Tatum, Samuel

Culturing Microbes from a Winogradsky Column

Hypothesis: The addition of lactose in a Winogradsky column will provide favorable conditions that will
select for particular microbial growth; the added lactose column will select for heterotrophic microbes
that are able to metabolize lactose.

Methods: A soil sample was acquired in close The wet mount and gram stains were further
proximity to a pond. Water from the benthic evaluated with bright field microscopy.
zone in the pond was taken. Soil clumps were
broken up and homogenized. A container was Results: It was seen and is demonstrated in
filled to around half its volume with soil. A Figure 1 that growth occurred more rapidly in
sulfur source and carbon dioxide source were the added lactose column. The images in
mixed in with the soil (about 1 to 2% of the final Figure 1 was taken after two weeks of growth.
mass). Then an equal volume of cellulose was This was concluded from the film at the water-
also mixed into the container. The water soil interface. There was a slight color change
sample taken from the benthic zone of the in the soil (from a red to reddish grey) in the
pond was mixed into the container. The lid was lactose added column; however, the image
placed on the top of the container a quarter of exaggerates this trend.
a turn from fully tightened. This process was
repeated to make a one control and one
deviation for comparison. The deviation
considered the addition of 2 grams of lactose
added to the soil with the other nutrients. The
columns were placed a few inches away from a
low heat/high intensity light source (less than
60 watts). Every week the columns was
considered for changes and contrasted.

From the columns depicted in Figure 2, 100

microliter samples from the top of the water
column and from the benthic zone were taken
and serially diluted (10-1, 10-2, and 10-3 mL). The
three dilutions were plated onto TSA and
wrapped in parafilm. Plates were allowed one Figure 1. Winogradsky column control (left) and
week incubation at room temperature. Also, column with 2g added lactose (right) after 2
100 microliter samples of the water column weeks
and benthic zone were taken to conduct a
Gram stain and a wet mount of each sample.
Tatum, Samuel

Figure 2. Winogradsky column control (left) and

column with 2g added lactose (right) after over
a month of incubation

In Figure 3 samples from the Winogradsky

benthic zone and water column from the lactose
containing column were compared. A greater
presence of microbial growth was seen in the
benthic zone of at the soil water interface
compared to the water column. Diversity of
microbial life was also compared between the
benthic zone and water column; however,
conclusions about which region produced
greater diversity was unclear.
 Tatum, Samuel

Figure 3. Bright field microscopy at 1000x magnification of organisms from Winogradsky benthic zone
(left) and water column (right)

Figure 4. TSA plates from 10-1, 10-2, and 10-3 mL dilutions (from left to right, respectively) of lactose added
samples taken from the water column (top) and benthic zone (bottom)
 Tatum, Samuel
Figure 5. TSA plates from 10-1, 10-2, and 10-3 mL dilutions (from left to right, respectively) of samples
(control) taken from the water column (top) and benthic zone (bottom)
Discussion: It was seen after over a month of
growth, the Winogratsky column with added
lactose produced significantly different growth
when compared to the column with no added
lactose. The lactose addition promoted
favorable growth for a specific type a microbe
(one which turned the water black). It is
hypothesized based on evidence that the
microbes responsible may be sulfur reducing
bacteria. The reduced sulfur and iron in the
solution react to darken the water. In order to
confirm this, the water column should be tested
for sulfur content before and after the color
change.
For future evaluation in order to more closely
monitor the changes in the column less lactose
should be added. Using half the lactose as used
in the methods may give an interesting
comparison. Since such a significant response
was shown by the sulfur producing bacteria the
addition of lactose may be a useful method of
selecting for sulfur reducing bacteria for
particular applications.
Distinct differences were seen between the
plates taken from the control and the lactose
added column. In comparison the plates with
lactose added seemed to grow microbes that
grew more densely. With only one exception,
the lactose added microbes spread completely
across the plate and formed thick excretions.
Perhaps these microbes had a higher metabolic
rate than those in the control sample. It could
also be that the microbes in the control were
not as equipped to utilize the TSA plate
 Tatum, Samuel

nutrients as the microbes from the lactose plate. This sort of inhibition was not seen in the
column. Or it may be that the microbes from plates from the lactose sample. This may be a
the lactose column are anaerobic (more so result of the lactose column having microbes
than the control) and their secretions are to that release similar products.
reduce O2 contact.
It should also be noted that the microbes from Reference
the lactose column were more homogenous
than those of the control. Particularly in the Rogan B, Lemke M, Levandowsky M, Gorrell
water column 10-2 mL sample of the control, it T. Exploring the sulfur nutrient cycle using the
was seen that antibiotic were produced by one Winogradsky column. Am Biol Teach.
colony that inhibited the larger colony on the 2005;67(6):348–56