The distribution of microscopic organisms in three samples of pond water taken at different depths.
Introduction
About 350 years ago, lens grinder and scientist Anton van Leeuwenhoek peered into his microscope
at a sample of pond water and discovered a whole world of creatures too small to see with the unaided eye.
He thought these creatures were animals the size of molecules, so he termed them “animalcules”. Later
scientists have determined that these animalcules are not animal at all but are mostly one-celled organisms of
the kingdom Protista. The protists that swim or crawl around in pond water are considered “animal-like”
protists and are grouped together as protozoa, which means “early form of animal”. Other common
microscopic organisms are considered “plant-like” and are classified mostly as algae. To scientists,
microscopic organisms are often just called microbes, but microbes can also refer to viruses and bacteria,
which are far too small to see with the compound light microscopes used in this investigation.
The purpose of the current investigation was to peer into van Leeuwenhoek’s world of protists and
determine if the microbes were even distributed within a sample of pond water. The hypothesis was if a
sample of water was taken just under the surface of the pond, then there would be a greater number of
protists present than there would be in samples at mid-depth or touching the bottom of the pond because of
the oxygen and light near the surface. The independent variable is the depth at which the pond water sample
was taken and the dependent variable was the number of microbes seen under the microscope.
Materials and methods
A sample of pond water was collected in an approximately 12” x 16” x 6” plastic container. The
sample was intentionally collected to mimic a real pond with pond weeds and algae in the middle of the
sample and a thin layer of detritus and mud on the container bottom. When allowed to settle, the sample had
clear water throughout except for the pond weeds and the bottom muck. Using a plastic pipette, a small
sample of water was sucked up from immediately underneath the surface of the sample and a drop
approximately ½ the diameter of a dime was placed on a clean glass microscope slide. A clean glass cover
slip was placed over the drop and the slide was studied under a compound light microscope starting at low
power (40x magnification). Higher magnification was used only to confirm suspect “dots” to see if they
were microbes or not. Otherwise low power was used for all scanning of the slide. For the purpose of this
investigation, a microbe was counted if it was moving or had an obvious “animal-like” appearance. Because
many plant-like microbes are difficult to separate from the muck, they were intentionally ignored.
Additionally, looking for moving objects greatly speeds up the process of searching the slide. Data from
each slide was collected as a raw count of microbes from the entire slide. Subsampling or estimating was not
needed due to the low numbers of organisms encountered.
A second sample of water was taken with a rinsed pipette from a depth approximately halfway down
from the surface, taking care to sample in the open water away from any noticeable pondweeds. A drop of
the same size as the first sample was applied to a cleaned slide, observed with the microscope and its
microbes were counted following the same criteria outlined above.
The third sample was collected with the pipette tip placed in contact with the detritus layer on the
container bottom. Care was used to minimize disturbing the detritus prior to sucking it into the pipette. The
sample was placed on a clean slide and counted similar to the first two samples.
Results
The sampling revealed that the microbes were not evenly distributed in the pond water sample. The
sample taken just under the surface had two microbes, the sample in the mid-depth had five microbes and the
sample taken in the bottom detritus contained 12 microbes. This data is also presented in the graph on the
following page.
Discussion
The hypothesis that the greatest number of microbes would be located just under the surface was not
supported in this investigation. The greatest number of microbes was found in the bottom sample. In the
hypothesis it was stated that the light and oxygen at the surface would attract a greater number of microbes
but based on these results, that does not seem to be the case. It seems likely that the detritus at the bottom
probably is a food source for the microbes, thus more microbes are found there.
There are some reasons to believe in the validity of this experiment. The results are not very close;
the surface sample had only two microbes compared to 12 in the bottom sample. Additionally, the results do
make sense, based on needs of living things. Microbes, just like any other organism, will be close to a food
source. Since the experiment was replicated only once, it is hard to have complete confidence in the validity
of the results. Repeating the same experiment several more times would increase the confidence in the result
if a similar pattern in the data was observed. The sampling pipette was difficult to use consistently. The
drops that came from it differed slightly each time, which could change the number of microbes placed on
the slide. If a pipette that gives a carefully measured drop was used, more confidence in that portion of the
experiment would result.
The same experiment could be tried with water from other ponds. Perhaps ponds that are deep
enough to have reduced light or oxygen near the bottom would show a different distribution of microbes. In
these conditions the need for light and oxygen might be greater than the need for the food source found in
detritus and more microbes might be concentrated near the surface. Samples could also be taken from middepth very near the pond weeds that were present in the sample container. The pond weeds may be the
“perfect habitat”—food, oxygen and light—perhaps even better than the sample from the bottom. A future
experiment should be conducted to investigate this possibility further.
Number of microbes observed at three sample depths of pond water
14
12
10
8
# of
microbes
observed
6
4
2
0
surface
middle
bottom