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Galileo had now an influential following, many of his pupils being themselves professors by this time and aiding in the spread of his ideas. But on the other side were very many more enemies, not only the professors who pinned their faith to Aristotle, but also the Jesuits who had supported Scheiner's claims, certain Churchmen who feared the progress of free thought, and many other people who objected to anything of the nature of an innovation. These now about the year 1612 seem to have decided to join forces and choose a new line of attack, being unable to disprove Galileo's facts and arguments by meeting him on his own ground. They raised the popular cry that the Church was in danger, and that Galileo's astronomical views were contrary to Scripture. The leading authorities at Rome did not initiate this. Several of them in fact, including the before-mentioned Cardinal Barberini, openly expressed their admiration of Galileo's work. But an Aristotelian professor suggested to the Grand Duke's mother, on a certain occasion when Galileo's pupil Castelli had been extolling his master's astronomical discoveries, that these discoveries were doubtless genuine, but that Galileo's inference that the earth rotated on its axis and revolved about the sun could not be true, as it was contrary to Scripture, which plainly states that God "made the round world so fast that it cannot be moved". Castelli objected to bringing the Bible into the question, but being challenged, he took up the position that the new views were not contrary to Scripture, winning over some of those present to his side, though the Dowager Duchess Cristina appeared unconvinced. It was in consequence of this that Galileo wrote his famous letter to Castelli, taking the stand that the language of the Scriptures is such as is suited to the intelligence of those for whom they were written, and that the interpretation must necessarily be revised in the light of new facts. He also maintained that the Copernican system raised no more difficulty than the Ptolemaic, when it was pinned down to verbal explanation of literal statements in the Bible. In short his view was that the Bible was intended to teach men morals, and not science, and that it could not be regarded as a court of appeal in scientific matters, many of its expressions being vague, and others misunderstood by commentators. He concluded with a discussion of Joshua's miracle, laying stress upon the fact that the important point was the prolongation of the hours of daylight, and that literal acceptance of the account in the Bible in its primitive language would involve absurdities in relation to the "primum mobile," which, according to the Aristotelian philosophers, carried the sun with it.

Castelli was delighted with the argument in the letter and circulated copies of it. But it provided just the handle that Galileo's enemies were seeking, in order to embroil him with the ecclesiastical authorities. Sermons were preached on both sides, the Copernican view being supported publicly by a Jesuit preacher in the Cathedral, for it must be understood that by no means all of the Jesuits were united against Galileo. A copy of the letter to Castelli was sent to the Inquisition with an unsigned denunciation of those who held the doctrine that the earth moves and not the heavens. This was of course aimed at Galileo though it did not mention his name, presumably because he was known to have influential friends at the Vatican. The Inquisition tried to obtain the original letter but failed to do so, and the copy was submitted to expert opinion, which pronounced that on the whole the document could not be said to belie Catholic doctrine. Accordingly fresh evidence against Galileo was sought, and it was alleged that his religious opinions were suspect, because he was in correspondence with Germans and others, possibly heretics, and was a member of the Academy "dei Lincei". Galileo heard that his letter to Castelli was being used to secure a condemnation of the Copernican doctrine, so he sent a copy to Rome to be submitted to Cardinal Bellarmine and other prominent men, promising to follow it with a fuller treatise, since the first letter was only written on the spur of the moment. His friends, including Prince Cesi, founder of the Academy "dei Lincei," recommended him to avoid theology altogether in his argument about mathematical and physical matters, but the advice was already too late. The promised fuller treatise, the letter to the Grand Duchess Cristina, would not affect the position, as the Inquisition was already preparing its attack in secret, based on the evidence already received. Galileo, not knowing how far things had gone, thought his best plan would be a personal visit to Rome, remembering, how well he had been received on his first journey thither. He went accordingly in December, 1615, but, though cordially received to all appearance, he soon found the task of defence a bigger one than he had expected, as he himself as well as the doctrines was being attacked. It might have been well for him to confine himself to the personal attacks as he might have succeeded in disposing of them. But he felt bound to persevere with his efforts on behalf of the Copernican theory, after obtaining apparently satisfactory assurance in regard to his own orthodoxy. His feeling of security did not last long, for in February, 1616, the Qualifiers of the Holy Office reported on two statements from Galileo's work on sun-spots. These embodied the Copernican doctrine that the sun is the fixed centre of the world; which they declared to be false and absurd philosophically, and formally heretical, the companion doctrine that the earth rotates and also revolves round the sun meeting with practically the same condemnation. Cardinal Bellarmine sent for Galileo in accordance with these findings and warned him of the error of these opinions. Galileo probably regarded the whole matter as a mere formality as far as he was concerned. At the same time the book of Copernicus himself and another setting forth the same theory were suspended "until correction," while a third work in which a Carmelite, Foscarini, argued that these doctrines were not contrary to the Bible, was prohibited altogether. It appears clear that Galileo was not asked to recant his opinions, and he actually obtained a certificate from Cardinal Bellarmine to this effect three months later, but something which happened at the citation in February had very far-reaching results. The plausible explanation is that the admonition of the Cardinal was noted in writing by a secretary, but that the Commissary of the Inquisition, who was present in virtue of his office, took upon himself to "dot the i's and cross the t's" by amplifying the Cardinal's admonition, and telling Galileo that he must not hold, teach, or defend the opinions in question; this officious comment was also entered in the minute, though Galileo, who was only concerned with the Cardinal's pronouncement, took no notice of it, and perhaps did not even hear it. His bow of acknowledgment to the Cardinal's statement seems to have been interpreted by the secretary as indicating acquiescence in the other matter also, Galileo's consent being noted accordingly in the minute. The probability of this view, which is practically that of Professor Favaro, the most diligent student of Galileo at the present day, is much increased by the fact that the minute was not signed. In the event, as we shall see, this weakness of evidence was ignored.

Galileo was assured by the Pope himself that his fears of continual persecution were groundless, but the Tuscan ambassador felt that Galileo was doing himself no good by staying on at Rome and arguing with people, so he gave a hint to his master, and Cosmo recalled Galileo to Florence.

While at Rome Galileo had found time to write to Cardinal Orsini a treatise on the tides, afterwards amplified in his great work on the two systems of the world, to which we shall refer later. He also made definite proposals to the Court of Spain upon a new method of determining longitude, which would be of great importance to a seafaring nation. Ever since his discovery of Jupiter's moons, with their frequent eclipses, he had kept in view the possibility of accurately predicting these phenomena, and had observed for six years with a view to perfecting his tables of their movements. His idea was that local time being determined by observing the sun at noon, the further observation of the time of one of these eclipses in Jupiter's system, compared with that given in the tables, would give the difference of longitude between the observer and the place for which the tables were computed. The negotiations with the Spanish Court led to nothing, owing partly to the difficulty of obtaining the observations on a moving ship, and to the absence of reliable chronometers to carry on the local time from noon till dark. The Grand Duke tried the method in the Tuscan Navy, but it was practically a failure.