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Julia Baca and Ha’Liegh Sapp
Dr Stephen Baron
22 October 2015
Biology 400
Isolation of Human Skin Bacteria From Used Cosmetic Brushes
Introduction
Cosmetic products can be found in almost every bathroom in the United States, and though
seemingly innocent, these products can be teeming with bacteria. Over the past month, research was
conducted to determine the specific types of human skin bacteria present in used cosmetic brushes. It was
hypothesized that Staphylococcus epidermidis and Pseudomonas aeruginosa would be present in the
brush part of used cosmetic brushes due to daily application of makeup onto human skin. Though many
bacteria can be present on cosmetic brushes, two that are most commonly found include S. epidermidis, a
Gram-positive cocci, and P. aeruginosa, a Gram-negative, rod-shaped bacterium. Bacteria are naturally
picked up when applicators are used on the skin, and if not cleaned properly can cause serious health
issues. These health issues can range from mild to severe infection and could include corneal ulcers (15).
The ideal goal of this research was to isolate and identify one Gram-negative and one Grampositive bacterium from the used brush part of cosmetic brushes, with the overall goal of isolating and
identifying two different bacteria. Through a series of microbiological processes, unknown bacteria
became “known” as biochemical tests were performed in the laboratory.
Materials and Methods
The method used for identifying and isolating specific types of bacteria from cosmetic brushes is
modified from a study performed by Estrin (6). In the study conducted at Bridgewater College, bacterial
samples were collected from five used cosmetic brushes. In order to obtain bacteria from the cosmetic
brushes, each was placed into a test tube containing 9 ml of sterile saline and sat for five minutes. This
extracted the bacteria out of the brushes. 1 ml of sterile saline from each test tube was spread evenly onto
its own TSA (trypticase soy agar) plate using the spread plate technique as described in Microbiology:
Laboratory Theory and Application, Brief (11). Furthermore, a second technique was used to isolate and
identify bacteria. Sterile swabs were rubbed around and in the brush part of the cosmetic brushes. The
swabs were each rubbed onto a TSA plate and streaked out using the quadrant streak method (11). After
the bacteria was inoculated, the TSA plates were incubated at 37°C for 48 hours and checked for colonies.
Colonies had not grown after 48 hours on either the spread or streak plates, so therefore a
different approach was taken for plating out and growing the bacteria. During the second attempt, the
cosmetic brushes were applied directly onto the TSA agar in hopes of yielding better growth. After the
brushes were applied to the TSA agar, each sample was streaked out using the quadrant streak method
(11). In addition, better aseptic techniques were used when inoculating the spread plates and the amount
of solution plated onto each of the TSA plates was decreased (0.5 ml of solution was used instead of 1
ml). After incubation at 37°C for 48 hours, bacterial colonies successfully grew.
Two different, well-isolated bacterial colonies that grew on brush number five’s TSA streak plate
were plucked using aseptic techniques and plated onto separate TSA plates. Another well-isolated colony
that looked different from the other two was plucked from brush one’s spread plate and streaked onto its
own TSA plate. These three plates incubated at 37°C for 48 hours to allow colonies to grow. After this
time, each colony was observed to have its own distinct characteristics and morphologies, indicating that
each was pure and isolated. From here, each colony was transferred onto its own TSA slant using the
procedures as described in Microbiology: Laboratory Theory and Application, Brief for preservation in
the refrigerator at 4°C using aseptic techniques (11).
For further characterization, simple Gram staining methods were performed on isolate 1 (the
white colony) and 2 (the yellow colony) from brush five (11). After the staining procedures, the bacteria
were observed under a light microscope at 1000x power under oil immersion.
A catalase test was performed on both isolate 1 and 2 (11). A loopful of growth of both isolates
were transferred to a clean microscope slide from the TSA slants. Two drops of hydrogen peroxide were
put onto the bacteria and the formation of bubbles was observed.
Based upon the findings of the Gram stains and catalase test, isolate 1 was plated onto an MSA
(mannitol salt agar) plate using the quadrant streak method (11). The MSA plate was allowed to incubate
for 24 hours at 37°C and bacterial colonies were observed.
Isolate 2 required more testing to determine its identity. A nitrate reduction test was performed on
it using procedures described in Microbiology: Laboratory Theory and Application, Brief (11). Indole
nitrite broth was inoculated with isolate two and incubated at 37°C for 24 hours. After the incubation
time, 1 ml each of reagents A and B were added to the tube containing the inoculated indole nitrite broth.
The tube was allowed to stand for ten minutes. After ten minutes, a pinch of zinc dust was added to the
tube, which stood for another ten minutes. This test determined if isolate two could reduce nitrate or not.
In order to determine if isolate 2 fermented glucose, it was inoculated onto a TSI (triple sugar iron
agar) slant. For further testing, phenol red broth was inoculated with isolate 2 to determine whether it was
able to ferment carbohydrates or not. Both procedures were performed as described in Microbiology:
Laboratory Theory and Application, Brief (11). Both specialized tests were incubated at 37°C for 48
hours.
16s rDNA amplification tests were run in order to sequence parts of the DNA from the unknown
bacterial isolates as well as to compare them to the DNA of already sequenced microbes. Samples of each
unknown bacteria were DNA isolated and run through a polymerase chain reaction (PCR) in order to
amplify the DNA. The amplified DNA was run through agarose gel electrophoresis to purify the DNA
sample. The amplified DNA was then extracted from the gel and sent to a lab to have the 16s rDNA
amplified (1).
Results and Discussion
The very first set of inoculations (both the sterile swab streak and one ml sterile saline solution
spread) of the cosmetic brushes onto the TSA plates did not grow after their initial incubation time of 48
hours. All ten plates were placed into biohazard and new plates were inoculated using slightly altered
methods. The dirty brushes were each pressed directly onto their own TSA plate in the second round of
inoculating and streaked out using the quadrant streak method (11). This allowed for the bacteria within
the old makeup residue in the brushes to be placed directly onto the TSA agar. After 48 hours of
incubation at 37°C, colonies successfully grew. The first time the spread plates were inoculated, too much
saline solution containing the makeup brush bacteria was spread onto the TSA agar, and improper aseptic
techniques were used, potentially causing unwanted bacteria to grow on the TSA spread plates. The
second set of plates grew successful bacterial colonies after incubation at 37°C for 48 hours.
Isolate 1’s colony morphology was characterized by a circular form, convex elevation, scalloped
margin, solid consistency, and a creamy white color (4). These isolates were Gram positive and occurred
in grape-like clusters, characteristic of Staphylococcus (see Figure 1). Due to the grape-cluster shape,
isolate one was inoculated onto an MSA plate because the agar contains 7.5% NaCl, which selects for
human skin bacteria such as Staphylococcus. Pathogens ferment mannitol to acid products, turning the
phenol a red yellow color (11). Non-pathogens do not ferment mannitol and therefore the phenol stays a
red color, which is what occurred after isolate 1 grew on the plate. The bacterial colonies grown on this
plate were white. Staphylococcus does not select for mannitol formation, as seen on the MSA plate. A
catalase test was performed on isolate 1 and was catalase positive. This means the isolate contains the
enzyme catalase that breaks down hydrogen peroxide into water and oxygen (11).
After consulting with Bergey’s Manual of Determinative Bacteriology, it was hypothesized that
isolate 1 belonged to the phylum Staphylococcus and the genus Epidermidis. This was confirmed when
the 16S rDNA test results came back. The results for isolate 2 were inconclusive and therefore could not
be compared to the BLAST homology database. Isolate 1 received good amplification and sequencing
results, and a portion of its DNA sequence was able to be run through the BLAST homology database
(13). Isolate 1 best matched DNA stretches from Staphylococcus epidermidis with an E value of 97% (see
Table 1). Based on these results it was concluded that isolate 1 bacterial colonies were in fact S.
epidermidis, and no further tests were performed on this bacterium.
S. epidermidis is most commonly isolated from healthy human skin (5). It is beneficial to humans
because it inhibits the growth of pathogenic Staphylococcus aureus (5). If a strain of S. aureus is
methicillin-resistant, it causes MRSA, a serious infection in the bloodstream (7). However, if S.
epidermidis grows underneath the skin and enters the bloodstream, it becomes pathogenic. Furthermore,
S. epidermidis is responsible for 30% of hospital-acquired infections (5). This bacterium can form a
biofilm on medical devices used to treat humans, such as catheters. The real harm comes when cells from
the biofilm enter the bloodstream, causing disease and even death (5).
Isolate 2 was a Gram positive cocci that occured in tetrads (see Figure 2). Its colony morphology
was characterized by circular form, convex elevation, scalloped margins and solid consistency (see Table
3). After consulting Bergey’s Manual of Determinative Bacteriology, it was hypothesized that isolate 2
belonged to the phylum Micrococcus and the genus Luteus. Since the 16S rDNA results were
unsuccessful for this isolate, tests were performed based upon a dichotomous key (10). Since the
bacterium was in fact Gram positive, occurred in tetrad-occurring groups, was catalase positive, and had a
yellow colony pigment, a dichotomous key suggested performing a glucose fermentation test (10). This
test was negative as expected, which is characteristic of M. luteus. After comparing other Micrococcus
species to M. luteus in Bergey’s Manual of Determinative Bacteriology, it was decided that the nitrate
reduction, starch hydrolysis, and phenol red glucose tests would be performed to rule the bacterium as M.
luteus (9). Due to the results of these tests (see Table 3), the final bacterium was in fact M. luteus.
Members of the Micrococcus phylum are common soil and water contaminants (3). M. luteus has
also been found to commonly grow on human skin, usually around the head, legs, and arms (11). These
bacteria are generally strict aerobes, and they usually produce pigments of a yellowish color called
carotenoids (2). Though M. luteus is a common skin bacterium and rarely pathogenic, it has been
implicated in cases of septic arthritis, meningitis, and endocarditis (14). M. luteus can perform as a
pathogen in the bloodstream, and can be resistant to antibiotics (12).
Figure 1. Pictomicrograph of isolate one. Note the purple cells characteristic of gram positive bacteria.
Cocci cells arranged in grape cluster formation typical of Staphylococcus.
Figure 2. Pictomicrograph of isolate two. Note the purple cells characteristic of gram positive bacteria.
Cocci cells in tetrad formation.
Figure 3. PCR reaction gel for the 16s rDNA sequencing. In lane one is isolate 1 and in lane two is
isolate 2. The band in lane one is exactly at 1kb PCR product.
Table 1. Results of the BLAST homology database search for contiguous 16S rDNA sequence from
isolate number one.
Accession
Description
Max
Score
Total Score
Query
Coverage
E-Value
Max Indent
KF088329.1
Uncultured
Bacterium Clone
1842
1842
97%
0.0
99%
1840
1840
96%
0.0
99%
1840
1840
97%
0.0
99%
1838
1838
96%
0.0
99%
1838
1838
96%
0.0
99%
ncm52h02c1
GQ158789.1
Uncultured
Bacterium Clone
16slp91-2b11.p1k
EU071608.1
Staphylococcus
epidermidis strain
EHFS1 s06Hc
KT152827.1
Bacterium
msa1-1
KT221537.1
Staphylococcus
sp.
TT31
Table 2. Tests performed and results observed for isolate number one.
TEST PERFORMED
RESULT FOR YOUR UNKNOWN #1 (white isolate)
Colony morphology on (indicate
medium)
TSA
Form
circular
Elevation
convex
Margin
Scalloped
Consistency
solid
Pigmentation or diffusible
pigments
Creamy white
Gram stain
Gram positive, grape clusters
Catalase
positive
Characteristics of MSA plate
Agar color
Red
Colony color
White
Mannitol Fermented?
No
Table 3. Tests performed and results observed for unknown 2, the yellow isolate.
TEST PERFORMED
RESULT FOR YOUR UNKNOWN #2 (yellow isolate)
Colony morphology on (indicate
medium)
TSA
Form
circular
Elevation
convex
Margin
scalloped
Consistency
solid
Pigmentation or diffusible
pigments
Mustard yellow
Gram stain
Gram positive
Cell morphology
tetrads
Phenol red glucose
No reaction (broth is red/orange color, no gas formed in tube)
Catalase
Positive
Starch hydrolysis
No reaction
Nitrate reduction to nitrite
No nitrite formed, nitrate still present
Literature Cited:
1. Baker, G.C., Smith, J.J. & Cowan, D.A. (2003). Review and re-analysis of domain-specific
16S primers. J Microbiol Methods 55, 541-55
2. Benson, H.J., (1998) Microbiological Applications: Laboratory Manual in General
Microbiology Seventh Edition. Boston: McGraw Hill.
3. Cappuccino, J.S., Sherman, N., (2014) Microbiology: A Laboratory Manual Tenth Edition.
Boston: Pearson.
4. Colomé, J.S., Kubinski, A.M., Cano, R.J.& Grady, D.V. (1986) Laboratory Exercises in
Microbiology First Edition. Minnesota: West Publishing Company.
5. Conlan, S., Mijares, L.A., Becker, J., Blakesley, R.W., Bouffard, G.G., Brooks, S.,
Coleman, H., Gupta, J., Gurson, N., Park, M., Schmidt, B., Thomas, P.J., Otto, M.,
Kong, H.H., Murray, P.R. & Segre, J.A. 2012. Staphylococcus epidermidis pan-genome
sequence analysis reveals diversity of skin commensal and hospital infection-associated
isolates. Genome Biology. Retrieved from http://www.genomebiology.com/2012/13/7/R64
6.Estrin, N.F. The Cosmetic Industry: Scientific and Regulatory Foundations. New York: M.
Dekker, 1984. Print.
7. Foster, T.J. 2004.The Staphylococcus aureus “superbug.” The Journal of Clinical
Investigation. Retrieved from http://www.jci.org/articles/view/23825
8. Holt, J.G., Krieg, N.R., Sneath, H.A., Staley, J.T. & Williams, S.T. (1994) Bergey's Manual
of Determinative Bacteriology Ninth Edition. Philadelphia: Lippincott Williams & Wilkins.
9. Holt, J.G., Krieg, N.R., Sneath, H.A., Staley, J.T. & Williams, S.T. (1994) Bergey's Manual
of Determinative Bacteriology - Identification flow charts. Philadelphia: Lippincott
Williams & Wilkins.
10. Kloos W.E., Musselwhite M.S., 1975. Distribution and Persistence of Staphylococcus and
Micrococcus Species and other Aerobic Bacteria on Human Skin. Applied Environmental
Microbiology. 30:381-395.
11.Leboffe, M.J. & Pierce, B.E. (2012) Microbiology: Laboratory Theory and Application,
Brief Second Edition. Colorado: Morton Publishing.
12. Marples, R.R., Richardson, J.F. 1980. Micrococcus in the Blood. Journal of Medical
Microbiology. 13:355-362.
13. National Library of Medicine. (n.d.). Nucleotide BLAST. Retrieved from:
http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn&BLAST_PROGRAMS=mega
Blast&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome
14. Seifert, H., Kaltheuner, M., Perdreau-Remington, F. 1995. Micrococcus luteus
Endocarditis: Case report and review of the literature. Zentralblatt fur Bakteriologie.
282:431-435.
15. Wilson, L.A. & Ahearn, D.G. 1977. Pseudomonas-induced corneal ulcers associated with
contaminated eye mascaras. American Journal of Ophthalmology. 84(1):112-9.