4 1gKCl 0.5gMgSO47H2O 0.5gFeSO47H2O 0.01gDistilled water 1L

Addition of Yeast-Extract (10% of the volume) to speed up the growth of the mould.10United States Department of Agriculture, Agricultural Research Service (ARS), His-

tory of the ARS Culture Collection; http://nrrl.ncaur.usda.gov/T heCollection/; Dr. C.Thom started with some investigations about the microbiology of Roquefort and Camem-bert Cheese at Connecticut Experiment Station in 1904. During his work he was ableto acquire several hundred of mould cultures. Later in 1913 he was relocated to Wash-ington D. C. and he brought his strain collection with him which was later called theThom and Church collection. In 1940 when the Northern Regional Research Laboratory(NRRL) was opened Dr. Kenneth B. Raper was chosen to lead the Culture CollectionSection what was a part of the fermentation division and Raper who had worked withDr. Thom brought about two thousand of the Thom and Church collection with himto Peoria. Other collections of deposited strains in Peoria were the citric acid-producingaspergilli and some bacterial strains. Just about 2 years later Florey and Heatley wentto Peoria to the NRRL to start and to develop the large-scale industrial production ofpenicillin with deep-tank fermentation technology with the support of the research teamfrom Peoria with Dr. K. B. Raper.

11Czapek Dox Broth, Czapek Solution Agar; https://www.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/233910.pdf

12The ingredients of the medium are from the unique collection of the original andhandwritten documents from Norman G. Heatley about the development of the productionprocess of penicillin and the manufacturing manuals of the used equipment at the "SirWilliam Dunn School of Pathology" in Oxford in 1940.

by C. Schwedes 14


The mould can grow and produce the Penicillium spores on a variety ofdifferent types of culture media, but not every medium is suitable for thePenicillium fungus because the fungus will produce the spores only understress in a particular synthetic environment. Before continuing with the de-scription and explanation about the culture medium which they have finallyused for the growth of the Penicillium fungus it is useful to briefly describethe use and importance of different types of agar plates which were used byAlexander Fleming and the research team in Oxford to try to find a suitableculture medium for the growth of the Penicillium fungus.

The name agar comes from “agar-agar” what is the Malay/Indonesianword for “red algae” (Gigartina, Gracilaria, agal-agal). Agar is generallyused to support the growth of several types of microorganisms and it is veryoften used in petri-dishes and as culture medium for microbiological pro-cesses. Agar is a kind of gelatine, it is tasteless and indigestible for manyorganisms. These properties makes it to a perfect agent for the use of aculture medium because it does not affect the growth of the microorganism.Agar is more appropriate and more stable than other kinds of gelatine. Ithas a melting temperature at 85◦C and solidifies between 32-40◦C (humanbody temperature 37◦C).

Blood Agar Plate:A blood agar is usually prepared with horse or sheep blood and has a bloodconcentration of about 5-10%. Blood agars are in the position to isolate fas-tidious (temperamental) organisms and to detect a hemolytic activity. Allin all the blood agar has two main functions:

- to support the growth of bacterial colonies such as streptococci species

- to determine the type of haemolysis-activity of the microorganism

Haemolysis describes the breakdown of red blood cells. The blood agar isused to determine particular microorganisms with this ability (such as strep-tococcal species). There are different types of haemolytic-activity:

Alpha-hemolysis: Describes a partial lysis and digestion if the red bloodcells and the cell membrane is still intact. The agar underneath the colonyappears in a dark greenish and sometimes brown colour.Beta-hemolysis: Shows the lysis and the complete digestion of red bloodcells and not just locally but even in the surroundings of the colonies. Anexample microorganism is the streptococcus haemolyticus. The area aroundthe colony appears in a lightened yellow and transparent colour.Gamma-hemolysis: Is the lack of hemolytic activity (non-hemolytic)

by C. Schwedes 15


Contains:enzymatic digest of casein 15g/Lenzymatic digest of animal tissue 4g/Lyeast extract 2g/Lcorn starch 1g/Lsodium chloride 5g/Lagar 14g/L

Final pH: 7.0 +- 0.2 at 25◦C

Corn-Steep Liquor by A. J. Moyer and R. D. Coghill (finally devel-oped in 1946): 150-200 u./ml.

A development for the large-scale industrial production of Penicillin in Peo-ria, U.S.A.. The microorganism was able to produce huge amounts of Peni-cillin units per ml. with the use of corn-steep liquor and the deep-tankfermentation technology with permanent stirring and agitation technique(see also the explanation at the beginning of this section). One of the firstsubstances added to the broth, to increase the production of the activeagent, was yeast. However, it was not as effective as they had expected, touse it hopefully for a large-scale industrial production. Later, they addedcorn-steep liquor to the medium which is an extensive growth-promoter infermentation. The important addition of glucose and mineral salts to thecorn-steep-liquor and to this finally “modified Czapek-Dox solution” was dis-covered by Moyer and Coghill in Peoria who noticed that the addition ofthese substances could greatly increase the yield of Penicillin.13 [14]Contains:

NaNO3 3gMgSO47H2O 0.25gKH2PO4 0.5gGlucose monohydrate 2.75gZnSO47H2O 0.044gMnSO44H2O 0.004gLactose monohydrate 44gCorn steep liquor 100gWater to make 1L

Initial pH 4.613The Legend of Moldy Mary: A technician assistance from the NRRL Ag. Lab. named

Mary Robertson had the brilliant idea to collect some moldy fruits from some fruit standsfrom the marked area in Peoria with the hope and the luck to find there more efficientsources of Penicillium mold. And indeed she had the luck to find a strain from a cantaloupewhich could produce 25 times more Penicillin than the original Fleming strain and themost important fact was that it produced Penicillin when grown in submerged cultures(deep-tank fermentation). This strain become the superior Penicillin producer and thelegend of “Moldy Mary” was born. ; http://www.peoriahistoricalsociety.org/!/Exhibits-PenicillinMoldyMary

by C. Schwedes 16


Figure 2: Relation between assay value and concen-tration of penicillin in solution; Median vertical sec-tion through assay cylinder; dimensions in mm.

It is most recom-mended to use agar-plates such as horse-blood agar, mannitol-salt agar or espe-cially the CzapekSolution agar for theassay and the exam-ination to determinethe yield of Peni-cillin. The func-tionality of the as-say method to de-termine the yield ofPenicillin is the fol-lowing. An ordi-nary nutrient agarplate was used withseeded staphylococci

aureus bacteria as testable microorganism on it. Short glass cylinders fromglass-tubes or vitreous porcelain cylinders were prepared with an internalbevel at the lower edge so that the cylinder could sink into the agar andmake a water- and bacteria-tight seal. The shape and measurements of thecylinder can be seen in figure 2.

Figure 3: The original incubator which wasused for experiments from the laboratory inPeoria with three metal-shelves in it [15]

To test the yield of Penicillinthe cylinders are filled withthe Penicillin broth and in-cubated for 12-16 hours at37◦C (figure 3 shows theincubator which was usedin Peoria, U.S.A.). Af-ter the incubation phasemost of the fluid in thecylinders had disappearedand in the area immedi-ately around the cylindersno bacterial growth was no-ticed. The diameter orzone of inhibition immedi-ately around a cylinder wasalso called “assay value”.This value was the most im-portant gauge to say something about the yield of Penicillin as the largerthe diameter, the higher the yield of Penicillin of the tested broth. The

by C. Schwedes 17


assay value also varies slightly with different batches and agar plates andwith the density and the homogeny distribution of the bacterial colonies atthe beginning of the incubation phase. In table 2 (the assay of antibioticsin body fluids) it can be seen the result of the cylinder plate method at thesection of the diffusion methods. [1] Noteworthy is column 8, the materialdoes not need completely sterile conditions for a successful assay. [3;5]

Figure 4: Bed-Pan from Hospital

Before they decided to use the rect-angular porcelain vessels as culturevessels (see fig. 5) for the fungus,many different shapes and materi-als were tried, from conical glassflasks (Erlenmeyer flasks) to porce-lain bed-pans from hospitals (which,incidentally, turned out to be themost suitable shape). The choice ofthe culture vessel was the rectangular porcelain vessel, which gave a largesurface to the fungus which proved to be was advantageous for the Penicillinproduction. The company Messrs. J. Macintrye Co. made 600 special rect-

by C. Schwedes 18


angular porcelain culture vessels with a side-arm and a volume of 1 litre (seefigure 5) (altogether they had about 700 vessels, Howard Florey mentionedonly 600 in his Antibiotics Volume, Norman Heatley collected 174 vesselson December the 22nd and the 23rd in 1940 in a borrowed van, on Decem-ber the 23rd 172 of them were washed, filled with 1 litre of culture mediumand autoclaved, and a second van load was collected by Norman Heatley onJanuary the 6th in 1941).[19]

Figure 5: Earthenware Culture Vessel Above.–Vessels stacked for autoclaving.

These vessels could bestacked in an incubatoras well as in the au-toclave machine. Heat-ley brought these vesselsto the Sir William DunnSchool of Pathology inOxford and the first sow-ing of Penicillium nota-tum was at December the24th in 1940. The firstincubated broth was ableto be harvested a fewdays later. [2;6][19] Theprepared culture mediumneeds to be sterilized byautoclaving before the actual growth process of the Penicillium fungus canstart. Autoclaving describes a process where the culture medium is boiledfor one hour at 121◦C and 15 psi of pressure.14 [11] This sterilization processis absolutely necessary to remove impure organic substances from the cul-ture medium such as from the contamination of air that the growth processcan go on uninterrupted. To initiate the growth process, it is necessary toadd some Penicillium spores to the sterilized culture medium. The additionof spores helps to start the growth of a layer of the Penicillium notatumfungus on the surface of the medium. In the early time of development itwas also usual to add some yeast extract to the medium to speed up theprocess. That was normally about 10% of the volume of the prepared cul-ture medium.15 (Gladstone and Fildes, 1940) [13] It was observed that the

1414.695 Psi is 1 atmospheric pressure, https://en.wikipedia.org/wiki/Atmosphere_%28unit%29Pressure_units_and_equivalencies

15Incidentally, the added extract was fresh pressed yeast from the Morrells Brewery onSt. Thomas’s Street in Oxford what is today “The Lion Brewery”, it was just like a miraclethat we found this information as we (Stephen and I) have visited the Welcome Library inLondon at November in 2014 because the Volume VII of Heatley’s handwritten documentswere actually stored in a closed stack until January the 1st of 2045, but fortunately thisvolume was mixed in a box with all the other volumes which we have ordered and wehave had the chance to photograph the original and handwritten Penicillin-productionscheme from N. Heatley with the information about this brewery on it. Furthermore, they

by C. Schwedes 19


addition of yeast extract accelerates the growth but does not affect the yieldof Penicillin. [3;4] 1 Litre of this medium filled the vessel to a depth of1.7 cm. A shallow fluid layer with a relatively large surface is the perfectcondition for the growth of the Penicillium fungus. The fill process to bringthe medium into the culture vessel should happen under sterile conditionswithout any kind of contamination otherwise the growth process would beinhibited and the fungus produce smaller amounts of Penicillium spores.It is absolutely necessary to avoid contamination through air, however thefungus cannot grow anaerobically16, as it needs oxygen for the metabolismand its existents.17

Just 24 hours after the sowing process of the Penicillium spores a verydelicate, fluffy, gauze-like growth can be seen with some difficulty on thebottom of the vessel. For a successful harvest to produce as many spores aspossible the Penicillium fungus needs a particular temperature of 24-25◦C.If the temperature is lower or higher the growth of the spores can be de-layed or completely destroyed. The fluffy growth becomes more voluminousduring the next days and after the 3rd day, if the liquid layer is not morethan 1cm thick, the fungus will reach the surface of the medium and throwsup dry and white mycelium particularly around the sides of the vessel. Themycelium is the vegetative and asexual reproduced part of the Penicilliumfungus. That means that a new organism can arise without the productionof seeds or spores. [10] The mycelium is the part of the fungus which isthe carrier of the Penicillium spores and after the fungus has thrown upthe white mycelium on the surface of the medium it starts to release thePenicillium spores to the liquid medium (liquid layer, underneath the Peni-cillium fungus). Another side-effect can be seen after the 3rd day if themedium has been rocked or shaken during the 1st and the 2nd day. Afterthe sowing process the medium should remain undisturbed without move-ments otherwise the gauze-like growth tends to conglomerate into balls andropes and those parts will be covered by a secondary growth a few dayslater. The white mycelium layer starts to expand its size and to move tothe centre of the vessel and, usually after the 4th or 5th day, the surface ofthe medium is completely covered with white mycelium and about 24 hourslater it begins to turn into a bluish-green layer. That is seen at first at thecentre of each vessel and starts to spread rapidly outwards. After the 6th

or 7th day of growing the mould is expanded to a very thick layer with an

added glucose (fruit- or grape-sugar) and some mineral salts to the used liquid medium toincrease the yield of penicillin; Norman G. Heatley, original notes, Scheme for Productionof Penicillin March 1941; The Wellcome Collection, Wellcome Library London, SpecialCollections, PP/NHE/A/2/1/5, p.64/65

16H. W. Florey, FURTHER OBSERVATIONS ON PENICILLIN p.417for refill and harvest methods see: Development: Howard Florey’s Surface-Culture

Method; 2. Downstream Processing, 2.1 Harvest the Culture Medium, changing pistol;

by C. Schwedes 20


upper and a lower surface. The upper surface is in a dark greenish-bluecolour and cannot or should not be wetted by water with any disturbanceof the vessel otherwise the fungus will stop the release Penicillium sporesinto the liquid medium (liquid layer). The lower surface of the mould layeris wetted and brownish-yellow and slimy. After 8-10 days this large mouldlayer (mycelium layer) with an upper and a lower surface can be seen. Ifthis above mentioned period of 8-10 days (which represents the incubationphase) is finished the colour of this mould layer have faded and grey. Thathappens usually on the 8th day or shortly thereafter. [3;1]

The changes in the pH-value is the most important gauge of the wholegrowth and production process of Penicillin. This value gives informationabout the antibacterial activity of Penicillin and its changes are accompa-nied with the different phenomena in the colour and the shape of the mould.The pH-value starts normally with 6 or 7 on the 1st day and falls down to avalue of 3 by the 3rd day, when the dry mycelium is formed on the surface ofthe culture medium. During the next days it rises and reaches a value of 5during the greenish-blue colour phase. At this stage a faint yellow colour canbe seen in the medium for the first time and slight traces of Penicillin canbe detected. The rise of the pH-value increase continuously and the yellowcolour and the yield of Penicillin both increase rapidly. At the last phase,when the colour becomes more faded and grey, the pH-value is increasingmore and more slowly and the maximum of the production of Penicillin ofthe Penicillium fungus is mostly at a pH-value of 7 very seldom exceed itvalues of 8.8 or higher. If the growth is finally prolonged the pH-value staysusually constant at about 7 for some days or starts to fall again rapidly. [3;3]

After a period of 7-8 days the Penicillium fungus releases a lot of sporesto the liquid medium. After a time period of about 8 to 10 days the fungushas produced enough spores and is now ready for the harvesting processwhich needs to be done under sterile conditions. The crude medium hadusually contained an activity of 1 and 2 units per c.cm. and even the driedand purified material for therapeutic purpose (1941 and later) had just anactivity of 40-50 units per mg. It had never been possible to obtain prepara-tions from the surface culture methods of a degree of purity higher than 300to 400 units per mg. by the simple solvent transfer purification method. In1941, the Penicillin product had an average activity of about 50 units/ml.and just one year later (industrial production in the United States, 1942) apreparation contains more than 10 times of this activity. [2;8]

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1.2 Sterilization (Autoclaving)

Sterilization describes generally the process of inactivating or the remov-ing of all living organisms from substances or objects. Most fermentationvessels such as the rectangular culture vessels with cotton plugs are opensystems and they need to be sterilized permanently to reduce the influenceof contamination by microorganisms. A system can only be sterile if it hasno openings or inputs for other organisms.18

There are different sterilization methods for different series of processes. Thesterilization can be done by heating, radiation, chemicals or filtration. Ster-ilization with heating uses steam, moist or wet heat. Autoclave-Machinesuse steam for the sterilization process with a heat of 121◦C and 15 psi ofpressure.19 [11] The research team in Oxford used Autoclave Machines forthe sterilization process [3;3] because it is a good acceptable and cheapmethod of sterilization and getting enough monetary support from donorswas a very big problem during war time in 1940.20 One litre of crude culturemedium will fill a vessel to a depth of about 1.7 cm and the addition of 10%of the fluid volume of yeast extract will speed up the growing process duringthe incubation phase. [3;6] The prepared medium needs to boil for about1 hour with the above-mentioned temperature and pressure. Otherwise theinfluencing microorganisms are still active after the sterilization process anda sufficient sterility can’t be guaranteed. The rectangular culture vessels areproduced with a specific design and measurements to give a big surface tothe Penicillium fungus for the growing process but it is also to put a stackof vessels into the autoclave machine as well as for the sowing process of thePenicillium spores. Fig.5 shows that each of the cotton plug is well sepa-rated from the other so that no bench space is lost. If the medium is boilingin the autoclave the plugs are unlikely to be wetted. [3;6]

Another sterilization method is the sterilization by filtration. This methodis used for the Penicillin production in stage 2 (see 2.1 Harvest the CultureMedium). The refresh process of sterile air inside of the culture vessel acotton filter is used to protect the medium against contamination by air.The supply of fresh and sterile air inside of the culture vessel is importantto support the spore production of the Penicillium fungus throughout thegrowth process.[3;6] The cotton filter needs to be sterilized before it can beused for the refresh process.

18Saritha Pujari; Sterilization: Common Contaminants and Sterilization Proce-dures; http://www.yourarticlelibrary.com/essay/sterilization-common-contaminants-and-sterilization-procedures/33629/

1914.696 psi is 1 atmospheric pressure, https://en.wikipedia.org/wiki/Atmospheric_pressureStandard_atmosphere

20See chapter: Financial Facts, to get more information about the funding of the researchgroup in Oxford

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1.3 Penicillium Spores

The culture medium is now sterilized and free from any contamination byother microorganisms. The Penicillium notatum fungus can grow uninhib-ited inside of the rectangular culture vessel and the production of sporeswill not be influenced or disturbed by other organisms. The addition of afew drops of spore-suspension to the culture medium will start and speedup the growth of the fungus. The strains of Penicillium used in this workhave been obtained from Prof. Alexander Fleming of St. Mary’s Hospital inLondon. [3;6] In later assays and investigations about the spore productionthey have found out that the addition of yeast extract doesn’t influencedthe production of the Penicillin spores but it just speeds up the incubationprocess.

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2. Downstream Processing

2.1 Harvest the Culture Medium

For the introduction part of the stage of the harvest process of the incubatedculture medium it should be noted that the work under sterile conditionsand with non-contaminated material was the most important fact of all butunfortunately it was far from easy to have these high standard condi-tions during the whole production process and through all of the productionstages because the used equipment was mostly self-prepared with very sim-ple materials. The introduction chapter with the “Historical Introduction”and the “Financial Facts” chapter explained in rich detail how difficult itwas to get high quality materials for scientific and research purposes, evenfor scientists in Oxford, England, during the time of the Second World War.This is also the reason for this relatively impure result of the PenicillinSodium Salt after the last step of production. However, the research teamwith the mastermind of Lord Howard W. Florey, Ernst B. Chain, NormanG. Heatley, et.al. did excellent work to find an extraction method for themassive production of Penicillin and with this huge investigation work theywere able to save more than 82,000,000 lives, up until the present day.21

Figure 6: Changing Trolley with 4 wooden slabs and 4 rectangular culturevessels on it.

21http://www.scienceheroes.com/; gives a very informative summary in the shape of anumber about the scientists who have the most saved lives in history

by C. Schwedes 24


The equipment for the harvest process was a changing trolley (see fig. 6)with a wooden board A, and 4 wooden slabs B and 4 rectangular porcelainculture vessels on it. The slabs are not hinged with the board A but theyhave a slight angle to it to support the harvest process. On depressing thehandle D it was possible to raise the angle slightly so that the fluid inside ofthe vessel drains more to the spout on the right corner. It was also possibleto put the vessel into the sloping part around C at the end of the woodenslab to raise the angle of the vessel to 45◦what is seen in N in order to makethe harvest and refill process easier. The laboratory assistant could operatewith both hands on the changing trolley.

Figure 7: vessels and volumina, 1 Batch with the volume of about 1 Gallonfits with 1 collecting bottle.

One hand to hold the changing pistol and the other hand was to control theposition of the cotton plug to reduce the influence of contamination duringthe harvesting process.The volume of one batch is divided into the volume of 4 culture vesselswhat fit with the volume of the collecting bottle L and the filtration bottleK what is all in all a little bit less than 1 gallon. (see fig. 7)The culture medium will be drawn off with the help of a special device Ecalled a changing pistol. The pistol has two glass tubes F and G and workswith pressure and suction controlled by the metal trigger H. The first glasstube F with sterile cotton-wool in it has the function of a filter and workswith pressure. F is responsible for flooding the culture vessel with fresh andsterile air with a little bit faster rate than the fluid is drawn off by tube G.The harvest process needs to be done under strictly sterile conditions with-out the influence of any contamination by other microorganisms. For thisstep the filtration as sterilization method is used and the filter (cotton-woolfilter) itself needs to be in a sterile condition before it can be used. The ster-

by C. Schwedes 25


ile air is very important to support the spore production of the Penicilliumfungus.The second glass tube G is responsible for the harvest process and is work-ing with suction to draw off the incubated culture medium from underneaththe layer of the Penicillium fungus. The pistol is also working with a re-placement technique, meaning that the medium can be replaced with freshincubated culture medium. For this replacement the function of tube Gneeds to be changed from suction to pressure to flood the vessel with freshmedium underneath the Penicillium fungus. If the Penicillium notatumlayer is once wet, the fungus stops producing Penicillium spores and needsto be removed from the culture vessel.The addition of yeast extract with a volume of 10% of one batch will speedup the growth of the Penicillium fungus but it does not affect the yield andthe activity of Penicillin.22 The first prepared batch contains 10% of it andthe replacement medium contains only 2.5%. That is because the spore pro-ducing fungus is already fully grown after a time of about 8 to 10 days andthe replacement medium does not need that high amount of yeast extractto keep the layer of the fungus fresh and active. After the incubation timeof about 8 to 10 days the medium can be replaced with fresh medium at 7day intervals to produce more Penicillin in about half the time. [12] Thisrefresh cycle can be repeated for about 14 times until the fungus stops toproduce Penicillium spores.The plugged cotton-wool of tube F needs to be changed after every twobatches and tube G is changed after every batch of 4 vessels.Further the pistol is connected with a special filtration bottle K with asilk-filter bag in it to remove solids and other impurities from the harvestedmedium. The filtrated medium will be finally collected in a separate collect-ing bottle L.

22The addition of yeast extract is also mentioned on page 13, section 1.1 CultureMedium, Corn-steep liquor

by C. Schwedes 26


Overview - Changing-Trolley:The Harvest- and Refresh-Method:

A. a wooden plank for the four hinged wooden slabs (B)

B. the four wooden slabs for the 4 culture-vessels of one batch

C. raised rim of the slab, runs into a handle D, it was possible to put thevessel into C to raise the angle to 45◦and to make the harvest- andrefresh-cycle easier

D. handle of the wooden slab to raise the angle to A slightly, the fluiddrains more to the spout near the right corner of the culture vessel

E. the special changing-pistol to draw off the medium of the culture vesselby suction, E is carrying two detachable tubes (F,G) and a metaltrigger H

F. a tube plugged with sterilised cotton-wool, acts like a filter to reducecontamination if the vessel is flooded with sterile air, happens a littlebit faster than the fluid is drawn out by tube G, needs to be changedafter every 2 batches (1 batch are four vessels)

G. a tube for drawing out the medium from the culture-vessel as well asfor the replacement of fresh medium, the tube needs to be changedafter every batch of four vessels

H. a metal trigger controls suction and pressure of the pistol of tube FandG, both tubes were sterilized and wrapped with paper before theywere used to reduce the influence of contamination

K. the harvested medium passes from the pistol into the inverted bottle,the medium is filtered in it with a silk-filter bag to remove solids andother impure substances from the harvested medium

L. the harvested and filtered liquid fromK flows into the collecting bottle,this bottle needs to be changed after every batch of four vessels

M. pin, if the pistol is not in use

N. refilling, the vessels are tilted at about 45◦, a fitting on each slabenables the vessels to be wedged in this position

by C. Schwedes 27


2.2 Separate the Culture Medium

(a) Counter-Current Apparatus - Primary Separation

Figure 8: Counter Current Apparatus, basicextraction unit [2;7]

The counter current appa-ratus was the heart andthe most complex part ofthe entire early develop-ment of Penicillin in Ox-ford. With the raising ofthe scale of the productionof Penicillin and the brew-ing of more and more cul-ture medium the researcherin Oxford needed to developbigger and more efficientextractors to increase theamount of the Penicillin pro-duction significantly. Thenew counter current appa-ratus had a weekly out-put of about 500 litre ofcrude broth and a capac-ity of about 12 litres perhour. [2;7] Norman Heat-ley built most of the usedequipment for the Penicillinextraction by himself andeven ordered parts such asvitreous collecting bottlesfrom elsewhere around Ox-ford. [17]

Unfortunately the originalapparatus from 1941 was de-stroyed because of an acci-dent which happened a cou-

ple of years later. Norman Heatley decided to create a replica because theScience Museum in London had interest of this apparatus as well as theBritish Broadcasting Corporation had interest to include it to an upcomingTV-show to this point of time. [18] The counter current apparatus had anumber of weak points and produced a few biological difficulties. The in-fluence and the contamination by air was a complete disaster and destroyeda lot of the active substance because some air organisms have produced an

by C. Schwedes 28


enzyme which destroys the Penicillin substrate

Figure 9: Constructionplan, counter current appa-ratus, basic extraction unit[16]

and they could not detect any activity of Peni-cillin in the metabolism fluid. [2;7] The basicconstruction of the counter current apparatusis mainly divided into 4 sections. The first sec-tion is the crude broth section which is thepart for the culture medium. This is wherethe 1 gallon collecting bottle L from step 2 isto be found (section 2.1. Harvest the CultureMedium). Figure 14 shows a Buckner funnelwhich was used to have a control of the fluid ofthe crude broth. The second section is the 10%phosphoric acid section with a 1 gallon bot-tle as well. Figure 15 shows a special funnelwhich was prepared without parts of rubberto control the flow of the phosphoric acid andthe amyl acetate. The phosphoric acid will bemixed with the cooled crude broth to an acid-ified solution with a pH value of about 2.The third section would be the amyl acetatesection with a smaller bottle, with a volume ofabout a half-gallon.23 The amyl acetate is im-portant to extract the precious Penicillin sub-strate from the acidified solution. This extrac-tion will happen inside of the extraction unitD, the fourth and the most important sectionof the counter current apparatus. In the fol-lowing points I want to explain the function-ality of each of these four sections in detail.The culture medium needs to have a tempera-ture of 24 -25◦C if it is in a not acidified con-dition. Higher or lower temperatures wouldmean that the broth would lose too much ofits activity and would be worthless for furtherextraction and separation steps. The Penicillinis a quite sensitive substance and it is neces-sary to have the right temperature and theright pH-value during the extraction processbecause the Penicillin remains only with someparticular pH-values to some particular tem-peratures in a stable condition. Fortunately

23I could not find any information about the exact volume of this bottle but its volumeis definitely smaller than the other ones.

by C. Schwedes 29


the Penicillin can deal with unstable conditions for a very short time ofsome seconds and sometimes minutes. Before the crude broth is mixed withthe phosphoric acid to a pH-value of 2 it needs to be cooled to 4◦C withthe help of the cooling coil (see figure 8, counter current apparatus, coolingcoil). It was observed that the activity of the crude-broth-phosphoric-acidmixture (acidified solution) would fall rapidly if the solution is not cooled.

Figure 10: Counter Current Ap-paratus, main extraction unit

The cooled broth is led to the point O(see figure 9 and 16) through a 6 man-ifold side-arm for each of the 6 parallelworking extraction columns. The phos-phoric acid is led through a six mani-fold side-arm as well to each of the 6vitreous jet needles O and broken upinto a spray of fine droplets before itis mixed with the crude broth (culturemedium) to an acidified solution. (seefigure 16, jet needles) Further the solu-tion will be led to the extraction unitwhich can be seen in figure 9 part Nand figure 10 from Norman Heatley’sconstruction plan for the counter cur-rent apparatus which shows every partin very rich detail. Construction plan:part I-VIII (figure 9-16).24 The acid-ified solution is led to a further glasstube M which is connected with plat-inum foil with some very tiny holes init. Immediately above the foil is a thinlayer of cotton wool strains. This actslike a sieve to avoid the platinum foil be-coming blocked up with solids and otherimpurities from the crude broth. (see

figure 11)24Norman Heatley gave a lot of effort to his personal notes to make them as rich in

detail as possible. That is shown by all of his 7 volumes and descriptions about thework of the development and purification of Penicillin. Every step and experiment,every part of the used equipment is described very rich in detail. Because of hisvery high quality notes it was possible to reconstruct the devices and the equipmentto a later point of time. Unfortunately the original counter current apparatus wasdestroyed through an accident a couple of years later after they had use of it inOxford in 1940. However, Heatley suggested to the science museum in London tocreate a replica of this apparatus in 1985. He ordered old and typical equipment andyou can find the apparat in an air force base in the north-west of London today;http://www.sciencemuseum.org.uk/online_science/explore_our_collections/objects/index/smxg-61195

by C. Schwedes 30


Figure 11: Counter Cur-rent Apparatus, glass tubewith platinum foil.

The number and the diameter of the holesis crucial for a successful Penicillin extractionfrom the acidified solution into the amyl ac-etate solvent. Only a certain amount of solu-tion may pass into the organic solvent during acertain time. The platinum foil breaks up thesolution into droplets with a uniform size andthese droplets fall through a column of organicsolvent (amyl acetate). The organic solvent inthat column (see figure 9 part A) comes fromthe amyl acetate section. Glass tube A hasan internal diameter of about 18mm. [20] Theamyl acetate flows to the same time throughanother system of tubes than the acidified solu-tion. I want to remain for the moment with theacidified solution at the point when it’s bro-ken up into droplets through the platinum foiland would like to jump/skip to the flow of theamyl acetate. The amyl acetate section is com-pletely separated from the crude broth- andthe phosphoric acid-section. There is anothersix manifold side-arm which distributes the es-

ter (amyl acetate) to each of the six parallel working extraction columns.

Figure 12: Counter Cur-rent Apparatus, jet nee-dles, to control the velocityof the flow of the acidifiedsolution.

Furthermore the amyl acetate is led down-wards with tube H into the bottle Fand then it is led upwards through col-umn A to the main extraction unit ofthe counter current apparatus. (see fig-ure 9 for the described parts H and Aand figure 10 for the main extraction unit)This unit is the most important part ofall of the counter current apparatus be-cause it is the point where the acidifiedsolution which is spread into droplets isfalling into the organic solvent (amyl ac-etate). A look at the densities of the dif-ferent liquids and the explanation of thesolvent-transfer method will reveal a betterunderstanding of the layer-building and thedraw-off technique of the Penicillin-rich so-lution (solvent-layer) which will be the fi-nal result of this separation and extractionstep.

by C. Schwedes 31


Densities:amyl acetate: 0.87 g/mlwater (4◦C): 1.0 g/mlphosphoric acid (liquid): 1.87 g/ml

A short moment after the acidified solution and the amyl acetate are broughttogether, you will see the amyl acetate in the upper part of the extractionunit because its density is the lowest one (see figure 10 part B). (It’s gatheredespecially around the area of the hole B which is drilled inside of the glasstube A to pull off the Penicillin-rich solution with amyl acetate) The crudebroth with the phosphoric acid will just fall down the column A and willbe collected in the bottle F which has a separate waste tap I to remove oldwatery and acidified solutions, this bottle was also called: “solvent-waterinterface”. (see figure 9) [20] The apparatus had a permanent support ofthe organic solvent through the rising column A and needs to be calibratedvery exactly because each of the fluids and chemicals need to have the rightvelocity in each part. [20] Otherwise, the final result and volume of thisextraction would be reduced. Some parts of the active Penicillin substratewould be destroyed because it remained for too long in an unstable con-dition, or the drawn off Penicillin-rich solution with amyl acetate is mixedwith parts of the broth mixture which are too impure because too much ofthe broth could escape through the hole B. [20]

‘Penicillin has not yet been tried in war surgery [for contaminated wounds]and it will not be tried until some chemist comes along and finds out whatit is and if possible manufactures it.’ (Fleming, 1940e.)

The most interesting step of all is the extraction of the Penicillin substance.It is important to know that the active Penicillin substance needs to beextracted from the acidified solution. The amyl acetate is in the positionto extract the Penicillin substance from the acidified solution because thePenicillin starts to move into the amyl acetate layer if you bring both ofthese solutions together. The Penicillin itself is an acid which is more sol-uble in organic solvents such as ether or amyl acetate, whereas salts aremore soluble in water than in organic solvents. “Small molecules such ascitric acid or Penicillin are extracted with organic solvents such as one of thehigher amines or butyl acetate. Such mixtures can separate large molecules,including proteins.” (a part of the description of the extraction process atdownstream processing in biotechnology). [21] When the watery and acidi-fied solution is mixed with the organic solvent both start to separate them-selves into two layers because they are not soluble and you will find thePenicillin substance in the upper layer (solvent layer) if the mixture is acidand in the lower layer (watery layer) if the mixture is neutral or alkaline.That was discovered and described with the “Solvent Transfer Method”.

by C. Schwedes 32


Solvent Transfer Method:The most of the Penicillin-rich solution will be found in the upperlayer (solvent layer) when the mixture is acid. (result of point 2.2.with the counter current apparatus)

The Penicillin-rich solution will be found in the lower layer (wa-tery layer) when the mixture is neutral or alkaline. (result of point2.3. with the separating funnels)

The aqueous solvent from hole B runs into a trap which is formed throughtube D and D’. (see figure 10) The aqueous solution is collected at thebottom of the interspace of tube A and D and droplets of the remainingcrude aqueous solution can be drawn off from time to time through tube E.(see figure 9) In figure 10 you can see an alternative version of the tube E.The drilled hole at the top of the tube is important to create an equilibriumwith E and D and just the crude and aqueous solution will be drawn offautomatically. The Penicillin-rich solvent will be finally drawn off at the topof the glass tube D through the separate side-arm C. (see figure 10) Thesolvent will finally be collected into a 1 gallon collecting bottle. [20]In a later version of this apparatus a special alarm system with some light-bulbs and a bell on a wooden board was added to the counter current ap-paratus to inform the scientists about full or empty bottles which neededto be replaced and to make the extraction process more automatic and self-sufficient. [20]However, this apparatus was in the opinion of some who used it as temper-amental as a prima donna but nonetheless it was a huge step forward to alarge-scale extraction of Penicillin and it gave a good service. [2;7]

“It is the technologist who is transforming at least the outward trap-pings of modern civilization and no hard and fast line can or shouldbe drawn between those who apply science, and in the process makediscoveries, and those who pursue what is sometimes called basicscience.”

Lord Howard Walter Florey (1898-1968)

Additional to the explanations of the harvesting process and the countercurrent extraction apparatus in Table 1 is shown the stability of Penicillinat several pH-values and temperatures and the dilution of Penicillin at thecylinder plate assay method. Noteworthy is that the Penicillin substance ismost stable if it’s extracted into ether in an acidified condition at a pH-valueof about 2.0.

by C. Schwedes 33


Figure 13: Counter CurrentApparatus, vitreouse bottlewith Buckner funnel to controlflow of culture medium.

Figure 14: Counter CurrentApparatus, construction of theBuckner funnel.

Figure 15: Counter Cur-rent Apparatus, special fun-nel without rubber parts, forphosphoric acid and amyl ac-etate, flow control.

Figure 16: Counter CurrentApparatus, jet needle to breakup the phosphoric acid intoa fine spray before it willbe mixed with the culturemedium.

by C. Schwedes 34


by C. Schwedes 35


(b) Separating Funnels - Secondary Separation

“Without Fleming, No Chain or Florey; without Florey, No Heatley;without Heatley, No Penicillin”

Sir Henry Harris – 1998, researcher groupSir William Dunn School of Pathology

Figure 17: Separating funnel, to separate andto draw off the watery penicillin-rich solutionfrom amyl acetate with the help of an alkalibuffer; solvent–transfer principle.

The extraction of the Peni-cillin substance from theamyl acetate is quite an easystep. A wooden frameworkwas used (see figure 17)with 2x 1 gallon glass bot-tles and a little separatingfunnel in the middle area.The upper bottle is fromstep 2.2. (the extraction ofPenicillin with the countercurrent apparatus) and con-tains the Penicillin-rich so-lution with amyl acetate.Water (usually one fifth ofthe volume of the originalbroth) and an alkali bufferare added. The mixture isgently shaken and allowedto separate again into twolayers with a hard bound-ary in between. The ad-dition of the alkaline bufferis vital to increase the pH-value of the mixture to avalue of more than 7 andto bring it into an alka-line condition. The sameprinciple of solvent trans-fer which is explained inthe previous step is usedhere to purify the Peni-cillin substance from theamyl acetate solvent into aPenicillin-rich watery solu-tion.

by C. Schwedes 36


Solvent Transfer Method:The most of the Penicillin-rich solution will be found in the upper layer(solvent layer) when the mixture is acid. (result of point 2.2. with thecounter current apparatus)

The Penicillin-rich solution will be found in the lower layer (waterylayer) when the mixture is neutral or alkaline. (result of point 2.3. withthe separating funnels)

The wooden framework is constructed that the whole apparatus can beshaken using the small, hinged wooden board in the middle, which allowsthe two separate layers to form faster. The Penicillin substance starts tomove into the watery layer and can be drawn off from the lower layer intothe lower glass bottle. There is another very similar construction plan ofthis step with the same wooden framework but with a tap in between thetwo glass bottles to block the liquid until the two layers are formed. The useof the Buckner-funnel is not very efficient for the purification of Penicillinbecause even in this step the drawn-off watery Penicillin solution would beinfluenced through the contamination by air-organisms.

by C. Schwedes 37


2.3 Freeze-Drying, Crystallization

The freeze-dry process was developed during World War II because of hugeproblems with the supply of vaccine and serum, and keeping them chem-ically stable without the need of refrigeration. [23] It was mainly used tofreeze organic or inorganic materials as well as liquid or fluid substances.Usually this process is done in a vacuum and evaporation chamber withlow temperature and very low pressure to create an environment with theoptimal conditions for the sublimation phase, to turn a frozen and solidmaterial immediately into gas. (sublime means evaporate) E. P. Abrahamsuggested in 1941 that the only way to evaporate the aqueous solution ofPenicillin to dryness and without loss of activity was to use the freeze-dryingmethod. (evaporation “in vacuo” from the frozen state)[2;9] The frozen wa-tery Penicillin-rich solution will evaporate at a very low pressure inside ofthe vacuum chamber. The massive amount of water vapour which resultscan escape very fast with the help of a vacuum pump and an under-pressurein the vacuum-chamber to the evaporation-chamber.

Freezing-Process:The freezing process is the most important step of the entire freeze dryingprocess as it affects the volume of the final product. The first step wouldbe to lower the temperature very slowly to about -50C. [23] The slower therate at which the freezing process is undertaken, the bigger the crystals pro-duced and the larger the amount of remaining Penicillin sodium salt. Theword “crystal” comes from the Greek “krustallos” and meaning both “ice”and “rock crystal” from “kruos”, “icy cold” or “frost”. Crystallization orsolidification describes the process when the crystal formation of a liquidis growing up. The crystal formation is the most significant point at thefreezing process. A formed crystal should have a nearly perfect and periodicarrangement of atoms for a perfect crystalline structure. The more struc-tured the arrangement of the atoms are the bigger the remaining crystals.[24]

Primary-Drying:A decreasing of the pressure to just a few microbars is the next step to passthe range of the sublimation phase. About 95% of the frozen water will besublimated (evaporated) during the first drying process. [23]Secondary-Drying:It is necessary to remove the unfrozen water molecules additionally with asecond drying-process. The remaining content of water of the finally freeze-dried product is about 1-4%. [23]

The remaining Penicillin Sodium Salt in the shape of a white powder isnow ready for storage and administration.

by C. Schwedes 38


2.4 Storage, Penicillin Sodium Salt

The very early development and extraction of Penicillin was a very small,impure and very low-active yield after all these efforts of purification andextraction, but it was nonetheless the start of a new era in medicine with amassive influence on the human standard of living.Tiny amounts of orange and yellow Penicillin Sodium Salt were collectedinto small bottles. In the first place the Penicillin was tested on mice bothbecause their body is 2000-3000 times smaller than a human body and theydon’t need that much Penicillin to heal a life-threatening infection wound,and, more especially, to see if the extracted Penicillin salt was strong enoughto act like the drug which was expected from the scientists.

The first human patient administrated with an intravenous injection wasa policeman of the age of 43 admitted December 12th, 1940. [3;7] He hadseveral infected wounds and scratches on his face because of an accidentwith a rose bush and the infection had begun to spread through the tissueinto his shoulder and lungs.[22] The first administration was quite success-ful with a total amount of 800 mg. Penicillin in 24 hours. A cessation ofthe scalp-discharge, and a diminution of his right-eye suppuration and theconjunctivitis was observed [3;7] but they didn’t have enough Penicillin, andeven with the re-extraction of Penicillin from his urine, the supply ran outand he died two weeks later. [22]

Most of the next patients were children in the hope that they would needsmaller amounts of Penicillin to be successfully treated and cured. Unfor-tunately only a few survived because either the Penicillin was too weak orthey didn’t have enough material for a successful administration. Most ofthe Penicillin was rapidly destroyed with an administration by mouth be-cause of the acid in the stomach, but it was possible to carry it throughthe stomach by raising the pH-value. A strongly antibacterial concentrationof Penicillin was observed in one of the cases and it was maintained in theurine for about 7 days with an administration by mouth and the additionof sodium bicarbonate. This particular effect was the reason why they wentso far as to re-extract the Penicillin from the urine of some patients to useit again for mouth administration.[3;7] The first successful administrationswere highly motivating, however the support of a commercial company wasimmediately needed to produce enough material in large-scale industrial pro-duction. That was finally found in July in 1941 in the Northern RegionalResearch Laboratory of the Department of Agriculture in Peoria, Illinois,U.S.A. and just a few months later the production of millions of mega-unitsof pure and white Penicillin Sodium Salt was possible especially for the pur-pose of war surgery.

by C. Schwedes 39


One of the usual arguments between Ernst Chain and Norman Heatley:

“Heatley, I’m telling you Penicillin is . . .Yellow!

Yellow!Yellow!”

(Ernst B. Chain). . .

“Chain . . . , your Penicillin is White.”(Norman G. Heatley)

by C. Schwedes 40


Financial FactsThe laboratory in Oxford where the early work of development and pro-duction of Penicillin was done (about 1940) was primarily supplied by theUniversity of Oxford. It was a big issue to find financial sponsors at thispoint of time because all money was invested into materials for the war,with the result that the main part of the required materials and salarieswere paid by Oxford University and even a part of this money was given bygrants and other private companies.

- The Medical Research Council contributed £8,287 in the years of 1939to 1945.

- The Rockefeller Foundation contributed £6,140 in the years of 1940to 1945.

- The Nuffield Provincial Hospitals Trust contributed £5,646 in theyears of 1943 to 1945.

For a whole researcher team, this was a real lack of financial support. Andmakes clear why the materials used in Oxford for the production of Peni-cillin were so primitive and sometimes even completely hand-made. Thebest example is the framework used for the Counter Current Apparatuswhich was an old bookshelf from the Bodleian Library. This may not havebeen the nicest solution for academic work in Oxford, but it did the job. Thework of the researcher team was never held up even by the lack of funds. [2;5]

Nobody knows anything about the supplies at other centres or researchgroups in Great Britain, but in a statement to the press on the 1st May of1946 the Minister of Supply said:

‘The expenditure on premises and plant in the U.K. has been in theneighbourhood of £3,000,000, of which more than two million has beenGovernment money.’

The expenditure of money on the American side is of course not compa-rable because there was an industrial large-scale production running whichwas supplied by Coghill and Koch (1945) but nonetheless it could be quiteinteresting to mention some facts:

‘The Penicillin program represents an investment of approximately$25,000,000 in buildings and equipment. Individual commercial plantshave ranged in cost from $350,000 for small surface culture facilitiesto over $3,000,000 for large installations employing the deep-tank cul-ture method. That the government financed less than one third of thistotal investment indicates the firm and continuous basis anticipated byindustrial management for Penicillin production.’ [2;5]

by C. Schwedes 41


Thanks to the large industrial production of Penicillin in the United Statesthe costs of Penicillin-units per ml. have been reduced continuously un-til today. The cost of the first produced Penicillin was $20 per 100,000Units and around 1949 the cost was already reduced to $0.60 per 100,000Units. There exist an international standard for Penicillin Units declaredfrom the Department of Biological Standards, National Institute for MedicalResearch in London in 1950. The department was authorized by the WHOExpert Committee on Biological Standardization to declare the Second In-ternational Standard for Penicillin. Recrystallized sodium salt of PenicillinG was used for the assay. The Second International Standard contained fi-nally 1,670 International Units (IU) per mg, describing an activity containedin 0.0005988 mg Penicillin Sodium Salt.25 [6]

The entire production of Penicillin in 1944 was worth of $35,000,000 andin 1945 because of the constantly price reduction even (only) $60,000,000.The price of Penicillin in hospitals in Great Britain in November 1947 was6s.4d. (£0.316) for 1,000,000 Units for ‘commercial’ Penicillin and 13s.4d.(£0.665) for ‘crystalline’ material. [2;5]

Figure 18: Penicillin production in the U.S.A., Great Britain, and Australia

25The Second International Standard for Penicillin: Declared in 1950 (Manuscript re-ceived in December 1952), Department of Biological Standards, National Institute forMedical Research, London, Humphrey, Mussett, Perry

by C. Schwedes 42


“Without Fleming, No Chain or Florey; without Chain NoFlorey without Florey, No Heatley; without Heatley, NoPenicillin”

Dr. Eric Sidebottom – 2015, Sir William Dunn School ofPathology, University of Oxford

by C. Schwedes 43


Bibliography

[1] H. W., Florey; E. B., Chain; N. G., Heatley; et al.: ANTIBIOTICS– A Survey of Penicillin, Streptomycin, and other antimicrobial sub-stances from fungi, actinomycetes, bacteria, and plants; Oxford Medi-cal Publications, Vol. 1 & 2, Geoffrey Cumberlege, Oxford UniversityPress, London, New York, Toronto, 1949

[1;1] Vol. 1, pp.1-2[1;2] Vol. 1, p.2[1;3] Vol. 1, p.2

[2;1] Vol. 2, pp.631-632[2;2] Vol. 2, p.666 ff.[2;3] Vol. 2, p.669[2;4] Vol. 2, p.691[2;5] Vol. 2, p.670[2;6] Vol. 2, p.643[2;7] Vol. 2, p.644 ff.[2;8] Vol. 2, p.650[2;9] Vol. 2, p.737

[3;1] H. W. Florey; E. B. Chain; N. G. Heatley; et.al: FURTHER OB-SERVATIONS ON PENICILLIN, The Sir William Dunn School ofPathology and the Radcliffe Infirmary, Oxford; THE LANCET, Au-gust 16, 1941; p.1 ff.

[3;2] Earthenware culture vessel, made by J. Macintyre and Co.; p.7

[3;3] H. W. Florey; E. B. Chain; N. G. Heatley; et.al: FURTHER OB-SERVATIONS ON PENICILLIN ; p.3 ff.

[3;4] H. W. Florey; E. B. Chain; N. G. Heatley; et.al: FURTHER OB-SERVATIONS ON PENICILLIN ; p.4

[3;5] H. W. Florey; E. B. Chain; N. G. Heatley; et.al: FURTHER OB-SERVATIONS ON PENICILLIN ; p.5

[3;6] H. W. Florey; E. B. Chain; N. G. Heatley; et.al: FURTHER OB-SERVATIONS ON PENICILLIN ; p.7.ff.

[3;7] H. W. Florey; E. B. Chain; N. G. Heatley; et.al: FURTHER OB-SERVATIONS ON PENICILLIN ; p.24.ff.

by C. Schwedes 44


[4] Prof. H. W. Florey, Investigation of War Wounds, Penicillin, APreliminary Report to The War Office and The Medical ResearchCouncil on Investigations concerning The Use of Penicillin in WarWounds, BRIGADIER HUGH CAIRNS, F.R.C.S., R.A.M.C. Con-sulting Neuro-Surgeon to the Army, Received 13th October, 1943

[5] Dr. P. Ravisankar;penicillins-power point-History,mechanism of action, classification, chemistry, SAR,Nomenclature, uses, sideeffects-Medicinal chemistry;http://de.slideshare.net/banuman35/penicillins-power-point-medicinal-chemistry?next_slideshow=1, p.32

[6] J. H. Humphrey, M.D.; M. V. Mussett, B.Sc.; W. L. M. Perry,M.D.; et.al: The Second International Standard for Penicillin,https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2542105/

[7] Corn Steep Liquor, used by Andrew J. Mojer during research workwith penicillin, https://en.wikipedia.org/wiki/Corn_steep_liquor

[8] Norman G. Heatley, original notebooks, handwritten documents; Vol-ume 5, p.61, The Wellcome Collection, Wellcome Library London,’Oxford Vol V’, PP/NHE/A/2/1/3

[9] https://bd.com/europe/regulatory/Assets/IFU/Difco_BBL/233910.pdf

[10] http://nrrl.ncaur.usda.gov/TheCollection/

[11] https://en.wikipedia.org/wiki/Atmospheric_pressure#Standard_atmosphere

[12] Norman G. Heatley, original notebooks, diagrams showing the prepa-ration of Penicillin, tracings and photographs of flow charts and di-agrams depicting Penicillin production. (Quite probably diagramsreproduced after the 1940s); The Wellcome Collection, Wellcome Li-brary London; section: PP/NHE/B/1/15

[13] Norman G. Heatley, original notebooks, handwritten documents; Vol-ume 7, scheme for production of Penicillin, March 1941; p.64/65; TheWellcome Collection, Wellcome Library London, ’Oxford Vol VII’,PP/NHE/A/2/1/5

[14] R. Winston Liggett, H. Koffler; CORN STEEP LIQUOR IN MI-CROBIOLOGY,https://www.ncbi.nlm.nih.gov/pmc/articles/PMC180696/pdf/bactrev00003-0030.pdf

by C. Schwedes 45


[15] http://www.peoriahistoricalsociety.org/assets/images/content/Penicillin_incab20130703_083056_%28350x263%29.jpg

[16] Norman G. Heatley, original notes, construction plan counter currentapparatus; The Wellcome Collection, Wellcome Library London, Spe-cial Collections, PP/NHE/C/1/5/1

[17] Norman G. Heatley, original notes, order of glass bottles; TheWellcome Collection, Wellcome Library London, Special Collections,C/8/1

[18] Norman G. Heatley, original notes, Letter Science Museum, equip-ment and glass bottle recollection; 1984 accession list; The WellcomeCollection, Wellcome Library London, Special Collections, C/8/1;C/8/1 ScM

[19] Norman G. Heatley, original notes, report: WORK ON PENI-CILLIN, OCTOBER 1st 1939, equipment and glass bottle recollec-tion; 1984 accession list; The Wellcome Collection, Wellcome LibraryLondon, Special Collections, PP/NHE/C/1/1; p.7

[20] Norman G. Heatley, original notes, APPARATUS FOR THECONTINOUSE EXTRACTION OF PENICILLIN; The Well-come Collection, Wellcome Library London, Special Collections,PP/NHE/C/1/5/1; p. 1-12

[21] J.A. Wesselingh, J. Krijgsman; Downstream Processing in Biotech-nology;Delft Academic Press / VSSD, first edition 2013; p. 59

[22] Dr. Eric Sidebottom; Penicillin 50 – Oxford Fights Disease; printedby Francis Lomas Limited, University of Oxford, 1991; p.10 ff.

[23] https://en.wikipedia.org/wiki/Freeze-drying

[24] Crystal, crystallization, crystalline structure;https://en.wikipedia.org/wiki/CrystalOrganigenic_crystals

by C. Schwedes 46

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