Julia Baca and Ha’Liegh SappDr Stephen Baron22 October 2015Biology 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 Gram-

positive 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

ScoreTotal Score Query

CoverageE-Value Max Indent

KF088329.1 Uncultured Bacterium Clone

ncm52h02c1

1842 1842 97% 0.0 99%

GQ158789.1 Uncultured Bacterium Clone

16slp91-2b11.p1k

1840 1840 96% 0.0 99%

EU071608.1 Staphylococcus epidermidis strain

EHFS1 s06Hc

1840 1840 97% 0.0 99%

KT152827.1 Bacterium

msa1-1

1838 1838 96% 0.0 99%

KT221537.1 Staphylococcus sp.

TT31

1838 1838 96% 0.0 99%

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

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