Vintage Visits | Doubting Leeuwenhoek: Microscopical Observations in Pepper Water

In a previous issue of Nanographia, I reported on a dispute between Nicolas Hartsoeker and Christiann Huygens, which was captured in an exchange of letters between the two scientists in the Journal des Sҫavans in 1678. In one of the letters, Huygens references the microscopical observations of rainwater and pepper water made by Antonie van Leeuwenhoek using his famous simple microscope.

Leeuwenhoek’s original microscopic observations in pepper water are now considered to be the first known microscopical observations of bacteria. Pioneering discoveries often invite skepticism and doubts, and Leeuwenhoek’s unprecedented findings to the members of the Royal Society were no exception.

I decided to take a deeper dive into Leeuwenhoek’s pepper water experiments, making my own pepper water infusions following his methods and observations to compare notes with Leeuwenhoek.

A Matter of Taste

Leeuwenhoek’s original pepper water observations were received on October 9, 1676 by the Royal Society and published as an 11+ page article, in the Philosophical Transactions titled:

“Observations, Communicated to the Publisher by Mr. Antony van Leewenhoeck, in a Dutch Letter of the 9^th of Octob. 1676. Here English’d: Concerning little Animals by him Observed in Rain-Well-Sea- and Snow water; as also in water wherein Pepper had lain infused.”

Several pages into Leeuwenhoek’s article, a subheading appears with the title, “Observations of Water, wherein whole pepper had layn infused for several days”. Here, Leeuwenhoek describes his reasons for wanting to examine pepper water with his simple microscope, along with his pepper water preparation methods:

“Having several times endeavoured to discover the cause of the pungency of Pepper upon the tongue, and that the rather, because it hath been found, that though Pepper had lain a whole year in vinegar, yet still retained its pungency; I did put about 1/3 of an ounce of whole pepper in water, placing it in my Study, with this design, that pepper being thereby rendered soft, I might be able the better to observe what I proposed to my self.”

Preparing a pepper water infusion following Leeuwenhoek’s recipe is as easy as it sounds—add peppercorns and water to a container and then let them sit for a few weeks. And that’s what I did. I weighed 1/3 of an ounce (9.4 grams) of whole peppercorns, placed them in a 250 ml beaker, and filled the beaker with enough bottled water to cover the peppercorns.

After a short time, the water became discolored, turning a deep burgundy color. Like Leeuwenhoek, I monitored the water level over the new few weeks, and if needed, I add more bottled water:

“This pepper having lain about three weeks in water, to which I had twice added Snow-water, the other water being in great part having been exhaled.”

The term “exhaled” was commonly used in the 1600s and refers to the evaporation of water.

Leeuwenhoek also discusses his thoughtful precaution of not contaminating the pepper water with the snow water:

“I took about 2 ½ ounces of Snow-water, which was about three years old, and which had stood either in my Cellar or Study in a Glass-bottle well stopped. In it I could discover no living creatures.”

Using his simple microscope, Leeuwenhoek confirms there is nothing living in his supply of snow water.

Leeuwenhoek’s Observations in Pepper Water

Reporting over a series of many days, Leeuwenhoek describes the types of “animalcules” moving about under his simple microscope and estimates how many microorganisms are present in a small drop of pepper water:

“…I judged, that if a 100 of them lay one by another, they would not equal the length of a grain of coarse Sand; and according to this estimate, ten hundred thousand of them could not equal the dimensions of a grain of such coarse Sand.”

Leeuwenhoek’s claim to the number and size of the microorganisms contained in a single drop of his pepper water infusion raised some eyebrows at the Royal Society. Members of the Society had doubts about Leeuwenhoek’s observations and told him so in two separate letters. Leeuwenhoek followed up with a letter of his own to the Royal Society explaining how he arrived at these estimations.

Counting Sheep

In Leeuwenhoek’s follow-up letter to the Royal Society dated March 23, 1677, he explains how he arrived at the great number of organisms observed in a single drop of his pepper water infusion, first mathematically and then with an analogy of counting a flock of sheep.

I won’t get into the weeds on Leeuwenhoek’s mathematical reasoning behind his estimations, but I did find his analogy of counting sheep easier to understand:

“Tis true, my calculus is not, nor can be, so exact, as precisely to determine the number: But I proceed, as those do, who intending to number a flock of Sheep running confusedly one among another, make an estimate by the breadth of the front, and length of the sides of a flock, how great the number of Sheep may be.”

Here, Leeuwenhoek’s admits his method of estimating the number of organisms can never be completely accurate. Also, in this passage, you get a sense of mild frustration as he tries to convey quantitatively the large number of microorganisms moving haphazardly in a drop of pepper water.

In the same letter, Leeuwenhoek references a microscope only he has access to, which enables him to see a greater number and much smaller “animalcules” in the pepper water.

“Amongst other Spectators, I shew’d it to a not ordinary person…he saw more than a thousand living animals…he wondered much more, when I said, I saw in it two or three much smaller Animals besides, which did not appear to him, because I saw them by another Microscope, which I still reserve to my self alone.”

Leeuwenhoek’s reputation for secrecy was known within the scientific community and his special microscope for “his use alone” is just one example. His Royal Society’s colleagues were unable to replicate his pepper water infusion observations due to what scholars believe was Leeuwenhoek’s use of, at the time, an unknown contrast method, giving Leeuwenhoek a microscopical advantage unavailable to others.

Uncommon Ground

Clifford Dobell in his book “Antony van Leeuwenhoek and his Little Animals: Being Some Account of the Father of Protozoology and Bacteriology and His Multifarious Discoveries in These Disciplines” makes the supposition that Leeuwenhoek used dark-ground or darkfield microscopy as a contrast method to observe the smaller microorganisms he reported to the Royal Society. This would explain why members of the Royal Society could not replicate Leeuwenhoek’s observations in pepper water.

Below are two images from my pepper water infusions after soaking the peppercorns in water for several weeks. The first image was made using brightfield; the second was made using darkfield. Each image is of the same field of view.

Dobell points to a passage in an earlier letter from Leeuwenhoek to the Royal Society dated January 22, 1675, as supporting evidence that Leeuwenhoek sometimes used darkfield microscopy. In the letter, Leeuwenhoek assures the editor that his observations, this time of “globules in blood”, are real:

“Sir, be assured that my manner of observation has not, for aught I know, misled me and that the little glass tubes formerly used in my observations were only made immediately before use when I wanted to make a careful observation, and they were not allowed to touch by hand or finger on the spot.”

Here Leeuwenhoek also eliminates the possibility of seeing artifacts from his own fingerprints rather than small, microscopic structures. Dobell notes that Leeuwenhoek, who was a draper by trade, adds an analogy that perhaps only a draper could have come up with:

“…I can observe the globules in blood as clearly as if we saw with our eyes without the help of glasses the grains of sand on a piece of black taffeta.”

The black taffeta in Leeuwenhoek’s analogy represents the darkfield microscopy observations Leeuwenhoek routinely used. In the end, Robert Hooke repeats Leeuwenhoek’s pepper water infusion experiment and confirms his findings to the Royal Society, along with a follow-up letter to Leeuwenhoek in 1677.

Comparing Notes with Leeuwenhoek

Unlike Leeuwenhoek, I did not have the willpower to wait three weeks before looking at my pepper water infusion, peeking occasionally during the twenty-one-day soaking period of the peppercorns. After four days of soaking the peppercorns, I could see, using darkfield illumination, thousands of microorganisms moving about in my pepper water infusion.

I ran Leeuwenhoek’s pepper water infusion experiment several times, and each time recorded similar results to his observations: a vast array of microorganisms moving about in a single drop of pepper water.

Pepper of a Different Color

Revisiting the Huygens/Hartsoeker letters mentioned at the beginning of this article made me recall a passage by Huygens describing his use of different peppercorns for his pepper water infusions rather than the black variety used by Leeuwenhoek:

“A very small drop of water taken from a glass in which pepper has been left to soak for two or three days, being thus enclosed, appears like a large pond, in which one sees an infinite number of tiny little fish swimming. What I have observed in particular about this pepper water, to avoid repeating what has already been published in the Journal, is that not all types of pepper give the same kind of animals. Those from a certain pepper are much larger than those from others, whether this comes from the age of the pepper or some other cause that can be discovered over time.”

Huygens makes no mention of checking the water he used for his pepper water infusions to make sure it was free of living material, but since he referenced Leeuwenhoek’s infusion experiments, I assume he took the same precautions.

Huygens’ claims of observing different microorganisms when using different varieties of peppercorns for his infusions sounded intriguing, so I repeated Leeuwenhoek’s pepper water infusion experiment, but used “rainbow” peppercorns.

After soaking the rainbow peppercorns for several days in bottled water, they yielded similar microorganisms as those I observed in the black pepper infusions. I did not see any obvious difference as reported by Huygens. However, one significant difference with the rainbow pepper water infusions was a substantial amount of mold growth.

Below are images showing the abundance of mold growth over the top of each of my rainbow pepper water infusions. In one case, the mold growth began less than one week and eventually took over the entire surface of the rainbow pepper water infusion.

In one instance, the mold layer became so thick I was able to remove it whole. Below is an image of the removed mold layer compared to the thickness of a U.S. nickel. Perhaps this mold is what Huygens was observing in his pepper water infusions?

Getting a Read on Leeuwenhoek

Overall, I was surprised by how such a simple and inexpensive experiment brought together so many interesting facets of microscopy, and a good bit of science history, too. I was equally surprised by how few examples of Leeuwenhoek’s pepper water infusion experiments can be found online. There are loads of websites and video demonstrations of the common surface tension of water experiment, which uses ground black pepper and dish soap, but only one regarding Leeuwenhoek’s work with pepper water infusions, which was a journal article behind a paywall.

I was fortunate to have access to the complete collection of Leeuwenhoek’s letters compiled in a nine-volume set. Each book contains edits, illustrations, and annotations by a committee of Dutch scientists. Leeuwenhoek’s letters in this printed format are presented in Dutch on the left page, with an English translation on the right page. This collection of Leeuwenhoek’s letters runs from 1676 through 1699.

Finding the printed volumes of Leeuwenhoek’s letters can be difficult, though, and individual volumes are expensive. A more accessible source is the Lens on Leeuwenhoek website, which holds a complete downloadable listing of Leeuwenhoek’s microscopical correspondence.

With Leeuwenhoek’s writings in hand, you too can follow his line of discovery and compare notes with this pioneer of microscopy one letter at a time.