1. Arthrobacter: Isolation and Identification Maes 1
Aurora Maes
Microbiology Lab 352L-002
November 22, 2015
Arthrobacter: Isolation and Identification
Introduction
The purpose to design an experiment to isolate and identify a bacteria genus
based on results obtained, lead to an interest in Arthrobacter. The diverse genus
Arthrobacter is in the kingdom Bacteria, the phylum Actinobacteria, and the family
Micrococcaceae (Busse et al. 2014). Arthrobacter is a genus characterized by being
Gram-positive, catalase positive, an aerobic organism, with a changing morphology
during its lifetime based on conditions, and is white and large while growing on media
(Arthrobacter 2015). The most common morphology change of Arthrobacter species is
from a coccus to a rod shape (Luscombe et al. 1974).
Other qualities of Arthrobacter are in their colonization and their metabolism.
Arthrobacter are usually in biofilms with Proteobacteria—Gram negative rods,
Bacterioidetes—Gram negative rods, Cyanobacteria—filamentous bacteria, other
Actinobacteria, and Nitrospirae—spiral shaped bacteria (Arthrobacter 2015). The
primary metabolism of Arthrobacter is carbohydrate metabolism through oxygenic
respiration, however a few species have been characterized as facultative anaerobes
(Levering 1981). The facultative anaerobic Arthrobacter species can grow as a result of
oxygen poor conditions in which they utilize nitrogen compounds such as nitrate to
reduce to usable nitrogen compounds for energy (Eschbach et al. 2003).

2. Arthrobacter: Isolation and Identification Maes 2
Arthrobacter is a non-pathogenic and non-virulent bacterium— with exception to
producing irritants to humans—that is primarily found in soil and in clinical specimen
(Luscombe et al.1974). Some Arthrobacter species degrade pesticides thus affecting
the agricultural industry and Arthrobacter is considered an opportunistic pathogen
(Eschbach et al. 2003). More studies on the pathogenicity of Arthrobacter and the
consequences of exposure are being conducted (Eschbach et al. 2003). The sample of
Arthrobacter obtained was from a swab of the faucet spout in the bathroom, in the
basement of the University of New Mexico’s Biology Building, Castetter Hall. The
sample originally targeted was Streptococci, but was changed due to growth of pure
cultures of Arthrobacter.
Materials and Methods
The media used for growth in this experiment were Trypticase Soy Agar (TSA)
plates and Sheep Blood Agar plates, due to the complexity of the metabolism of
Arthrobacter; colony types are more distinguishable by growing on Blood Agar
(Arthrobacter 2015). Blood Agar grows organisms such as Streptococci (the original
target organism) that have a more sophisticated metabolism and will utilize the complex
media and grow (Arthrobacter 2015). TSA plates are standard growth media— in which
bacteria use the nutrients within the media for metabolism and grow into colonies—
which were used as a control media to compare to Blood Agar plates. These were
incubated in 37 degrees Celsius, aerobic environments, after samples of the unknown
were placed on TSA plates and Blood Agar.
The many tests conducted were based on metabolic strategies of the organism,
special characteristic of the target organism (genes dealing with respiration), and DNA

3. Arthrobacter: Isolation and Identification Maes 3
sampling. A Gram-stain was the first test conducted will differentiate between all other
bacteria phyla, found with the uniquely Gram-positive rod-like unknown bacteria, is in a
Gram-negative environment with obviously different morphology non-rodlike (Busse et
al. 2014) Crystal violet is added to a sample of the bacteria isolated, as well as use of
counterstains such as safranin and iodine (Vesbauch-Takacs 2015). If it is Gram
positive, staining purple—a bacteria colony with a thick peptidoglycan layer will
appear—or Gram negative, staining pink—a bacteria colony with thin peptidoglycan
layer for a cell wall will appear (Vesbauch-Takacs 2015). After staining, the bacteria was
observed under a microscope to determine the result (Vesbauch-Takacs 2015). Another
test conducted will confirm if the organism is Gram positive and a carbohydrate
fermenter with Mannitol Salt Agar (MSA) plates. MSA plates are a selective and
differential media, MSA selects for growth of only Gram-positive organisms, and
differentiates between Mannitol (carbohydrate) fermenters (Vesbauch-Takacs 2015). If
fermentation of the mannitol occurred a yellow region should appear indicating, a drop
in pH from the red indicator, showing that the bacterium is a mannitol fermenter; if no
color change then the bacterium is not a mannitol fermenter (Vesbauch-Takacs 2015).
A sample colony of unknown bacterium was obtained and a streak plate was made and
incubated in an aerobic environment at 37 degrees Celsius (Vesbauch-Takacs 2015).
More carbohydrate fermentation tests were conducted using four sugar media
that were placed in tubes containing Glucose, Lactose, Sucrose, or Mannitol. The
fermentation test was used to determine metabolic pathways of the isolated organism
(Vesbauch-Takacs 2015). If fermentation occurs, then a color change will appear
changing the medium from a red liquid to a yellow liquid (if the unknown bacterium

4. Arthrobacter: Isolation and Identification Maes 4
ferments all the way to carbon dioxide then a gas bubble will also be present), if no
color change then the bacterium is not a fermenter of the sugar. These were incubated
at 37 degrees Celsius and in anaerobic environments (Vesbauch-Takacs 2015).
The final two tests conducted were a nitrate reduction test and a catalase test.
This test will differentiate unknown into species as a few species have this capability to
utilize nitrate during anaerobic conditions by testing its metabolic strategies (Vesbauch-
Takacs 2015). A nitrate reduction medium was used and inoculated with bacteria and
incubated at 37 degrees Celsius in an anaerobic environment, and reagents A and B
were added to media after incubation (Vesbauch-Takacs 2015). If upon addition of
reagents A and B there is a color change then the nitrate was reduced to nitrite, if a
bubble is present in a yellow liquid then the nitrate was reduced to nitrogen gas, if
yellow still 10 grains of zinc were added, if red then no nitrate reduction if yellow then
ammonium or amide compounds were formed (Vesbauch-Takacs 2015). A catalase test
was conducted by taking a sample of unknown bacteria and placing on a slide, the slide
is placed under a microscope at 10x objective and a drop of 3% hydrogen peroxide is
added (Vesbauch-Takacs 2015). The catalase gene makes an enzyme specific to
aerobic bacteria to use oxygen and is a key characteristic of Arthrobacter respiration
(Wauters et al. 2000). If bubbles form then the microbe is catalase positive and is able
to use oxygen for growth (Vesbauch-Takacs 2015).
Additional research included a DNA extraction of sample bacterial isolate and
Polymerase Chain Reaction of 16S rRNA and a DNA blast—a search tool to identify
potential bacteria based on the portion of the 16S rRNA sequence (Vesbauch-Takacs
2015). This is used to identify the unknown phylogenetically. The protocol is a standard

5. Arthrobacter: Isolation and Identification Maes 5
DNA isolation with enzymes removing extracellular debris from isolating DNA and
running DNA through a gel and a basic search through a database.
Results and Data
The Gram-stain test revealed purple stained rods. Whitish-large colonies were
subcultured and used for further testing. The Mannitol Salt Agar plates had growth but
remained red, no color change. The carbohydrate fermentation tests did not have a
color change and had an orange-red tint. The nitrate reduction test showed no color
change or gaseous products. The catalase test showed bubbling when hydrogen
peroxide was added to the slide of Arthrobacter. The DNA extraction showed no
utilizable results. The DNA blast showed results of previous sequencing of Arthrobacter
of the 16S rRNA gene.
Discussion
The results of the Gram-stain were purple stained rods, meaning the morphology
was Gram-positive and rod shaped as predicted. The whitish color and large colonies
are typical of Arthobacter colonies on Trypticase Soy Agar (TSA) and Blood Agar
plates. The carbohydrate fermentation test did not have a strong color change indicating
that in anaerobic environments this bacterium is not efficient at fermentation. This is
also indicative of Arthrobacter, as most Arthrobacter species are obligate aerobes. The
slight color change (orange-red) could be from some trapped oxygen in the tube
allowing for minimal growth of the bacterium and since the tubes are filled with
carbohydrate, the bacteria were able to somewhat use the carbohydrate.
The nitrate reduction test showed no color change, at first glance indicating that
the bacterium is a reducer of nitrate to ammonium or amide. A mistake in the procedure

6. Arthrobacter: Isolation and Identification Maes 6
in nitrate testing was made and identified as the procedure was viewed later, no zinc
was added. It is possible that the species of bacteria isolated is a nitrate reducer but
due to oversight this test is inconclusive. The result, if the color did not change is the
bacterium is a nitrate reducer and further identification of a species could be done.
Arthrobacter species, such as Arthrobacter globiformis and Arthrobacter nicotianae are
able to reduce nitrogen and if the test were positive might indicate that one of these
species is isolated. The catalase test with the formation of bubbles, means that this
species of bacteria contains the catalase gene and is able to use oxygen for respiration
and metabolic activity. This is indicative of most aerobic organisms but can be found in
nearly every species of Arthrobacter (Wauters et al. 2000)
The DNA PCR was not successful at getting results that were able to be
differentiated between genera. Thus these results were inconclusive due to the
complete accuracy necessary and some mistakes in procedure affected these results,
the equipment used included many transfers, and the DNA did not make it to the final
steps of the procedure (Vesbauch-Takacs 2015). The DNA Blast search was helpful for
determining what the 16S rRNA of Arthrobacter should look like, but without the tested
DNA, the sequence was not useful for comparison or identification of the genus of the
bacterium.
The intended isolate was Streptococci, however upon Gram-staining and
achieving non-cocci forms it was researched that within the environmental sample, the
only Gram-positive bearing rod shapes, is that of Arthrobacter (Wauters et al. 2000) The
tests were then developed around characteristics of Arthrobacter—being Gram positive,
rod shaped, aerobic, catalase positive, not effective at fermentation but able to use

7. Arthrobacter: Isolation and Identification Maes 7
carbohydrates, not able to reduce nitrate and whitish large colonies forming on Blood
Agar and TSA plates—to determine if the bacterium was isolated. (Wauters et al. 2000)
The results obtained support that this is an Arthrobacter species. The Arthrobacter is
typically found in soil and humans are in contact with soil but is not virulent or
pathogenic to humans, thus it is not improbable to find it in a sink of a bathroom, in the
Biology Department in Castetter Hall (Luscombe et al. 1974). Most Arthrobacter
Arthrobacter species have not been completely described thus far as part of the
mucosal flora more research is being done to understand the relationship between
Arthrobacter and humans (Wauters et al. 2000).
Conclusions
The purpose to identify and isolate a microbe was successful. Many tests were
performed to support that this is an Arthrobacter species. Arthrobacter is a bacterium
that is mostly aerobic and has genes for respiration such as catalase, Gram positive,
and rod shaped. Arthrobacter poses potential danger to the agricultural industry and is
not know to be pathogenic in nature to humans. (Arthrobacter 2015) A bacterium was
isolated satisfying the purpose of the experiment, however to confirm that the bacterium
is Arthrobacter more studies will need to be done.

8. Arthrobacter: Isolation and Identification Maes 8
Works Cited
Busse, H., & Wieser, M. (2014). The Genus Arthrobacter. The Prokaryotes, 105-132.
Retrieved November 15, 2015, from
http://link.springer.com/referenceworkentry/10.1007/978-3-642-30138-4_204
Eschbach, M., Mã¶Bitz, H., Rompf, A., & Jahn, D. (2003). Members of the genus
Arthrobacter grow anaerobically using nitrate ammonification and fermentative
processes: Anaerobic adaptation of aerobic bacteria abundant in soil. FEMS
Microbiology Letters, 223(2), 227-230. Retrieved November 21, 2015, from
http://femsle.oxfordjournals.org/content/223/2/227
Levering, P., Dijken, J., Veenhuis, M., & Harder, W. (1981). Arthrobacter P 1, a fast
growing versatile methylotroph with amine oxidase as a key enzyme in the metabolism
of methylated amines. Archives of Microbiology Arch. Microbiol., 129(1), 72-80.
Luscombe, B., & Gray, T. (1974). Characteristics of Arthrobacter Grown in Continuous
Culture. Journal of General Microbiology, 213-222. Retrieved November 16, 2015.
Arthrobacter. (2015, September 14). Retrieved November 13, 2015, from
https://microbewiki.kenyon.edu/index.php/Arthrobacter

9. Arthrobacter: Isolation and Identification Maes 9
Wauters, G., & Charlier, J. (2000). Identification of Arthrobacter oxydans, Arthrobacter
luteolus sp. nov., and Arthrobacter albus sp. nov., Isolated from Human Clinical
Specimens. Journal of Clinical Microbiology, 38(6), 2412-2415.
Vesbauch-Takacs, C. (2015). Laboratory Protocols for Introductory Microbiology.
Retrieved 2015.