As part of the BIO220 Microbiology course at Rose-Hulman Institute of Technology, our group conducted a study isolating and classifying various soil microorganisms with suspected antimicrobial properties.

1. Isolation and Classification of Soil
Microorganisms with Suspected
Antimicrobial Properties
Michael DeBrota [CM1612] and Olivia Penry [CM 1861]
BIO220 Microbiology | Dr. O’Connor
February 19, 2018

2. Abstract.
Soil is a complex ecosystem, with multiple distinct layers and thriving populations of
diverse microorganisms that act as nitrogen fixers and decomposers. Some microorganisms
present in soil have antibiotic properties, and are able to eradicate pathogens growing near
them. Through analysis, experimental plating, and biochemical testing of microorganism
colonies present in a collected soil sample found near Speed Lake on the Rose-Hulman
campus, it was possible to isolate and identify bacteria that exhibit antimicrobial
properties.
Introduction.
Selection of a collection site for the soil sample depended largely on assumptions regarding
the diversity of the microbial population at the site, and level of influence of human
behaviors such as landscaping and fertilizer use. A site on the bank of Speed Lake was
chosen, as it was in an area relatively free from fertilizer and close maintenance, and was
also close to a water source, making it an ideal bacterial growth area. The collected sample
was homogenized and diluted with water to make it easier to deposit on the agar plates.
Successive further dilutions were made from the original dilution with the goal of producing
plates with a countable number of colonies after incubation. This aided in the process of
calculating colony-forming units (CFUs) per gram. The counted colonies were analyzed for
their morphology, and a patch plate was made of the colonies from the first countable
dilution plate.
Given that the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and
Enterobacter species) were deemed too dangerous to work with, the isolated colony was
tested against safer ESKAPE relatives (Enterococcus raffinosus, Bacillus subtilis,
Staphylococcus cohnii, Escherichia coli, Acinetobacter baylyi, Pseudomonas putida, and
Enterobacter aerogenes). In this experiment, only Acinetobacter baylyi and Enterobacter
aerogenes were used.
The colony from the patch plate that was observed to exhibit antibiotic properties was
plated next to safe ESKAPE pathogen relatives (Acinetobacter baylyi and Enterobacter
aerogenes) to further confirm its antibiotic behavior. The isolated colony was then
characterized through a series of metabolic tests (motility, hemolytic activity, catalase,
triple sugar iron slants, Simmons’ citrate, mannitol salt agar), whose results were entered
into the ABIS Online database to help identify the species of the isolate.

3. Materials and Methods.
Materials.
The materials used in the experiment were as follows:
● Prepared 10% Tryptic Soy Agar (TSA) plates
● Spatula
● 50 mL conical centrifuge tube
● Sterile water
● Volumetric pipette
● Micropipette and tips
● Test tubes
● Hockey stick bacteria spreader
● 95% ethanol
● Bunsen burner
● Vortex mixer
● Analytical balance
● Weighing paper
● Inoculating loop
● Incubator
● Permanent marker
● PCR thermocycler
Methods.
Approximately 200 g of soil was collected from the site in a 50-mL conical centrifuge tube
using a spatula. The sample was collected with care to exclude small rocks or grass. A
balance and weighing paper were used to weigh approximately 0.5 grams of the soil into a
new 50-mL conical centrifuge tube. Using a volumetric pipet, sterile water was added to the
new soil tube up to the 50 mL mark. The tube was then capped and vortexed to homogenize
the sample into solution. 100 µL of the solution was deposited on a 10% Tryptic Soy Agar
(TSA) plate using a micropipette, and spread using a hockey stick sterilized using 95%
ethanol and a Bunsen burner. Four 1:10 serial dilutions of the master soil solution were
prepared, in four separate test tubes containing 0.5 mL of the previous dilution with 4.5 mL
of sterile water added to each. Each of these tubes were then vortexed, and 100 µL of each
dilution was spread plated on a new 10% TSA agar plate in an identical manner to the
master solution. The plates were then labeled and incubated at 37 degrees Celsius. After
incubation, the plates were inspected, and the colonies on the first countable serial dilution
plate were enumerated visually and characterized based on morphology. The amount of
CFUs per gram of soil was then calculated based on the number of colonies present.

4. A master patch plate was then created from the first countable serial dilution plate. A 10%
TSA agar plate was gridded and numbered with a permanent marker using a 32-box grid
template. 24 colonies from the dilution plate were then individually transferred to the
gridded plate using sterilized toothpicks. This patch plate was incubated at 37 degrees
Celsius.
To test colonies for antimicrobial properties, two different top agar solutions were
inoculated with safe ESKAPE pathogen relatives, one with A. baylyi and one with E.
aerogenes. Each top agar solution was poured onto its own new 10% TSA agar plate, and
the inoculated agars were allowed to dry. The plates were then gridded and numbered with
a permanent marker, and 18 colonies from the master patch plate were individually
transferred to each plate with sterilized toothpicks. The ESKAPE patch plates were both
incubated at 37 degrees Celsius and observed after a day to determine if the isolates had
demonstrated an antibiotic effect.
A colony that successfully showed antimicrobial properties was selected for biochemical
testing and sequencing. A standard PCR protocol was performed to amplify the 16S rRNA,
followed by gel electrophoresis to confirm amplification. A standard PCR clean-up was
performed and it was sent off for sequencing.
Results.
The colony morphology primarily consisted of colonies of circular form with convex
elevation and even margins. The second most abundant morphology present was colonies of
circular form with flat elevation and wavy margins. Two unique individual colonies were
also present: one being a colony of filamentous form and margin and flat in elevation, the
other having an irregular form with serrated/wavy margins and a very flat elevation.
45 colonies were counted on the 10-1 dilution plate. To calculate the number of colonies in
one gram of soil, the number of colonies was divided by 10-1 to find the CFUs on the
undiluted plate. Because the undiluted plate was a 10% TSA plate, the number was again
divided by 1/10, or multiplied by 10. To convert this number from CFU/mL to CFU/g, the
conversion factor of 50 𝑚𝐿/0.5 𝑔 was used. The overall calculation was as follows:
45 ∗ 10 ∗ 10 ∗ (50/0.5) = 450000 𝐶𝐹𝑈/𝑔
The viable cell count was calculated to be 450000 CFU/g.

5. Test Result
Gram Stain +
Hemolytic Activity (Sheep’s Blood) -
Triple Sugar Iron Agar Slants -
Mannitol Salt Agar (MSA) Media -
Catalase +
Motility +
Simmons’ Citrate
-
Figure 1. Metabolic test results for isolated colony. + indicates a positive result
and - indicates a negative result.
Figure 2. The isolate was positive for the motility test (left) and negative for the
hemolytic activity (center). The Gram stain was positive (right).
After using the ABIS online database and inserting the test results, one of the potential
isolates that ABIS listed matched with the test results and colony morphology with a
confidence level of 89%. The isolate was most closely identified as Viridibacillus neidei.
The sequencing of the isolate was inaccurate due to contamination by a strain of
Pseudomonas putida.

6. Discussion.
The experiment yielded an expected, countable number of colonies on the serial dilution
plate, indicating that the isolation and enumeration were successful. Possible sources of
error include the time the plates spent in incubation, as some colonies may not have grown
large enough to see and could have affected the number of CFUs per gram calculated.
Errors may have occurred when diluting the soil and water mixture. Because there were
many measurements to take, poor micropipette skills could have lead to a less accurate
viable cell count. When patch plating, microorganisms on both the source plate and the new
patch plate could have been easily contaminated due to air exposure every time the lids
were removed. The toothpicks used to transfer the cultures were originally sterile, though
some could have been exposed to air contamination due to the container being open for long
periods of time.
The colony morphology of the isolates had little variation. This was assumed to indicate
success in effectively isolating one singular species, or a group of closely related species,
from the soil sample. It was also possible that the source plate was not a valid
representation of the colony morphology of the undiluted plate’s cultures.
Determining that Viridibacillus neidei was indeed the identity of the isolate was based
entirely on the results of biochemical testing. This is because nucleotide sequencing of the
isolate was inaccurate due to contamination by a strain of Pseudomonas putida, which
became aerosolized and contaminated multiple plates. This rendered the sequence data
useless in identifying the isolate, because queries on BLAST showed only Pseudomonas
strains. Because the description of V. neidei in the ABIS Online Encyclopedia accurately
described the habitat, morphology, and microscopic structure of the isolate in question, it
was concluded that there was little error in the biochemical testing, and that the colony
morphology supported the conclusion.
References.
Holt, John G., and David Hendricks Bergey. Bergey’s Manual of Determinative Bacteriology.
Williams & Wilkins, 1994.
Small World Initiative: Research Protocols and Research Guide to Microbial and Chemical
Diversity Package.” XanEdu, Small World Initiative, www.xanedu.com/higher-
education/educators/custom-books-catalog/small-world-initiative-research-protocols-and-
research-guide-to-microbial-and-chemical-diversity/.