A Comparison of Chemical, Radiation and Physical
Techniques to Disinfect Used Toothbrushes
Kaitlin Zurawski
Bio 402: Senior Seminar
Final Draft
May 2, 2008
Table of Contents
Abstract
3
Introduction
4-10
J. S. Weese and J. Rousseau (2006)
5
Warren et al. (2001)
5-6
Devine et al. (2001)
6-7
Sato et al. (2004)
8
Mehta et al. (2007)
8-9
Methods
10-13
Week 1: Positive/negative control
10-11
Week 2: 3% Hydrogen peroxide
11-12
Week 3: Dishwasher
12
Week 4: Ultraviolet light
12-13
Week 5: Listerine® mouthwash
13
Comparison Tests
14
Results
14-16
Discussion
16-18
Acknowledgements
18-19
Literature Cited
20-21
2
Abstract
This study was performed to determine a specific method to easily remove
bacteria that may be adhered to a previously used toothbrush. Appropriate dental hygiene
is important in maintaining both good dental and systemic health as there are a great
number of bacteria that are able to thrive in such a dark, warm and moist place. These
bacteria are able to cause many problems, especially for oral health, for example dental
caries, or other bacterial diseases. Once one has brushed their teeth, these bacteria can be
found on the toothbrush. This produces the opportunity for these bacteria to be
reintroduced into the body. Four different methods of removing bacteria from the
toothbrush were studied; use of 2% hydrogen peroxide, UV light, a dishwasher cycle and
Listerine®. Upon completion of this study, the method of putting the infected
toothbrushes through a complete dishwasher cycle using generic dishwasher soap showed
to be the most effective method as it removed 100% of the bacteria involved.
3
Introduction
Appropriate dental hygiene is critical to maintaining both dental and systemic
health. There are a great amount of bacteria that are able to thrive in such a dark, warm
and moist place, which can cause many problems for dental health, for example dental
caries. Dental caries develop when the structure of a tooth, or teeth is damaged. This
occurs as bacteria present in the mouth convert food to acid. These both combined with
saliva make a sticky substance called plaque. This plaque sticks to teeth and then can
mineralize into tartar. Dental hygiene is not difficult to manage, but if incorrectly
controlled tartar and plaque present on the teeth can build up and cause irritation to the
gum tissues. This can lead to the development of dental caries, or cavities, as well as
other disorders such as gingivitis and periodontitis (Colgate, 2007). The American
Dental Association (2007) recommends that a person should brush their teeth at least
twice a day with fluoride toothpaste, as well as dislocate the bacteria located in between
teeth where the toothbrush cannot reach by flossing.
Streptococcus mutans and Staphylococcus epidermidis are prevalent bacteria that
can be found growing within the oral cavity. Streptococcus mutans initiates the caries
development of smooth surfaces and fissures of crowns of teeth (Tanzer et al., 2001).
Staphylococcus epidermidis can cause oral infections if not controlled (O’Gara et al.,
2001). The common procedure used to remove these bacteria consists of brushing the
teeth with a toothbrush. After use, a toothbrush is contaminated with the bacteria it is
designed to remove, and when used again it may reintroduce the bacteria into the oral
cavity. These bacteria grow in the dark, damp places of the mouth and therefore are able
to grow in the damp place of the used toothbrush head in the dark bathroom. In 1920 a
4
dentist by the name of Dr. Cobb was concerned that toothbrushes, full of bacteria, was
causing the continuously occurring infections in the mouths of his patients as he would
frequently report that the mouth infection extended to the throat (Warren, et al., 2001;
Sato, et al., 2004; Mehta, et al., 2007). Dr. Cobb suggested soaking the toothbrush in
alcohol, as alcohol was a known bactericide, and the patient would recover. In order to
reduce the possibility of infection, a technique should be devised that is easily
accomplished to remove the bacteria from the toothbrush.
Many studies have been conducted on contaminated meat, but according to Weese
and Rousseau (2006), it has not yet been widely studied the potential of the bacteria
remaining adhered to the food bowl after it is used, as well as after it is cleaned. They
tested the persistence of Salmonella spp. on the bowl after specific cleaning activities
which included; rinsing with warm water, rinsing and scrubbing, scrubbing with soap,
soaking in bleach, in a dishwasher, and scrubbing along with soaking in bleach. The
most efficient method was the bleach soak after scrubbing. Their dishwasher result was
the second best of the different methods introduced with only 67% of the bacteria
surviving or remaining (Weese and Rousseau, 2006). Due to their results, I tested this
dishwasher method on infected toothbrushes to see how well it disinfects the brushes.
The soap used and the high water temperature of the dishwasher should be able to kill
most of the bacteria found on the toothbrush as seen in Weese and Rousseau’s study
(2006).
Warm, wet toothbrushes occupying the dark are viable environments to the
bacteria that reside upon its bristles. Warren et al. (2001) decided to see if triclosancontaining toothpaste would aid in the inhibition of the microbial contaminants.
5
Triclosan is an antimicrobial agent used in the health field to disinfect instruments (Jones
et al., 2000). It is bacteriostatic, which means it is able to inhibit bacterial growth against
a wide range of both gram-negative and gram-positive bacteria. Warren et al. (2001)
chose to use anaerobic bacteria because of their ability to survive on the toothbrush and
become reestablished within the oral cavity. They had twenty patients brush their teeth
with no toothpaste, regular toothpaste or triclosan-containing toothpaste. In order to
remove the bacteria and measure the amounts they placed the brushes in prereduced
peptone-saline diluent and agitated the toothbrushes within to remove the bacteria. After
plating and incubating they found that toothpaste in general reduced the residual
microbial contamination of the toothbrushes, though it was not statistically significantly
different between the two and thus they concluded that toothbrushes should regularly be
replaced. This shows how perpetual bacteria are and the necessity of disinfecting a
toothbrush before and/or after use. The results of their test showed that anaerobic
bacteria survived even with the use of the triclosan-containing toothpaste. This suggests
that persons susceptible to periodontal diseases need to regularly exchange their
toothbrush for a new one or find a proper way to disinfect their toothbrush. This study
further shows how there is a need to have a high quality way to dislocate the bacteria
from a toothbrush in order to prevent contamination and infection. This is the reason I
chose to test different methods of sanitizing toothbrushes using a common disinfectant.
Cross-contamination due to contaminated instruments is a great concern in the
health world. When toothbrushes are left out in the open within a bathroom, many
bacteria can come in contact with the head and bristles, thus contaminating the
toothbrush. Devine et al. (2001) were interested in a procedure of general
6
decontamination that is cost-effective and effective. Ultraviolet radiation has been shown
to kill microbes and thus was studied by Devine et al. to test its reliability. They used a
quartz beaker with mercury vapor located in the enclosed hollow walls of this beaker.
The mercury vapor, when placed in a conventional microwave, transforms the
microwaves into ultraviolet waves (Devine et al., 2001). This device is an older version,
and I plan to use the newer version, for example the UV toothbrush sanitizer and holder
presently available on the market. This is where one places their toothbrush within
exposing the brush head to ultraviolet light and it is sanitized after a certain amount of
time has passed. Devine et al. (2001) used many organisms, such as bacteria, yeast and
viruses to evaluate the ultraviolet beaker’s ability to disinfect. They grew many strains of
each and placed into the beaker and the beaker was placed in a commercial microwave
from 15 to 120 seconds. They found the effectiveness depended upon the organism and
the quantity of the ultraviolet light produced by the microwave. In order to kill more
bacteria, more time within the microwave was needed. They found that the amount of
bacteria was more drastically reduced due to ultraviolet waves from all directions as
compared to when they removed the lid inhibiting ultraviolet waves through the top as
well as the sides and bottom. Thus I arrived at the intention to test the efficacy of
ultraviolet waves to disinfect a contaminated toothbrush. Their device was an older
version, and I used a newer version, for example the UV toothbrush sanitizer and holder
presently available on the market. I purchased and used an iTouchless UV-C ray
toothbrush disinfector as a method to test efficient bactericide to compare to the other
methods tested.
7
Sato et al. (2004) conducted a test to see if antimicrobial sprays would protect the
toothbrush from oral bacteria. The first spray was made up of “cetylpyridinium chloride
– CPC and basic formulation” which contained preservatives, a vehicle to transport the
bacteria and distilled water to contain the bacteria. The second spray contained “basic
formulation only” and the third was just water (Sato et al., 2004). For one week each
subject utilized a toothbrush and a spray, which was applied after the toothbrush’s use.
Each subject used one of the three sprays and then rotated sprays until each person used
all three sprays. These toothbrushes were placed in Letheen broth and agitated to remove
the bacteria. Phosphate buffered solution was then used to dilute the bacteria. The
solution was plated and incubated at 37° Celsius for one to two days to see if bacteria
were present and how effective each spray was, comparably. Colony forming units were
counted and it was found that the two sprays containing the antimicrobial solutions
helped to reduce the number of bacteria on the toothbrushes (Sato et al., 2004). The first
spray had a 16-20% positive result for bacteria and the second spray had a 20-50%
positive result for bacteria. The inactive solution, or water, had a 46-83% positive result.
This helps in understanding that there are different ways to disinfect a toothbrush that
may not result in completely eliminating bacteria, but it is useful in aiding in the
prevention of oral disease. In a similar way I used an antibacterial solution of hydrogen
peroxide to disinfect toothbrushes.
Many bacteria are present on the open counter surface of the bathroom. These
would include the bacteria left behind from previous oral care activities as well as
bacteria from the aerosol when the toilet is flushed and the area of it residing in the moist
conditions of the bathroom. Barker and Jones (2005) found that toilette aerosols are
8
spread throughout the area of the toilet after flushing occurs. A study proposed by Mehta
et al. (2007) suggested covering the toothbrush head with a plastic casing, as indicated by
some toothbrush companies, to prevent contamination. Mehta et al. also tested whether
chlorhexidine or Listerine® would be effective in purifying toothbrushes. Students were
used as subjects and participated in a study with three phases. During the first phase the
student used the toothbrush in the usual habit of brushing and then leaving the brush out
in the open space of the bathroom. For the second phase, the toothbrush was placed in
the chlorhexidine or Listerine® for a half of a day after use. For the final phase the
toothbrush had the cap placed on when not in use by the student (Mehta et al., 2007).
The cap placed on the toothbrush only promoted growth of bacteria as it retained a moist
environment in which bacteria flourish (Mehta et al., 2007). According to the results of
the tests run by Mehta et al. the chlorhexidine as compared to the Listerine® was the
more effective solution for decontaminating the used toothbrushes as it inhibited bacterial
growth in three of the five tests (2007). Chlorhexidine is also practically harmless to
people thus it would be the best option so far as a disinfectant. This study directly relates
to my own as they used Listerine® in their test and I also tested it to see its effectiveness
in decontamination as compared to the other options of ultraviolet light, hydrogen
peroxide and a household dishwasher.
Used toothbrushes are contaminated with the bacterial flora generated by the oral
cavity as shown by the tests completed by Mehta et al., Sato et al., and Warren et al. In
order to not reintroduce these bacteria into the mouth the next time one brushes, a method
of disinfecting the toothbrush bristles/head should be designed. To simulate this process
I infected toothbrushes with the bacteria Lactococcus lactis and let the brushes sit for an
9
hour inside the fume hood. The first disinfection test I used hydrogen peroxide and then
tested it for bacteria. I performed the same test with a dishwasher, an ultraviolet light
disinfector and finally Listerine® mouthwash. Because hydrogen peroxide is a strong
bactericide I believe it is the best option for toothbrush disinfection. I hypothesize that
household, low grade 3% hydrogen peroxide, which is commonly used as a disinfectant,
will be most effective in decontaminating a used toothbrush as compared to ultraviolet
light, a dishwasher, and finally, Listerine®.
Methods
Four different techniques of disinfection were tested on infected toothbrushes.
These specific tests were chosen to simulate an easy-to-do process that can be done at
home. A total of seven toothbrushes were used for each test. The toothbrush was
exposed to Lactococcus lactis and then subjected to the appropriate disinfection method.
Positive control
The first test was to see how much of the bacteria would normally grow on the
toothbrushes. This was the positive control in order to compare it to the different
disinfection techniques conducted. I incubated a 250mL Erlenmeyer flask with
Lactococcus lactis bacteria in nutrient broth at 37°C. L. lactis was chosen as it was
available as well as it is similar to oral bacteria because it is from the same family as the
Streptococcus spp. bacteria that are actually located within the oral cavity. It is in fact
very useful as it is commonly used in the production of buttermilk and cheese. L. lactis is
very relatable to humans, as according to Goyache et al. it is “more frequently found in
human and animal infections.” This helps to show how it is closely related to my study.
Seven toothbrushes, straight from the package, were dipped and lightly agitated in the
10
bacteria for approximately five seconds and then tapped against the inside glass to
remove excess liquid and placed inside the fume hood to dry for one hour.
In order to dislodge the bacteria, the toothbrush head was immersed in 10mL of a
previously prepared sterile phosphate buffered solution (PBS) at an approximate pH of
6.5 within a sterile tube, similar to Mehta et al. (2007). It was then mechanically agitated
using a vortexer for two minutes in order to release the bacteria into the solution. Ten
serial ten-fold dilutions were then made in the sterile media; this helped to visualize
distinct, single colonies that grew. Then 10μL of the solution were spread onto a nutrient
agar plate (Mehta et al., 2007). The plates were incubated at 37°C to promote growth for
three days (Warren et al., 2001). The plates were then examined to see if bacterial
colonies were present and how many as well as how dense they were in order to
understand the appropriate dilutions needed. The number of colonies was noted in turn to
compare to the other tests. This was done for each toothbrush with the intention to
compare to the next few weeks to determine which method is best to disinfect
toothbrushes.
Negative Control
As a negative control I used seven toothbrushes that were not used to brush at all,
directly from the package. This showed the difference between the bacteria present on an
infected brush and the new brush.
Test 1: H2O2
The first disinfection test I conducted was the method of dipping the toothbrush
head into 10mL of the common household solution of 3% hydrogen peroxide for 10
minutes. Again I dislodged the remaining bacteria in the PBS solution with the agitator
11
and plated 10μL of the ten dilutions on the nutrient agar. I incubated it at 37°C for three
days. The number of colonies was counted to see how much bacteria was thriving there.
This entire method of removing, plating and examining was done with each toothbrush.
Test 2: Dishwasher
The next method of disinfection I tested was the dishwasher method; as the high
temperature of water and the presence of soap sanitize very well. The approximate
moving water temperature of 85°C and soap, according to Weese and Rousseau, (2006)
should be able to kill or dislodge the bacteria present on the toothbrush. I used a GE
QuietPower 1 dishwasher on the normal wash cycle with the settings at heated dry, hot
wash and hot start, through one full cycle, which was timed at approximately two hours.
Kirkland Signature™ Dishwasher detergent liquid gel was added to the detergent cup.
The toothbrushes were carefully removed and placed in PBS solution which was again
used to dislodge any remaining bacteria with a two-minute agitation. The ten ten-fold
dilutions were made and then 10μL were plated on the nutrient agar. It was incubated at
37°C for one week. Observations were made, colonies were counted, and results were
recorded.
Test 3: UV Light
The third method of disinfection tested was the method of ultra-violet light. A
test conducted by Devine et al. (2001) showed that beakers that conduct ultraviolet light
greatly reduce the bacteria present. I used the ultraviolet light created from a UV-C
toothbrush sanitizer, which is a device specifically created to disinfect used toothbrushes.
The model I used was the iTouchless UV Toothbrush Holder which was obtained online
from Target.com. After being infected with L. lactis the toothbrush was placed within
12
the container and for the default time of seven minutes, the ultraviolet light acted upon
the bacteria. The toothbrushes were put through the program a total of two times which
is recommended by the company, so the toothbrushes were exposed to the ultraviolet
light for a total of fourteen minutes. After the toothbrushes were treated with the
sanitizer, I then agitated the brush heads in the PBS solution for two minutes in order to
dislodge the remaining bacteria. Then the ten ten-fold dilutions made were used in the
growth of the bacteria. I used 10μL of each dilution to plate on the nutrient agar. It was
incubated at the temperature of 37°C for one week. I then counted the number of
colonies to see how well the ultraviolet light killed the bacteria on the brush heads. I
observed the plates to see if bacteria survived on the toothbrush heads.
Test 4: Listerine®
The next test explored the disinfecting potential of Listerine® mouthwash. After
infecting the toothbrushes with L. lactis bacteria I placed and sanitized each of the
toothbrush heads in 10mL of Listerine® mouthwash for ten minutes, similar to the
hydrogen peroxide. They were each removed and placed into 10mL of PBS solution and
then agitated for two minutes in order to remove the bacteria. Ten serial ten-fold
dilutions were made. Then 10μL of the solution were spread onto a nutrient agar plate.
The plates were incubated at 37°C for three days. They were then removed and studied.
The colonies on the plate were counted to uncover the amount of bacteria present.
Throughout the entire lab I recorded all of the information I determined and observed.
13
Comparison Tests
The data recorded were tested for differences in the ability to disinfect and kill
bacteria on the toothbrush head. A one-way ANOVA test aided in determining the
differences and similarities between the four tests. A Tukey test was also performed to
see if there was a statistically significant difference between them. I compared the
primary week of no disinfection with each group; 3% hydrogen peroxide, the dishwasher
method, the ultraviolet light, and the Listerine® mouthwash. I then tested each test to
each other. This aids in understanding which method is better for disinfecting a
toothbrush by comparing how much bacteria is left upon toothbrushes after their specific
disinfection.
Results
In the first phase of the experiment, Lactococcus lactis was observed in great
numbers on each of the seven positive control plates (Table 1). This shows how much
bacteria was available on a toothbrush head when no disinfection occurred. The four
methods of sterilizing the toothbrush heads after infecting them with L. lactis included:
immersion of the toothbrush head in 0.2% hydrogen peroxide for 10 minutes, allowing
the infected toothbrushes to go through a full dishwasher cycle, fourteen minutes of UV
light, and the final test of soaking the toothbrushes in Listerine® mouthwash (Table 1).
The negative control showed no growth on the new toothbrushes directly from the
package.
14
Table 1. Number of colony forming units (cfu) after specific methods (* indicates
the two UV light plates that grew lawns and could not be counted).
Toothbrush 1
Toothbrush 2
Toothbrush 3
Toothbrush 4
Toothbrush 5
Toothbrush 6
Toothbrush 7
Positive
control
1753
972
2615
333
1041
333
59
Negative
control
0
0
0
0
0
0
0
H2O2
1
16
10
25
10
11
16
Dishwasher
0
0
0
0
0
0
0
UV light
24
12
1
12
*
20
*
Listerine®
12
11
9
12
5
1
1
Using raw data from the tests completed, the differences in treatments of
eliminating bacteria were compared using a statistical one-way ANOVA test which
shows a statistically significant difference between the positive control test and the four
sterilization techniques (F=7.91, DF=4, P=0.0001). The Tukey test was conducted with a
95% confidence level. The graph shows these averages of the different tests (Figure 1).
The error bars have a great magnitude as the inconsistency of the numbers from the
CFU
positive control give it a large variability.
2000
1800
1600
1400
1200
1000
800
600
400
200
0
pos itive
c ontrol
hydrogen
perox ide
dis hwas her
uv light
lis terine®
Figure 1. Average number of colony forming units (cfu per 10µL of solution) on
plates after inoculation of the Phosphate Buffered Solution with oral bacteria. Each
bar represents the mean number of colony forming units after treatment based on
seven toothbrushes. Error bars represent the standard deviation of the seven
toothbrush averages. (The UV light test only has an average of 5 toothbrushes)
15
According to the same Tukey test, there is not a statistically significant difference
between the four sterilization techniques with the same confidence level and P-value
when the positive control is included. It also shows the pairwise comparisons which
show the four tests all are statistically significantly similar (individual confidence level:
99.30%; P-value = 0.0001).
However, another one-way ANOVA test was performed comparing the four
disinfection tests to eachother which showed the treatments were statistically different
(F=7.24, DF=3, P=0.001). The Tukey test showed the dishwasher method was
significantly more effective at the 98.91% confidence level compared to the hydrogen
peroxide and UV light treatments. However, the Listerine® method was not statistically
significantly different from the dishwasher method (Figure 2).
25
CFU
20
15
10
5
0
hydrogen
perox ide
dis hwas her
uv light
lis terine®
Figure 2. Comparison of the remaining bacteria on the toothbrushes after the four
disinfection tests. Each bar represents the average cfu per 10µL of solution method
tested based on the seven toothbrushes. Error bars represent one standard deviation of
the the mean of the seven toothbrushes used. (The UV light test has an average of 5
toothbrushes).
16
Discussion
The results of my experiment indicate that the toothbrush disinfection method of
the dishwasher demonstrated to be far more effective than the predicted 3% hydrogen
peroxide method, which according to J.B. Linger et al. (2001) hydrogen peroxide
achieved the ADA goal of no more than 200 colony forming units of bacteria per
milliliter when they tested its capability of disinfecting dental unit waterlines. In my test,
the recovery of bacteria from the toothbrushes after cleaning them in the dishwasher was
0%. I expected to find that the hydrogen peroxide would be the most effective due to it is
generally used as a disinfectant, but hydrogen peroxide was not as successful in
eliminating bacteria as was predicted. This may be due to the great amount of surface
area located on the bristles as well as the brush was stationary while in the hydrogen
peroxide. A way to possibly improve my procedure would be to agitate the brush while
in the solution so that it would potentially have had a greater amount of contact with the
surfaces. More time than the proposed ten minutes within the solution also may increase
the bacteria removal. Other possible changes could include a larger sample size like
those used by Melita et al. (2007) or Warren et al. (2001) (10 and 20 respectively) to
decrease the comparison errors. Other methods could be tested as well, such as using a
microwave as it has the ability to effectively kill bacteria on food products and laboratory
equipment (Culkin and Fung, 1975; Latimer and Matsen, 1977, as cited by Conder and
Williams, 1983), or even boiling water, which uses high heat to kill bacteria.
Each method of sterilization tested showed great reduction in the bacteria as
compared to the total infection without any removal. As shown in Table 1, each test
greatly reduced the amount of bacteria as compared to the positive control. The negative
17
control, which was not infected with any bacteria, grew nothing on the plates. This was
desired as they came straight from the package and should not have had any time to
gather any bacteria. The positive control had such a variability, which helped to cause
the large error bar in Figure 1. The hydrogen peroxide also had a little bit of variability
as the first brush only had one colony, but the rest were an average of about fourteen
colonies. The dishwasher, from each and every toothbrush grew zero colonies. The
ultraviolet light as well as the Listerine® also had variance in their colony numbers to
give them their error bars.
Figure 1 shows how the positive control had a statistically significant difference
from the four tests showing that the methods were successful in removing bacteria. In
that same test from Figure 1, it is shown that the four methods, when compared to each
other, are not statistically significantly different. This indicates that each test is just as
successful in eliminating bacteria as the others comparably. When trying to distinguish
the four tests to each other, it is difficult, because the positive control dominates the
graph and minimizes the other data. This is why I created the Figure 2 to show the tests
in comparison to each other.
When Figure 2 is studied, it can be devised that there are differences within the
four tests when they are compared solely to each other. The figure suggests the
dishwasher method as the best solution to disinfecting toothbrushes as it shows absolutely
no bacterial growth. According to the Tukey test this method is not statistically
significantly different from the method of Listerine®, thus these two tests, both, are
highly effective in disinfecting the toothbrushes. The other two tests of hydrogen
peroxide and ultraviolet light are statistically significantly different from the dishwasher
18
test, even though they are not statistically significantly different from the Listerine®.
This denotes the tests of Listerine® and the dishwasher to be the most successful. It
seems that the ultraviolet light appears to be the least effective within the graph. This
may be due to light waves not being able to effectively reach all of the bacteria located on
the toothbrush or that the time the brushes were submitted needed to be extended.
The importance of this study relates to the health risks due to the bacteria
introduced to the body through the oral cavity. Bacteria from previous brushing would
be adhered to the brush as well as bacteria from other sources such as the toilet flushing
(Barker and Jones, 2005) or others’ near by coughs or sneezes. Bacteria located on the
toothbrush can be reintroduced into the body where they can cause health problems.
Toothbrushes that contain these bacteria should be disinfected. The dishwasher method
was the most successful in removing the bacteria in this study and I recommend
disinfecting toothbrushes using this technique.
Acknowledgements
I would like to acknowledge and thank Cheryl Guglielmo for her constant
assistance in locating instruments and chemicals throughout my experiment. I would also
like to thank Dr. Mary Jo Hartman in her fantastic attempt to help me obtain volunteers.
Dr. Aaron Coby deserves thanks for loaning the bacteria to me. Dr. Margaret Olney also
greatly contributed in answering my many questions and aiding me in procedures as well
as always pointing me in the right direction. Finally, I would like to express my gratitude
to fellow Saint Martin’s students, Enjoli Washington and Shannon Davis for
accompanying me during the after-school, late hours.
19
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