"It would be a huge waste of our resources to take a few drams of the contaminated wine and dilute them into a vat of broth," Mendel said passionately, "I appreciate the theory, but we don't have enough broth as it is, and how would we sterilize that amount?"
They were arguing, as they had for the past hour, on the merits of various methods of diluting the bacterial rods to such an extent that they were widely separated from one another. So widely separated, the theory went, that when a small amount of solution was placed on the surface of the solid broth in a bottle, only one or two single rods would grow on its surface.
"But that would be the only way of ensuring sufficient separation between the organisms," Brother Matthew insisted, as he had done for some time. "If we don't take that precaution, how will we know if growth on the solid media comes from one, pure rod contaminant, or from a whole cluster, such as we saw under the microscope?"
"I agree," Mendel concurred, "that we must achieve sufficient dilution, but we don't know what the dilution factor must be, or have a method of determining when we have reach a satisfactory degree."
"That part is easy," Brother Joseph told him, "we must experiment by transferring samples of diluted wine to the surface of the solid broth at varying degrees of dilution. Some will be wasted, those at the wrong dilution factor, but if we use a wide enough range of dilution factors, we are certain to get close to the right one. We will then repeat the process but over a more narrow range and closer to the correct one."
"That would work," agreed Mendel, "but the problem still remains that, to do the various dilutions you suggest would cost us more broth than all the beef in Brno. Look!" He picked up some paper and did the calculations.
"If we use the new metric system of measure, and take one milliliter of contaminated wine, and only dilute it by a factor of a thousand, scarcely enough under the circumstances, we would have to add that one milliliter to nine hundred and ninety nine milliliters of clean, sterilized broth to achieve a one to a thousand dilution. That is a liter of broth. To take the dilution on by a factor of ten would require adding a milliliter of wine to ten liters of pure broth, and if we wished to dilute the wine by a factor of one hundred thousand, we would need ONE HUNDRED liters of broth - that is crazy!"
Put that way, even Klacel shook his head. "But do we need to dilute the wine that much?" he asked, reluctant to give up on his side of the argument.
To which Brother Gregory replied with a simple demonstration. He took a sample of the wine and placed it once more under the microscope. "Look," he said after focusing the instrument, "there is just one cluster of the bacterius organisms, count them, how many do you see?"
With a bad grace, Klacel began to count, but quickly lost his way after reaching a number in the hundreds.
"And that was only a tiny sample," Mendel said, and took another drop of the contaminated wine. This time he placed one drop into a small flask and added two cups of ordinary water. He swirled the flask for a moment and then put a drop of this new mixture onto a fresh microscope slide. "Now look."
Klacel looked again. This time he saw a lot fewer bacteria and the clusters were significantly broken up, in one set he counted about two hundred, and in an adjacent one only fifty. "That was a lot easier," he told his companions, but there are still too many of them."
"Huhh!" snorted Mendel, and without thinking he picked up the watered down flask of bacteria and withdrew about a milliliter. This he put into a fresh flask and once again added two cups of ordinary water.
.
"Now look," he said, once again preparing a microscopic specimen of his watery preparation.
This time Klacel became quite agitated. "This may be it," he exclaimed after searching for several minutes. "I only see single rods, no clusters, and the rods are well spread out from one another. If we were to place this sample on the surface of our solid broth, each of these rods would be able to grow on their own!"
"See," Mendel almost shouted, "to get that kind of dilution in sterile broth would take ..." His voice trailed off as he looked at the flask in his hand, "... over a hundred liters of broth ...er..."
Both his friends looked at him.
"Wait a minute," Klacel exclaimed, "what did you just say?"
"Er ... we would need a hundred liters of broth ..." Mendel said, uncertainty creeping into his voice.
"But how much water did you just use to achieve about the right dilution?" Brother Joseph asked, the pitch of his voice rising and the excitement showing through.
Mendel looked up from the flask he was holding and stared into the faces of the two monks. "You know," he said, starting to laugh, "it is getting late, and we are all tired. With a fresher set of minds we would have arrived at this solution much quicker."
But Klacel was not going to let him get off that easy. "What solution?" he demanded, also beginning to laugh as he worked out the answer.
With a grin Mendel drew out the answer to their problems on yet another of his dwindling supply of Abbot Napp's fine notepaper.
"Look, we start with a series of small containers, such as apothecary flasks, and place into them ten milliliters of water. We then sterilize all these flasks using the Appert method, so that nothing grows in them." Everyone nodded, so Mendel continued, "At the first step we take one tenth of a milliliter of contaminated wine and add it to one of these flasks of sterile water."
"That would give us a dilution factor of one into one hundred," shouted Klacel, unable to contain himself.
With similar enthusiasm Brother Joseph could not help himself, "And then if we take one tenth of a milliliter of that dilution and add it to another, separate sterile flask of water, we will dilute by yet another factor of one hundred, or ..." he calculated quickly, "... one hundred multiplied by one hundred times!"
"That could be enough for our needs," Brother Matthew thumped on the table.
"A small sample at that dilution placed on the surface of the solid broth would have rod bacterius organisms sufficiently far apart, they would grow in isolation from each other," Mendel finished for them.
Unable to contain themselves, the three scientist monks hugged on another in celebration.
"I think we have it!" Brother Gregory exclaimed at last, "Let's get started."
"Wait a minute," Brother Matthew said, wiping a tear from his eye, "Look at the clock, it is late. Why don't we wait until tomorrow. The dawn will bring a fresh perspective. You go and teach your classes, and Brother Joseph and I will start preparing more bottles of solid broth and many small flasks of sterile water in which to dilute the wine. We will perform this dilution experiment with clear heads tomorrow evening."
It was such a good suggestion the three friends accepted it at once, and they separated to their respective rooms, well satisfied with themselves.