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
Jon-Michael Beasley, Bailey Craig, Michael DeBrota, Jake McClure
Introduction
Materials and Methods
Results Conclusions
References
Rose-Hulman Institute of Technology, Terre Haute, Indiana
Antibiotic resistance is an increasing problem in the areas of public
health and pharmaceuticals. To combat this resistance, research is
constantly being conducted in an attempt to discover novel antibiotic
compounds. Some naturally-occurring substances, including
microorganisms, have been found to exhibit antimicrobial properties. In
our experiment, closely modeled after the protocols of the Small World
Initiative: Research Guide to Microbial and Molecular Diversity,
microorganisms were isolated from soil samples taken on the Rose-
Hulman Institute of Technology campus, tested for antimicrobial
properties, and classified.
Materials
● 10% TSA agar plates
● Inoculation loop
● Bunsen burner
● PCR Thermocycler
● Microcentrifuge
● Gram staining materials
● Stereomicroscope
● Biochemical testing materials
Methods
Five different soil samples were collected from various locations on the
Rose-Hulman campus. These soil samples were serially diluted and
plated on 10% TSA plates. For each soil sample, 32 colonies were
selected from the first countable serial dilution plate and grown on a
patch plate. A new patch plate was created from the original, and a top
agar inoculated with a safe ESKAPE pathogen relative was poured
over the new patch plate. The new patch plates were then incubated
and inspected for zones of growth inhibition of the ESKAPE pathogen
relative, indicating which colonies exhibited antibiotic properties. Four
of the antibiotic-producing isolates were chosen for classification. PCR
was performed to amplify the 16S rRNA gene and gel electrophoresis to
confirm results. The isolates were classified by Gram staining,
microscopy, biochemical tests, and sequencing of the 16S rRNA gene.
Isolate 1 2 3 4
Gram Stain + - + -
Hemolytic Activity (Sheep’s
Blood)
- +Beta
Triple Sugar Iron Agar
Slants
- +(all)
MSA Media + +
Catalase + +
Oxidase + - - -
Motility - - + -
Simmons’ Citrate - -
EMB Media - -
Indole - - -
MacConkey’s Media - -
Isolate Bacterial Morphology
1 Circular, Flat
2 Coccobacillus
3 Bacilli, Central spore
4 Filamentous
Figure 4: The bacterial morphology was
determined by examining each isolate.
http://www.tgw1916.net/bacteria_logare_desktop.html
http://www.microrao.com/identify.htm
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/.
Figure 1: Gel elctrophoresis
reveals PCR was successful for
isolates 1 and 2.
Isolate Identification
1 Viridibacillus neidei
2 Acinetobacter baumannii
3 Bacillus cereus
4 Shigella sonnei
Figure 2: Isolates 1 and 3 (first and third images) were confirmed to be gram-
positive after a successful Gram stain, indicated by purple coloration. Isolates 2
and 4 (second and last images) were confirmed to be gram-negative, indicated by
pink coloration.
Figure 3: Table of biochemical test results for each soil isolate.
A combination of Gram
staining, viewing the bacteria
under a microscope, and
biochemical testing were useful
in classifying the antibiotic-
producing isolates.
The following are our final conclusions
for the antibiotic-producing species
contained in the soil samples:
Unfortunately, our PCR sequencing did
not yield results that supported the
other tests. A form of Pseudomonas took
over many of the plates, including
isolates 1 and 2, and the PCR
sequencing was unsuccessful for isolates
3 and 4.
Figure 5: FinchTV nucleotide sequence for
Isolate 2.
Location CFUs/g soil # Antibiotic-producers
Speed Lake 450,000 4
Hill, Mees/Union 520,000 6
SRC Creek 460,000 13
Base of tree, Scum Pond
location
310,000 1