Spontaneous generation experiments examined from the food microbiology viewpoint.

     Experiments carried out to study the spontaneous generation theory, involved  a kind of practices (vegetables infusions and organic solutions employed as culture media, sterilization and pasteurization processes, open/closed devices) belonging mostly to microbiological laboratory and food technology practices. They can then be suitably analyzed on the basis of the today food microbiology knowledge.
    The following analysis is applied to experiments carried out by three prominent investigators in the 18th and 19th centuries.
For centuries, it was commonly believed that some forms of life arose spontaneously from nonmoving matter. The origin of the theory can be traced back to the Scriptures, which state that bees were generated from the carcass of a dead lion (Bible). Several Greek thinkers, up to Aristotle, claimed that several animals grow spontaneously from putrefying organic matter. It was generally accepted that worms, beetles, frogs, and salamanders arise from dust, mud, or food scraps. It was widespread the belief that corruption of one being might result in the birth of another:  “.. the seed must die before the young plant springs from it.” (Saint Paul). The Middle Age's well known ‘recipe’ for the spontaneous production of mice prescribed old rags and husks of wheat to be placed in an open-mouthed jar and left open for about 21 days in the warm season. In the mid 17th century the hypothesis that maggots arise from dead organic matter was still acknowledged.
Francesco Redi, was the first to prove instead that maggots arise from eggs laid by flies by using the classic experiments of the two batches of flasks. He put pieces of meat in a series of flasks left open to the air, and in a second series of flasks whose mouth was covered with gauze. Within few days maggots appeared only in the open flasks which allowed flies to reach the meat and lay their eggs. The experiment yielded so clear-cut results that the covered uncovered batches spread as a reference method among many scientists afterwards: Malpighi (1628-1694), Swammerdham (1637-1680), Vallisneri (1661-1730) and Reamur (1683-1757), among others. What Redi demonstrated by his primitive, though highly probative experiment, is acknowledged still today.

    With the development of the microscope by Leeuwenhoek (1632-1723), many types of new life forms were being observed and no one knew whence these microscopic organisms came from. As a result, their existence seemed to add new evidence in favor of the spontaneous generation doctrine. Based upon the findings of Redi, Joblot (1645-1716) (Doetsch, 1976) tried to demonstrate that ‘infusorians’ (simple organisms found in vegetable infusions, mostly ‘ciliates’) were not generated in sealed vessels containing boiled medium, but only in open vessels. However, Buffon (1707-1788) and Needham (1713-1781) both doubted that Redi’s hypothesis could be applied to infusorial ‘animalcules’. Needham endeavored to put the question to an experimental test: “.. I took a quantity of mutton-gravy hot from the fire, and shut it up in a phial, closed up with a cork so well masticated, that my precautions amounted to as much as if I had sealed my phial hermetically.” (Needham, 1748, p. 637). Following some days storage at room temperature, Needham found his broths filled with microscopic organisms (‘animalcula’). He claimed  “.. there is a real productive force in Nature..There is a vegetative force in every microscopical point of matter.” (Needham, 1748, p. 645, 653), that ‘activate’ the generation of ‘animalcula’ from dead organic matter. Thus, following Needham’s experiments concerning organisms not visible to the naked eye, the theory of spontaneous generation was revitalized in the mid 18th century. Epigeneticists, led by Buffon, supported the spontaneous generation theory. By contrast, Spallanzani, along with Haller (1708-1777), Bonnet (1720-1793) and Voltaire (1694-1778), were the most famous preformationists who believed that organisms arose either from seeds, or eggs, or in some way from organized biological particles able to develop the complete organism (Adams, 1929; Doetsch, 1976; Strick, 2003; Wilkins, 2004).
The sake of Needham was to prove that microscopic organisms (‘animalcula’) grown in his flasks were generated from dead organic matter, and do not entered the flasks from outside. To achieve the purpose, Needham’s experiments should have meet two features absolutely critical from the methodological view point: (a) infusions should have been heated enough to kill all organisms inside the flasks (the sterilization process), to be sure that if organisms should arise from ‘dead’ matter during storage, they were ‘spontaneous’, not survivors;  (b) the flasks should have been closed reliably enough to prevent organisms to reach sterile infusions from the outside, during storage - yet van Leeuwenhoek’s opinion was that “animalcules.. may be carried.. by the particles of dust blown about by wind.” (Stein, 1931). Needham believed that his method of heating infusions was adequate to kill all organisms inside flasks “I neglected no precautions, even as far as to heat violently in hot ashes the body of the phial; that if any thing existed, even in that little portion of air which filled up the neck, it might be destroyed, and lose its productive faculty.” (Needham, 1748, p. 638). Whereas he never proved it experimentally. Just the same, he assumed that flasks were closed hermetically “They did not seem to be enascent..from a deposition of any extraneous spawn; for the phials had been closed with corks; “ (Needham, 1748, p.636); without proving it. Not even he perceived the very relevance of the closure: ”..upon three or four scores of different infusions of animals and vegetables substances.. all which constantly give me the same phenomena with little variations, and uniform in their general results,..phials closed or not closed, the water previously boiled or not boiled, the infusions placed upon hot ashes.. (or not).. appeared so nearly the same, that I neglected every precaution of this kind, as plainly unnecessary.” (Needham, 1748, p. 639). Such statement incontestably proves that both closures and heating applied by Needham were fully ineffective, inasmuch as both “closed and not closed”, and “boiled or not boiled” phials gave the same results. Finally, he confirmed his belief: “.. besides the precautions I took .. no supposed germs might either be conveyed through the air or the water, or remain adhering to the substances infused; I have often, for the purpose, made use not only of the broths, immediately closed up in a phial, but also of pure animal substances, such as urine, blood, etc., with the same success; and in these I believe, no one will suppose that germs, eggs or spawn, are precontained, if care is taken to close the phials immediately.” (Needham, 1748, p. 660). Both shortcomings instead - heat treatments size and closure of phials - are so relevant to invalidate completely the Needham’s experiments and the theory he drawn thereof. Rather, his results could be regarded as a proof of the inadequacy of either the heating process to kill the organisms inside his flasks or failure of the flasks closure, or both, even from the contemporaries view point.

    Lazzaro Spallanzani (1729-1799) indeed, tried to verify Needham’s experiments by taking into account both heat resistance of ‘animaletti’ (small animals) and reliable flasks closure.
(1) - Spallanzani performed the first, albeit rudimentary evaluation of microbial resistance to heat. To do this, he used several sets of nine flasks, each containing one kind of vegetable infusion, among which: lentils, broad bean, vetches, buckwheat, barley, maize, seeds of hemp, seeds of mallows and seeds of beet. The neck of the jars was flame fused and stretched up to a capillary size, left cooling to ensure that the atmosphere in the head space of flasks was the same of the environment, and then it was easily flame sealed. The infusions were then boiled for different times (from 0.5 to 60 minutes) and stored for eleven days at room temperature. Using this method, Spallanzani found that 0.5 minutes (“mezzo minuto”) boiling was enough to kill all the organisms of “maximum, intermediate and small size” (Opuscoli, p. 30) (seemingly protozoa and algae; that is the ‘organisms of the higher order’, following Bonnet’s suggestion’; Opuscoli, p.30), whereas two minutes were without lethal effect against the organisms “endlessly small, I will call afterwards of the smallest size” (doubtless bacteria) (Opuscoli, p. 30). Later on, the amount of surviving organisms visibly decreased as boiling time increased up to 30 minutes. After 45 minutes or more, no one infusion showed organism not even of the smallest size. Accordingly, infusions boiled for 45 minutes or more can be regarded as sterile (“.. 45 minutes boiling had the effect to give all infusions completely sterile of organisms.”, Opuscoli, p. 33).
 (2) – Having shown that ‘germs of animals of higher orders’ are very heat labile, being killed in less than 0.5 minute boiling, Spallanzani wrote that organisms of higher orders growing in boiled flasks “ .. undoubtedly have reached infusions from the environment, after the heat treatment ceased.” (Opuscoli, p.43). He was right, since he believed that heat treatments applied by Needham would be equivalent to about half a minute boiling al least. But if Needham’s phials were not hermetic, so that organisms of the higher order entered the cooling phials from the outside (‘post process contamination’), as rightly said Spallanzani, then organisms of any size had access to the flasks, and mostly the ‘infinitely small’ of the ‘last order’. However, the kind of organisms grown in Needham’s flasks were nearly the same ("microscopical animals of most dimensions, from some of the largest I had ever seen, to some of the least.” (Needham, 1748, p. 638), independent of treatments applied to phials: “.. phials closed or not closed, .. boiled or not boiled,..placed upon hot ashes.. (or not)..” (Needham, 1748, p. 639). Spallanzani’s experiments underlined the great relevance of the heating time (45 minutes boiling at least) required to reach the sterility of infusions. Therefore, it is quite likely that Needham’s phials were not sterilized, since he did not heated enough his infusions; however, his: “ .. infusions (were) placed upon hot ashes..”. According to Spallanzani’s observations about heat lability of organisms of higher orders, it could safely believed that Needham’s infusions did not even reached 100°C. Hence, if the mixture of organisms grown in phials was the same, both in open and in closed (boiled/heated) phials (Needham, 1748, p. 639), then the mixture could be originated from both failure of closure and/or inadequate heating, indifferently. This though logic conclusion comes directly from diagnostic criteria currently applied by food microbiologists.  It follows, that Needham’s experiments were so badly planned and erroneously executed that it is impossible to say if his phials were inadequately heated or inadequately closed, or both.  Though, his scant description of heating practices leads to be inclined to believe that phials were inadequately heated, with very high probability. Needham only observed that microscopic organisms were able to grow in his substrates; he proved nothing. Needham did not prove the spontaneous generation of microscopic organisms at all. Hence, the theory supported by Needham and Buffon was immediately and completely disproved by heat resistance studies done by Spallanzani. In absence of his results (heat lability of organisms of higher orders, and 45 minutes boiling required to reach sterility) heat treatments of any size would be regarded as producing sterile ‘phials’, and the growing organisms seen after storage, would be argued to be generated spontaneously.
    Moreover, contemporaries epigeneticists did not understand the logic though unmistakable of such evidence. They didn't understand such logic nor ‘heterogeneticists’ nor ‘preformationists’ of the following century. They behaved themselves as if spontaneous generation would be still undisproved.

(3) - However, Spallanzani repeated the first Needham’s experiments, by taking the following precautions: (3a) he employed eleven infusions of vegetables (kidney beans, lentils, broad bean, peas, vetches, buckwheat, barley, maize, seeds of hemp, seeds of mallows and seeds of beet) in flasks which were hermetically sealed by flame melting their neck, thus removing the chance of environmental organisms to enter the already sterilized flasks (the phenomenon food technologists now call ‘post process contamination’) (Lopez, 1987); (3b) he boiled the flasks for one hour, to ensure that all organisms were killed, and so achieving the flasks sterility. Following storage at room temperature, Spallanzani found that all flasks were devoid of surviving organisms. They were all sterile. And sterility was maintained throughout extended storage, inasmuch the spontaneous generation did not occur in any of the nineteen infusions. As a matter of fact, sterility and spontaneous generation are biological conditions absolutely antithetical, each other incompatible, mutually excluding, because sterility could never be reached nor maintained, obviously, if microorganisms of whatever kind should appear anew from inanimate, sterile organic matter.  Then Needham’s theory was disproved once more.

 (4) - Notwithstanding, it could be argued that microscopic organisms were unable to grow in flasks boiled one hour, because the prolonged heating affected too much the head space atmosphere of flasks. In effect, Needham claimed that spontaneous generation did not occur in Spallanzani’s flasks because ‘the air closed inside flasks was extensively jeopardized in his elasticity by fumes and hotness of fire ‘ (Opuscoli, p.13). This objection was identified (see Pasteur, p.215-217) with a modified air quality, i.e.; the head space gas of closed flasks would have had reduced oxygen content, supposedly removed through heat induced infusions oxidation. However, as it is well known now, heat treatments do not cause oxygen to disappear totally from vacuum sealed container's head space, even when temperatures very higher than those used by Spallanzani are employed (Lopez, 1987; Hamblin et al., 1987; Bertoli, 1990; Whiting & Naftulin, 1991). In fact, it can be expected that the series of nineteen vessels Spallanzani boiled for one hour, lost some oxygen. But in the series of more than fifty flasks subjected to microbial heat destruction experiments (whose mouth was flame fused and stretched up to a capillary size and then flame sealed when cooled, in such a way as to preserve inside the flasks the environmental oxygen level close to 20%), the oxygen tension was very high instead, with high probability, after heat treatments. It is well known that spore forming bacteria (spores are the single cell types which can survive the high temperatures reached by Spallanzani’s flasks during heat inactivation experiments – see below) can develop in a wide range of oxygen tensions. For instance, the reference heat resistant anaerobic (growing in absence of oxygen) bacterium Clostridium sporogenes and the facultative aerobic Bacillus stearothermophilus are both able to growth in vegetables hermetically canned under vacuum and treated some minutes at 121.1°C (Richards, 1968; Stumbo et al., 1950; Pflug and Odlaug, 1978; Stumbo, 1983; Casolari, 1994). Doubtless, Spallanzani’s flasks only reached the lower temperature of 115-118°C, after 30-60 minutes boiling (see below); but they do not stand more than few minutes at such temperatures. It follows that head space gas composition on both sets of Spallanzani’s flasks were, with very high probability, at oxygen tensions suitable to the growth of more heat resistant surviving organisms, both anaerobic and facultative.

 (5) - To disprove Needham’s claims that Spallanzani’s flasks did not show any organism, because one hour boiling could have ‘widely weakened and perhaps destroyed the vegetative force of infused matter' (Needham’s claims, from Opuscoli, p. 13), Spallanzani subjected four sets of eight infusions – seven of different seeds and one of egg yolk - to 0.5, 1, 1.5 or 2 hours. He left the flasks open: ".. because if prolonged boiling weakens or destroys the power of infusions to support microbial growth, the same result can be safely expected to occur in open as well as in closed flasks." (Opuscoli, p. 16). Within few days, microorganisms appeared in all heated flasks, showing that the ability of heated infusions to support microbial growth was unaffected, even after two hours’ boiling. Besides, Spallanzani showed that infusions of even burned, toasted and carbonized seeds or yolk were able to support microbial growth. Thus, Spallanzani disproved even so the existence of the hypothetical 'vegetative force' claimed by Needham to be affected by prolonged heating. So he added further weight to his evidences against Needham’s theory.

 (6) - It was suggested that the low power microscope used by Spallanzani compared with that used by Needham might have caused the disagreement between the two rivals: “Spallanzani, not Buffon and Needham, was the technically handicapped party in this debate…” (Sloan, 1992). Nevertheless, Spallanzani wrote of having used both a single lens and a compound microscope, “made by highly skilled craftsmen” (Saggio, 1765). He devoted an entire page of his “Opuscoli..” (Opuscoli, p. 32) to claim that he had considerable experience with microscopy and he was well aware of the difference between what we now call the Brownian movement of particles (organic molecules seen by Buffon?) and those organisms ‘infinitely small’ (Opuscoli, p. 34) now called bacteria. As well known, in the 18th century the microscopes have a magnification in the range of 100-150 diameters (Stein, 1931; Sloan, 1988; Casida, 1976). As well known to microbiologist, bacteria of the genus Bacillus can be readily seen by using a current microscope set at 100x magnification, even without phase contrast. Casida (1976) demonstrated that bacteria like E. coli, Bacillus and Arthrobacter could be viewed with a Leeuwenhoek microscope (100-150 diameters; a copy loaned by American Society for Microbiology) as well as with a now available compound microscope without the condenser. It follows that disagreement between Needham and Spallanzani could not be ascribed to their microscopes, with high enough probability. Moreover, the results of Needham as well as Spallanzani’s experiments could be regarded as positive or negative on the basis of the simple observation of flasks with naked eyes, because sterile broths are usually clear, while those filled with growing organisms have about one million organisms/ml or above (broths containing full  developed bacteria hold a number of particles close to billions/ml) are cloudy. Further, cloudy solutions usually have additional characters revealing growth, as smell, gas bubbles, modified pH, etc. A suspension containing less than about 300.000 bacteria/ml is not cloudy if viewed with naked eyes, and bacteria are hardly viewed even with 100x-200x microscope, mostly if of primitive type. Hence, suspensions not cloudy could anyway regarded as sterile, independent of microscopic evidence.
Anyway, the results obtained by Spallanzani do not require microscopic proof, because they can be unequivocally interpreted on the thermo-microbiology basis. The infusions, closed in hermetic flasks of fixed volume (being the flasks of glass with flame fused mouth), probably reached temperatures of more than 100°C (220°F) after just a few minutes, since Spallanzani reports: “… after a few minutes boiling many jars began to burst …and to avoid the bursting of all jars .. [I was able to obtain] flasks of glass thickness more suitable to withstand the fire [high pressure]..”.(Opuscoli, p. 33). It is highly likely that during heat treatment, and mostly after 30 - 45 minutes of boiling, temperatures as high as 115°-118°C were reached, in line with the gas state equation: T = P*V/nR. Spallanzani was well aware of this: “water boiling in closed jars gains more heat than boiling in open vessels, as is well known to physicists”(Opuscoli, p. 34). It follows that temperature raised minute after minute up to 115°C at least, so that the organisms found in flasks heated less than 45 minutes, doubtless were vegetative rods derived from germinated bacterial spores, because only bacterial spores could survive such high temperatures (Stumbo, 1973; Casolari, 1988; Lopez, 1987).

(7) - Discussing the survival of organisms in flasks boiled 45 minutes, Spallanzani distinguishes two kind of organisms: the ‘animaletti’ (small animals) and ‘germs’. He believes that the function of the ‘germs’ is to generate ‘animaletti’ of the last order, what we now call vegetative bacterial cells. So, he seems to foreshadow the existence of bacterial ‘spores’, identifiable with the ‘germs’. Such idea seems very clear as he discuss the probability that organisms ‘of the last size’ could have penetrated boiled flasks, from outside. Being ‘animaletti’ of a certain size - he says - the ‘germs’ must have a certain size too; and since air, water, and smelling molecules – certainly of lesser size - did not penetrate inside flasks, neither ‘germs’ nor ‘animaletti’ penetrated the flasks. And more, since after 30 minutes boiling the ‘animaletti’ did not grow in metal cans welded with the same metal – though having more pores than glass – not even ‘germs’ penetrated the metal container. Afterwards, Spallanzani adds, ‘animaletti’ derive from ‘germs’ present inside the flasks, able to resist “some time to the violence of fire, but in the end dying.” (Opuscoli, p. 42). From the present viewpoint, such ‘germs’ doubtless were bacterial spores.

Therefore, Spallanzani proved that Needham’s experiments were completely erroneous, and his objections to Spallanzani’s ones, were without foundations. His investigations allowed him to reach conclusions still valid more than two centuries later. However, Spallanzani’s experiments did not persuade completely his contemporaries, and some scientist of the following century, so that spontaneous generation was questioned anew.

“By the end of the 1850s there was virtual unanimity in the Académie des Sciences in Paris that spontaneous generation did not exist.” (Harris, 2002). Though, a member of the Académie, F. Pouchet (1800-1872), director of the Natural History Museum of Rouen, presented (1855) to the Acadèmie ‘experimental evidences’ of spontaneous generation. In 1859 Pouchet published ‘Hétérogénie’, a book devoted to support the spontaneous generation.  In 1860 a series of contribution of Louis Pasteur (1822-1895) of completely opposite opinion appeared in the Comptes Rendus. In the same year, the French Académie des Sciences announced the prize Alhumbert of 2500 francs to whoever would shed new light ‘by well conducted experiments on the question of the so-called spontaneous generation.’ A season of experimental approaches to check the generation theory was re-opened. The question to be answered was of ‘proving the negative’ (Pasteur, p.  295): “one cannot prove ‘a priori’ that spontaneous generation does not exist.” (Pasteur, p. 354). It can only be proved that spontaneous generation does not occur under defined experimental conditions. In effect: ‘we do not know: we can only guess. . The old scientific ideal of episteme – of absolutely certain, demonstrable knowledge – has proved to be an idol. . Every scientific statement must remain tentative for ever.’ (Popper, 1991).
Pasteur undertook his experiments to check spontaneous generation, when he was 37 years old and ‘.. he had only just entered the study of biological problems’. (Narasimhan, 2001), as production of vinegar and ‘pasteurization’ of wines. One can't argue about how such circumstances affected Pasteur’s investigations. Though, his experiments about spontaneous generation were completely flawed.

Pasteur employed experimental conditions decreasing microbial growth probability in general and of organisms arising by spontaneous generation too, if any: he used almost invariably (1) a single culture solution (‘eau de levure sucrée’), (2) which was acid, (3) which was boiled for 2-3 minutes only (Pasteur, pagg. 188, 234, 235, 237, 249, 253, 260, 310, 313; i.e.: pasteurized, not sterilized) and (4) incubated aerobically (Pasteur, 188, 190, etc.).

A – Microorganisms with all gradations of complexity in their requirements for the recognized nutrients are known. Some grow either in neutral or in acid substrates; some in presence or in absence of air; at high or low water activity and osmotic pressure; in substrates of poor or complex composition; or in intermediate conditions. By using a single culture solution in almost all experiments (in a very exiguous series of trials Pasteur employed urine, sugar beet juice, infusion of pepper, milk or his solution after neutralization), he disregarded the highest probability of microbial grow which could be expected to occur by using a multiplicity of culture media, having different pH, water activity, osmotic pressure, chemical composition, etc. Even Needham employed different substrates: “upon three or four scores of different infusions of animal and vegetables substances..” (Needham, p. 639); Spallanzani subjected to his experiments up to nineteen different types of infusions. Experiments of this kind, set out to prove if organisms of a wide as unknown physiological requirements can grow in ‘dead organic matter’, can not be carried out with a single culture medium. The minimum experimental condition required to employ a certain number of ‘general purpose’ substrates, namely meats, eggs, different kind of vegetables, etc.

(B) – “The control of pH, temperature and oxygen supply, is critical with every bacterial culture “ (Costilow, 1981); “Hydrogen ions concentration is among physico-chemical factors of major concern affecting microbial growth.” (Casolari, 1989); “.. there are always at least three factors controlling microbial growth: the pH, the water activity and temperature.” (Robert & Jarvis, 1983). Almost all microorganisms are able to growth in the so-called ‘general purpose media’, having neutral pH and intermediate oxygen tension. Only few types of organisms can grow in acid substrates. The single solution Pasteur employed in nearly all experiments was acid (Pasteur, pp. 233, 247, 248, 250, 350, 357) and incubated aerobically. Without doubt the acidity of the culture solution restricted microbial growth to yeasts and moulds, the microorganisms most fostered by acidic and aerated substrates. Indeed, nearly all observations reported by Pasteur show that yeasts and moulds prevailed among the microscopic organisms growing in his solution. Pasteur gives a detailed description of the organisms that he sees in his flasks (“.. bacterium,.. penicillium, des ascophora, des aspergillus..” (Pasteur, p. 189), “.. les mucoracées, les torulacées, les mucédinées..” (Pasteur, p. 244) and illustrates them. So, they include almost exclusively yeasts and moulds. At room temperature yeasts and moulds grow more slowly than bacteria. Thus, the prevalence of yeasts and moulds in Pasteur's solution provides further evidence of the low pH of his solution. Table 1 shows some of the microorganisms expected to be unable to grow in acid solution, as prokaryotes and eukaryotes unable to grow in environments of pH lower than 4 (the pH of the Pasteur's solution is not known); those preferring pH values close to neutrality; and microorganisms preferring anaerobic conditions. Acid environments are not privileged not even by eukaryotes of the protozoa type (Tremaine and Mills, 1991; Baldwin and Campbell, 2001). Thus, by using an acid culture solution, Pasteur chose to exclude the greatest part of microorganisms from growing – spontaneously or not – in his flasks.

(C) - The fact that Pasteur’s solution was acid is important, because the use of acidic (low pH) solutions to test the growth probability of the widest range of microbial organisms is wrong on principle, since: " pH is the most important factor that determines the degree of thermal processing needed to achieve product stability because of the inhibitory effect of acidity on survival and outgrowth of microorganisms." (Lopez, 1987). Pasteur always ‘stabilized’ his solutions – that is he obtained solutions devoid of organisms able to grow in his acid solution - by 2-3 minutes boiling (in practice, a pasteurization treatment). Pasteur achieved such a result, because both microbial heat resistance of microorganisms in general is low at low pH, and the acidophilic microorganisms, and mostly yeasts and moulds, are very easily killed by heat (i.e.: by pasteurization). Moreover, protozoa too do not survive heat treatments at temperatures higher than about 70°C (Rose and Sifko, 1999; ICMSF, 1999; Fujino et al., 2002).

(D) - Pasteur disregarded the nearly fifty years of experience accumulated by Appert (1810) who successfully sterilized a variety of products in hermetic containers by heating them for several hours (that is, according to Spallanzani’s figures). He carried out occasional experiments using milk and its usual acid solution previously neutralized with calcium carbonate, showing that higher temperatures (at least 112°C = 233.6°F) were required to keep them from spoiling: “weak acid infusions require a treatment at 100°C or less only, while neutral or weak alkaline liquids must be treated – as milk – at temperatures higher than 100°C.” (Pasteur, p. 351, and 357, 208). Therefore, Pasteur was aware of the different results obtainable by heating ‘weak acid liquids’ and neutral or alkaline liquids. He observed that the organisms that grew in milk and in his solution after neutralization were different from those that grew in his still acid solution. So he understood that neutral media require stronger heating (about 45 minutes boiling) than acid ones (2-3 minutes), because a greater number and type of organisms are able to grow in neutral media and possibly organisms of high heat resistance. And certainly, he understood that since more organisms can grow in neutral media, then it was in neutral media that the chance of organisms to grow, spontaneously or not, would have been tested. Notwithstanding, Pasteur chose that his acid solution was more suitable to his kind of experiments, and when M.V. Meunier said that Pasteur’s experiments can be explained by the nature of culture medium he used, Pasteur replicates “I believe it surely: such is a result belonging to me, and that I vindicate.” (Pasteur, p. 350). Summing up, since it is unbelievable that Pasteur did not realize that the sterility - of neutral media - was the single feasible and maintainable condition required to prove that spontaneous generation do not occur in common environmental conditions, why he chose to continue his experiments with his acid solution? Perhaps his position was ideological? He was religious, and spontaneous generation was considered a kind of materialism result (Harris, 2002). Certainly, he was well aware that the Académie des Sciences was unanimously adverse to the spontaneous generation doctrine.

(E) - Pasteur said: “By boiling I killed microorganisms that may have been  present inside the liquid as well as those on the inside surface of the vessel.” (Pasteur, pp. 341, 343). “.. the boiling practice destroys the ‘germs’ that substances or flasks carried out in the solution.” (Pasteur, p. 225). Nevertheless, he never demonstrated this experimentally. To show the killing effect of heat over the microscopic organisms, Pasteur ought to have shown that microscopic organisms were decreasing, with the increasing heating time. Spallanzani did it, showing that just 45 minutes boiling in hermetic flasks were necessary in the end to kill all organisms (to achieve sterility).

(F) – Pasteur very often obtained ambiguous results, since only a fraction of the experimental flasks of the same series showed the expected results: “Very often, within few days the solution spoils.. frequently it occurs.. that solution remain absolutely intact (without mucedinées et torulacées)..” (Pasteur, p. 199); “it often occurred that solution remained absolutely clear, without organisms,..” (Pasteur, p. 267)  “a number of flasks was not spoiled by any ‘animalcules’, or mould... (Pasteur, p. 344) and “.. eleven flasks out of twenty were without micro organisms,” (Pasteur, p. 203); “.. nineteen out of the twenty open close to the Mer de Glace (Mont Blanc)..” (Pasteur, p. 345); “.. fifteen out of twenty among those opened at the Jura Mountain, were without organisms” (Pasteur, p. 203, 345); “twelve out of twenty were without of organisms..” (Pasteur, p. 345);  “ten out of thirteen with organisms..” (Pasteur, p. 204, 345) ".. three out of thirteen..” (Pasteur, p. 345). Pasteur claimed that these negative results were proof that spontaneous generation does not exist; otherwise organisms should have filled all flasks, whereas positive flasks were contaminated by airborne organisms. Competitors, however, claimed that organisms grown in the positive flasks were from spontaneous generation. They said that if contamination had been aerial, all the flasks had been contaminated, since even the smallest quantity of air contains microorganisms. Pasteur claimed that a higher or lower airborne contamination level in different locations or situations (e.g. over Mont Blanc, during rainy weather, etc.) was the cause of the positive and negative results he obtained. In fact Pasteur’s assumption was right, although he never proved it by counting microscopic organisms in different locations / situations. It was not until approximately 20 years later that Koch conducted such counts of microorganisms (Salle, 1961). Pasteur also found that even flasks with twisted necks were sometimes not devoid of microbes: “I never said flasks with twisted necks succeeded one hundred percent.” (Pasteur, p. 351). His Competitors claimed that positive flasks with twisted necks were proof of spontaneous generation and negative flasks were caused by the defective arrangement of experimental conditions.

(G) – Pasteur write “Gas, fluid divers, électricité, magnétisme, ozone, choses connues et choses occultes, il n’y a absolument riens dan l’air atmosphérique qui ..soit la condition de la putrefaction ou de la fermentation des liquides ..“. (Pasteur, p. 263, and 191). But he only proved that oxygen does not affect the growth of microscopic organisms in his solution. He never demonstrated that different fluids, electricity, magnetism, ozone – and even less the ‘unknown things’ – affected microbial growth.  The statement that even ‘unknown things’ are not involved in biological phenomena of his flasks, it seems quite an ideological position.

(H) – Seemingly, Pasteur performed his experiments to prove that microorganisms epigeneticists thought to grow spontaneously, actually come from dust carried in the air: “All progress of my work is as follows…I showed that a boiled infusion..is spoiled merely by falling solid particles that are always carried in the air.” (Pasteur, p. 310).  At the same time, Pasteur said that “Needham, Schwann, Schulze and Schroeder demonstrated only the existence in the air of an unknown principle affecting the life of infusions.. so they claimed that this principle was necessarily a kind of ‘germs’, while they do not have any proof of their opinion..” (Pasteur, p. 222). (Needham, really, demonstrated anything about). But the same reasoning could be referred to Pasteur too. He only piled up a number of observations of the same kind of his competitors, that is suggesting that the ‘quid’ present in the atmosphere and destroyed by heat – or filtered on cotton wool - were, with high probability, the microscopic organisms growing in his solution. However, it was a common knowledge that “.. almost all naturalists acknowledged..  the old hypothesis of aerial dissemination of ‘germs’..” (Pasteur, p. 304; see also p. 205).  “.. everyone acknowledges that the smallest quantity of air inoculated into infusions, gives rise within few days to ‘mucédinées ou infusoires.” (Pasteur, p. 198). His own words were: “ When organic substances of infusions are heated, they become populated by infusorians and moulds... they must come solely from air .. do exist microscopic organisms in the air? Nobody deny it, because it is well understandable that it may not be otherwise.” (Pasteur, p. 225). In the following pages of the same chapter II, Pasteur says: “There are always, in the ordinary air, organized corpuscles very similar to the ‘germs’ of the organisms of lower order.” (Pasteur, p. 238)   Nevertheless, Pasteur itself was not sure about the true nature of such particles, nor if they were really ‘fertile’. And he wrote: “What should have been better to do, and more directly (to recognize the nature of such particles) it would have been to follow at the microscope the development of those ‘germs’. It was my intention; but the instrument I projected for the purpose did not reach me in due time.”  (Pasteur, p. 233). Then Pasteur saw microscopical particles ‘very similar to the germs’ in dust collected from air, but he was unable to prove ‘at best’ neither their nature nor viability.

Concluding remarks

    Needham’s experiments were flawed just in the outgoing, because he claimed of having destroyed ‘animalcula’ in his heated flasks, without proving it experimentally; nor he proved that his flasks were truly hermetic. Failing such control measures, fundamental to his experiments, both observations he done and the theory given thereof were flawed.
    Pasteur tried to show – but unproved it - that: (1) with some probability (see ‘ambiguous results’, section F) microorganisms able to grow in his acid and aerated solution (moulds and yeasts) were carried out into flasks from the open air; (2) probably often – but not always (see ‘ambiguous results’, section F) the spontaneous generation of yeasts and moulds did not occur in his single acid solution, stored in the presence of atmospheric oxygen. Anyway, with regard to spontaneous generation in general, Pasteur’s results cannot be extended to more commonly occurring environmental conditions, such as substrates with pH close to neutrality, with different oxygen tensions as it occurs in decaying organic matter, and anyway in ‘general purpose’ substrates allowing growth of all types of microorganisms. So, Pasteur’s experiments were technically and methodologically interesting, but were unsuitable to disprove the spontaneous generation.
    Spallanzani showed that microorganisms never arise from sterilized organic matter, even under the most suitable environmental conditions. He showed that organisms of “maximum, intermediate and small size” (protozoa and algae) are very easily killed by heat (within half a minute at boiling) whereas those “endlessly small” (doubtless spore forming bacteria) are very heat resistant. Sterility is reached after 45 minutes boiling in hermetic flasks and preserved during storage. He proved it using up to nineteen infusions. He showed that vegetables and egg yolk he employed would have supported microbial growth though after severe heat mishandling. At the same time, he proved that the ‘vegetative force’ hypothesized by Needham does not exist by using a variety of infusions. Spallanzani repeated Needham’s experiments and proved that they were wrong, using up to nineteen different substrates with pH close to neutrality, truly sterilized and very hermetic. His infusions, sterilized in hermetically sealed flasks, were kept sterile while at different head space oxygen concentration. He employed both a large number of flasks and repeated experiments several times, thus satisfying the highly valued statistical side of the scientific method. It follows that Spallanzani’s experiments, from the current micro biological viewpoint, were highly probative against both Needham’s experiments and the spontaneous generation theory. It follows that on the basis of "the probative value of the experiments themselves" (Harris, 2002) Spallanzani clearly and incontestably disproved the spontaneous generation of microorganisms a century before Pasteur.
 

                        A. Casolari

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Table 1.  pH and red-ox affecting microbial growth (Singleton & Sinsbury, 1995; I.C.M.S.F., 1996)

Microorganisms unable to grow at pH <= 4 : Acidaminococcus sp., Aeromonas sp., Azotobacter sp., Bacillus alcalophilus, B. amylolyticus, B. anthracis, B. brevis, B. cereus, B. circulans, B. firmus, B. licheniformis, B. megatherium, B. mycoides, B. pumilus, B. sphaericus, B. thuringiensis, B. stearothermophilus, B. subtilis, Campylobacter jejuni, Chromobacterium sp., Clostridium sporogenes, Cl. botulinum type A, B, and F,  Cl. perfringens,  Derxia sp., Desulfobacter sp., Desulfosarcina sp., Desulfuromonas sp., Erwinia carotovora, Jantinobacterium sp., Leuconostoc cremoris, Listeria monocytogenes, L. enterocholitica, Micrococcus sp., Micrococcus lysodeikticus, Proteus vulgaris, Pseudomonas aeruginosa, Rhizobium sp., Salmonella choleraesuis, Serratia marcescens, Streptococcus faecalis, Streptococcus lactis, Vibrio parahaemolyticus, Yersinia, Xanthobacter sp.; Schizosaccharomyces octosporus, Halomyces spp. (4,5)
Microorganisms having optimal pH close to neutrality (pH values are between brackets) :  Bacillus cereus (6 - 7), Bacillus spp. (6-7), Brucella sp. (7,3 - 7,5), Campylobacter (6,5 - 7,5), Clostridium botulinum (7,2), Clostridium perfringens (7,2), Escherichia strain (6 - 7), Listeria monocytogenes (7,0), Plesiomonas sp. (7,0), Salmonella sp. (7 - 7,5), Staphylococcus sp. (6 -7), Streptococcus sp. (7,0), Vibrio colerae (7,0), Vibrio parahaemolyticus (7,8 - 8,6), Vibrio vulnificus (7,8), Yersinia enterocolitica (7,2); Aspergillus flavus (5 - 8), Aspergillus parasiticus (5 - 8), Fusarium sp. (5 - 8), Penicillium citreonigrum (5 - 6,5), Penicillium citrinum (5 - 7), Penicillium islandicum (5-9), Penicillium verrucosum (6 - 7).
Some prokaryotes and eukaryotes preferring low red-ox: Actinomyces, Bacteroides, Carpediemonas, Clostridium, Desulfovibrio, Desulfotomaculum, Desulfuromonas, Desulfobulbus, Didinium, Diplomonads, Entamoeba, Enteromonads, Neocallimastix, Nictotherus, Parabasalia, Piromyces, Propionibacterium, Psalteriomonas, Retortamonads, Sarcina, Selenomonas, Spiroduceus, Succinivibrio, Trichomonas, Stentor, Trimastix, Veillonella, etc.