1

COMPARISON OF BACTERIA ISOLATED FROM UNCAPPED AND

CAPPED TOOTHBRUSHES STORED INSIDE AND OUTSIDE THE

BATHROOM AFTER 30 DAYS OF USAGE

BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE

ABO, LENARD

CALIBAYAN, KRIS

December 2020
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COMPARISON OF BACTERIA ISOLATED FROM UNCAPPED AND

CAPPED TOOTHBRUSHES STORED INSIDE AND OUTSIDE THE
BATHROOM AFTER 30 DAYS OF USAGE

An Undergraduate Thesis Presented to the Faculty

of North Valley College Foundation Incorporated
Lanao, Kidapawan City, in Partial Fulfillment
of the Requirements for the Degree

BACHELOR OF SCIENCE IN MEDICAL LABORATORY SCIENCE

ABO, LENARD

CALIBAYN, KRIS

December 2020
 3

TABLE OF CONTENTS

Preliminaries Page

Title Page i

Table of Contents ii

Chapter I

INTRODUCTION

Background of the Study 1

Review of Related Literature and Studies 4

Theoretical Framework 13

Conceptual Framework 14

Research Objectives 15

Significance of the Study 15

Scope and Limitation 16

Definition of Terms 17

Chapter II

MATERIALS AND METHOD

Sample Collection 20

Materials 20

Steps in Analysis 21

REFERENCES

APPENDICES
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CHAPTER I

INTRODUCTION

Background of the Study

Oral hygiene has an important contribution to the overall health and well-

being of an individual. It is a crucial routine and necessities that a person

should practice always, as it a way to prevent the predisposition to certain

illnesses that we might in contact, which can lead for our further development

on how we look for our safety, that is why, we use to toothbrush to help us

with these.

Even on early days, brushing one’s teeth was already practiced, such as

the use of feathers and bones as toothpicks dated back on Babylonian period

(Beavers Dentistry, 2020) and bamboo stick with attached stiff hog hairs as

bristles by early China’s civilization (Sembera, 2001). At the modern times,

the emergence of novel ways of cleaning, such as dental floss and electric

toothbrushes has gain popularity as these seems more convenient on

completely removing plaques and usually are better than ordinary

toothbrushes on preventing the formation of cavities (Oral-B, 2020). But even

with the evolution of various kinds of oral cleaning tool, the main sentiment

was that, oral hygiene is undoubtedly an essential practice for healthy living

which is something even the oldest civilizations understood.

Notwithstanding of these facts, that not only with the appropriate choice of

toothbrush as an efficient instrument for removing the oral biofilm and the soft

debris out of the mouth, proper maintenance, storage management, and

caring of toothbrush are crucial on having a better oral hygiene and health as

well, because although toothbrushes may seems not an ideal habitat of

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microorganisms for growth, it can still be contaminated and may provide a

favorable medium of life of microbes and may serves as fomites of other

pathologic microorganism if come in contact. As said by that, they become

contaminated because of different factors primarily on how one handles them.

Starting from the first usage of toothbrush, it is immediately contaminated by

the bacteria that are present in our mouth since human mouth contains

normal biota which are approximately 6 billion bacteria residing (Booth, 2019)

and after every further use, microbial colonization reaches higher level, but

none of this is cause for concern unless there is an unhealthy balance of

bacteria in the mouth which is can be a result of possible recontamination of

the mouth by using contaminated toothbrush without proper sanitation of it .

But other than normal biota contaminants to toothbrush, the environment,

hands, aerosol contamination, and even storage containers are also other

contributors that needs to be considered because the conditions by which the

toothbrushes are kept are of a big importance for the bacterial survival. In line

with that, according to Asumang, (2019) he discovers that toothbrush bristles

particularly on between the tufts serves as the predominant retention hub of

bacteria which can be pathogenic and can be a causative agents of various

diseases such as the findings in the study of G.N. Karibasappa et. Al (2011) it

can become contaminated with Streptococcus mutans which can cause

dental carries; Lactobacilli which can contribute to the progression of dental

carries, Candida, Pseudomonas which can cause suppurative otitis, eye

infections, UTI; Klebsiella which can cause pyogenic infections, pneumonia,

diarrhea, septicemia; Streptococcus pyogenes which can cause rheumatic

fever and glomerulonephritis as well as UTI; and Staphylococcus aureus

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which can cause subcutaneous infections; that is in agreement with the

findings of Willi-eckhard Wetzel et .al in the year 2005. Another study

conducted by Suma Sogi et al. finds out that toothbrush become

contaminated with E. coli when stored inside the bathroom that can lead to

the likelihood of predisposing to diseases such as diarrhea, septicemia, and

urinary tract infection. That is why according to ADA (2019), toothbrush

replacement every one to three or four months or more often if the fraying of

toothbrush becomes prominent is highly recommended.

Therefore, this study will be conducted to promote awareness of the need

for appropriate toothbrush storage management by identifying and comparing

the types of bacteria present in the capped and uncapped toothbrushes

particularly between the tufts of the bristles when it is stored inside the

household bathroom and when it is placed outside the bathroom after 15 days

and 1 month of toothbrush usage.

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Review of Related Literature and Studies

This study contains the different studies, books, and literature that are

important for the completion of this research paper.

Foreign and local studies

Toothbrushes serves as an important medium in maintaining good oral

hygiene which is widely used throughout the society. It typically reflects

indirectly and directly the overall well-being of an individual. And even with

earliest times, cleaning one’s teeth had become a tradition that many past

civilizations worldwide, has always been a practiced, by developing their own

unique and innovative kind of oral cleaning materials. Many early civilizations

use tools that are rudimentary in nature such as according to Beavers

Dentistry (2020), dated back to Babylonian period, Greeks and Romans uses

many mediums as a cleaning implements which includes chewing on bark or

sticks with frayed ends and utilization of feathers and bones which serves as

a toothpick, that was considered to be the first versions of brushing. According

to Sembera (2001), versions of toothbrush that has close approximation of

current’s type was the employment of a bamboo stick with attached stiff hog

hairs as bristles dated 1400’s, which was invented by China’s civilization as

well as the so called chewstick, and the idea of just by picking teeth

(Eichenauer et. Al, 2014). The discovery also in 1953, dug from tomb in

Laoning Province, the two toothbrushes made from ivory which is believed to

emerge during Liao-dynasty, leads to idea that China was considered to be

the place of origin where the earliest type of toothbrush with somewhat similar

appearance of today’s type of toothbrush was developed. At the modern times

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the emergence of novel ways of cleaning such as dental floss and electric

toothbrushes has been the tool of choice for most of high status individuals as

these are more convenient on completely removing plaques and on

preventing the formation of cavities which was supported according to many

studies (Oral-B 2020).

But notwithstanding of these crucial role of toothbrush on our health, it

can also serve as an important reason why one can acquire certain illnesses

and diseases just by brushing teeth. These detrimental effects on one’s health

is primarily the cause of various bacterial contamination on toothbrushes, as it

may serve as fomites where species of pathogenic bacteria can adhere such

as bacteria which causes most of dental carries and other related illnesses (S.

S. Bhat et al, 2003). In regards with this, there are various ways that a certain

toothbrush can be contaminated, either from different storage management or

by the improper or no sanitation at all of it and also with the addition of

continues reinfection by the normal flora of mouth brought by brushing on

daily basis.

According Filho et. Al (2000) newborns is free of bacteria and as we grow

there are many factors that predisposed us from the acquisition of bacteria

specially in our mouth as it is the most dominant medium of entry of

microorganisms that can be good or has an bad effect on our oral health

leading to dental health problems cause by the build-up of plaques by the

different causative agents on which toothbrushes can eventually contribute. In

regards with good bacteria, as we grow, normal flora will be residing our

mouth which according to Booth (2019) approximately there are 6 Billion

bacteria habiting, that is why it is not new for an idea that starting from the first
 6

usage of toothbrush, it is immediately contaminated by the bacteria that are

present in our mouth but not on the thought that from the initial usage which

pose less contamination, the microbial colonization riches higher level with

every further use of toothbrush and many studies had identified that

toothbrush bristles particularly on between the tufts is the predominant

retention hub of microorganisms, therefore, posing a reason for greater risk of

bacterial infection and associated diseases (Asumang, 2019). But other than

being contaminated with normal biota of an individual’s mouth, the way of

storing of toothbrushes has a great responsibility of why the increased

predominance of contamination might arose and might pose a threat to the

health safety of an individual (G N Karibasappa et al., 2011). Some ways can

contribute to this phenomenon such as the way the toothbrushes are being

kept which are a great bestowed to the survival of the bacteria specially to

those toothbrushes that are kept closed due to the creation of moist on which

most bacteria are likely to proliferate rather than kept exposed to air as

toothbrushes may become dry (Pesevska, 2016). The suitable environment

that the toothbrush with this kind of keeping stimulates bacterial growth which

can prolong the survival from 2 days up to one week leading to more

inoculation and increased risk for health, especially without inadequate

storage (S. Stefanovska, 2016). In addition, greater risk can acquire specially

to those toothbrushes that are stored inside the bathrooms, since bathrooms

can be a suitable place for the growth of various organisms that brings

illnesses such as Coliforms yeasts and enterics especially at home where

family members shares toilet (Sato, 2004). According to Komiyama et al

(2010), bathrooms are a suitable place for the growth of various bacteria that
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can be transmitted through aerosols which increased the likelihood that

toothbrushes stored in such place can become contaminated. One study

conducted by American Dental of Lagrange (2018) finds out that droplets

inside bathrooms floats as far as six feet from the toilet as you flush, which

was also supported by a study of Yadav (2015) that with the flushing on

lidless toilet, bacteria can flew as high as ten inches above the toilet which

can be a devastating fact, especially to those individuals that stores things

that are related to their hygiene maintenance as it typically can become

contaminated by fecal materials and with the likelihood of carrying bacteria

along with it. Furthermore, some factors can also contribute to the

contamination that may be suitable for their survival to the toothbrush such as

the arrangement of filaments at the head of the toothbrush specially to those

matted and frayed ones (W. E Etzel et al, 2005). In addition, the optimal

environment brought by the accumulation of the hard deposit on toothbrushes’

head as times goes by, after regular use can provide stimulation to bacterial

growth, even with the most pathogenic ones that can affects the overall health

to the person using it (Karibasappa, 2001). According to the study of R. M.

Abd-ulnabi (2012) there is likelihood that toothbrushes become contaminated

with the claims that 90% of used toothbrushes can be associated with

microbial growth particularly the species of bacteria such as Streptococcus,

Staphylococcus and Pseudomonas, which is considered to be the most

isolated bacteria from toothbrushes that are not properly stored (R. M. Abd-

ulnabi,2012). Particularly, these microorganisms can live in the toothbrushes

and can eventually cause diseases, but not only with that , organisms which

are not usually associated with our oral flora can grow also within the
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toothbrush such as the family of Enterobacteriaceae by several factors

particularly the place of storage (Peveska, 2016) such the bathroom which is

a shelter for most of this type of bacteria as according to Ferreira C.A, (2012).

Furthermore, the presence of Streptococci on toothbrush is primarily due to

that they are great mediator of bacterial transfer so is not surprising that they

can be isolated from the toothbrush as they are likely to originate from the

residual plaque on mouth and can cleaved in the toothbrush (Peveska, 2016)

which can eventually associated with the greater risk of formation of dental

carries specifically the Streptococcus mutans and Streptococcus pyogenes

which can eventually cause post sequelae diseases such as Reumatic fever

and Glomerulonephritis, and with the addition to the presence of imbalance

number of Lactobacilli population, dental carries can progress to more serious

complications (G.N. Karibasappa et. Al, 2011) as well as the presence of

Candida albicans which can contribute to further destructions of teeth

(Eichenauer, 2014). In regards to the presence of Staphylococcus, particularly

Staphylococcus Aureus, this also can be a significant finding since, although,

this is an oral flora, more attention should be put to consideration due that it

causes not only a number of oral diseases but also opportunistic infections

that belongs to the category of subcutaneous infections like carbuncles and

folliculitis (G.N Karibasappa et Al, 2011). In addition, growth of Pseudomonas

and Klebsiella are still relevant findings because they can cause suppurative

otitis, eye infections, UTI; and pyogenic infections, pneumonia, diarrhea,

septicemia, respectively. Furthermore, not only with the increased risk of

damage to one’s teeth due bacterial contamination of toothbrushes, this can

also leads to predisposition to other diseases such as heart illnesses and

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bone associated brought by other pathogenic bacteria that are likely to be

found in the toothbrushes’ bristles (Samuel et. Al 2015). In addition, there are

also other reports that toothbrush can be contaminated by hepatitis C virus

which cause the disease HCV-RNA which are delivered from other people

who has this or uses the same toothbrush. (G. Lock, 2016).

In regards with these bacteria that can be isolated from the contaminated

toothbrush, techniques of how to identify those microorganisms should be an

important knowledge to be considered to be able to have an accurate

detection of expected bacteria in the toothbrush and accurate information of

awareness to other people. Since bacteria are believed to survive at the hard

deposits between tufts of toothbrush bristles (Karibasappa et. Al 2001) cutting

the bristles and the use of swabbing is an appropriate method for the

acquisition of samples. According to the study of R. Niveda 2019, nutrient

broth such as the Tryptic soy broth (TSB) and Brain heart infusion broth can

be used to support the growth of these bacteria that can be isolated in the

contaminated toothbrush as this may allow the inoculation of a wide range of

non-fastidous organisms. Furthermore, Selective and deferential agar medium

can be used for identification and further isolation of these bacteria such as

the Mac Conkey agar which is used for culturing of bacteria that belong to

Family Enterobacteriaceae when allowing incubation aerobically for 37

degrees celcius for 24 hours; Blood agar plates and chocolate agar plates for

the identification of species belonging to Streptococcus and Staphylococcus

after incubation aerobically and anaerobically respectively for 37 degrees

celcius for 24 hours (Gauyam et. Al, 2017). Gram staining should be

performed for further aid in identification and are subculture on nutrient agar.
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Another method developed by Sheretz, Raad and Balani suggested the use of

streaking for isolation of this bacteria on which samples of toothbrushes is

placed on the Tryptic Soy Broth where bacteria can grow and a streak on the

plates containing sheep blood agar and 5 % MacConkey agar using an

inoculation loop and stored in bacteriologic incubator at 37 degrees celcius for

a period of 24 hours for microbial growth analysis (Ferreira, et Al 2012).

Notwithstanding of these contamination on toothbrushes, prevention of

these unlikely events that involves the inclination of risks of one’s health, there

are different ways, one can eventually achieve prevention of contamination of

toothbrushes such as replacing toothbrushes on monthly basis, ideal storing,

the use of sanitation and disinfectants, and even with the innovations by the

dentistry community regarding to the prevention of microbial contamination

such as the recommendation suggested by the American Dental Association

2019 that toothbrushes should be replaced approximately every one to three

or four months or more often if fraying of toothbrush becomes prominent since

according to C. A Ferreira (2012), toothbrushes might not be able to

adequately removed plaques with torn out toothbrushes especially with the

addition of contamination by bacteria from everyday use especially to

individuals who has underlying illnesses and who are immunocompromised

which should frequently change their toothbrushes as they can have high risk

of exposure to pathogens and can acquire more illnesses with the

contamination of toothbrushes (Warren et al, 2001). According to M.Y

Komiyama et al. 2010 even with people having healthy periodontium can be

exposed to various bacteria from contaminated toothbrushes with no proper

storage of it, how much more to diseased individuals who are more prone to
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increased risk as they are immunocomprised. Proper toothbrush management

and storage such as washing it properly can help reduce the likely hood of

being exposed to bacteria (Nourbakhsh et al., 2005).

Other than that, one of the most important necessities that an individual

should consider to avoid contamination of toothbrushes are proper sanitation

and disinfection. In the early days the use of alcohol as a disinfectant of

toothbrushes was prominent and later the emergence of the usage of sunlight

and table salt can aid in desiccation process of toothbrushes preventing the

growth of bacteria (S. Sato et. Al, 2014). But sanitation of toothbrushes has

not been a focus now a days leading to more exposure to pathogenic bacteria

that causes various health problems not only with teeth illnesses, to avoid so

the use of sterilization equipment can aid in preventing such acquisition

(Kozai et al. 1989). The use of proper sanitary products should be a must to

avoid exposing to risk brought by contaminated toothbrushes, not only with

washing it with water as bacteria might still proliferate (P. Nelson-Filho 2006).

There are different types of sanitary solution and products such as the use of

Brushstox and periogard and dentifrices can aid such prevention. The aid of

various solutions as for sanitation of toothbrushes is necessary such as

Virkon and Listerine that inhibit the growth of bacteria such as Mycobacterium

species and Streptococcus that may brought certain kind of diseases (S. D

Caudry et al, 1995). But the use of these sanitary product and disinfectant

might still have drawbacks, other than having a well storing and management

of toothbrush antiseptic protocols should be included since not all aids for

disinfect ions can prevent the growth of bacteria since resistant to a specific

antimicrobials and these sanitary agents might have happened (R. L. Merino-
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Alado, 2018). Various Dentifrices solution can be an aid for preventing

contamination of toothbrushes but is not widely use due to its disadvantage

such as time consuming and the production of unwanted residues that might

cleaved into the toothbrushes (M.V Thamke, 2018). The use of normal saline

will not aid in the prevention of contamination of toothbrushes as it may lead

to the growth of other resistant organisms such as streptococci, instead the

use of mouthwashes with antimicrobial properties such as clorhexedine or

sprays can be an aid in prevention (0. Ayşegül, 2007). Even with usage of

toothbrushes with antibacterial tufts, other resistant microorganisms might still

grow such as those that are cariogenic and periodontopathogenic that is why

it is still recommended to use solutions such as dentifrices (E.Y. Komiyama,

2010). various innovations have emerged to aid such resistant problems. With

the emergence of increasing population of bacteria being resistance to

various antibiotics, solutions for sanitation of toothbrushes is not enough, with

the use of UV light which has bactericidal properties such as violighta a

toothbrushes holder prevention of contamination can be achieved (R. Boylan,

2008). UV lights or by tetrasodium EDTA can have a better result when it

comes to decontamination of toothbrushes but is not determined yet in clinical

settings (M.V Thamke, 2018). In addition, toothpaste also can prevent the

growth of bacteria but not with toothbrushes with triclosan coated tufts on

which bacteria might still grow (M. Efstratiou, 2017). Behind all of this, it is

important to know also that even with the knowledge on proper storage

management and disinfecting toothbrushes bacteria can still exist, how much

more if a person has no toothbrushes storage management (L.K Rodrigues et

al, 2012)
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THEORITICAL FRAMEWORK

This study does not only aim to identify and isolate bacteria from

contaminated toothbrush but will focus and pose awareness to the public on

how storage management of toothbrush is significant to the level of safety of

using it. This study will be anchored on the study of Gautam et al. (2017) that

emphasizes on how the neglecting of proper toothbrush keeping and its

relationship to the microbial content is significant on the overall health and

well-being of an individual. Since the oral cavity contains a population of

different types of microorganisms it can be transfer to toothbrush when in

regular use, so it is not surprising that most isolated bacteria from toothbrush

comes from our mouth so it is possible to have a repeated recontamination of

toothbrush, but aside from this the typical storage conditions of toothbrushes

also may serve as a significant reservoir for re-introduction of potential

pathogens to the oral cavity. Moreover, G. N. Karibasappa et al. 2011,

proposed that that toothbrushes stored inside bathrooms with toilet does not

provide contamination on oral pathogens only but as well as general

pathogens since aerosols from toilet flushing can reach as far as six feet.

According Peveska, 2016, the time period of toothbrush usage may also be

connected with its contamination, since as the time of usage increases

accumulation of the deposit inside the bristles also increases which typically

serves as the predominant hub of the bacteria as explains by Karibasappa et

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al (2011) can be significant with this. In addition, Asumang 2019 explains also

that bacteria can eventually proliferate more on closed environments where

likely moist conditions are prominent in which toothbrushes with cover might

be inclined to. Thus, as said by this, contaminated toothbrushes have been

suggested to play a role in both systemic and localized diseases.

Conceptual Framework

Place of Storage Ways of storing and Improper sanitation

 Inside the other factors and disinfection
bathroom  capped  Washing only
 Within the sink  Accumulation of with water
 Under bed had deposits  No sanitation
between routine/plan
bristles
 Oral flora
 Period of
replacement

Bacterial contamination of
toothbrushes
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Research Objectives

The following research objectives will determine the extent on how

microorganisms contaminate toothbrushes, and because highly contaminated

toothbrushes may pose threat to one’s health, these aims of the study were

set to:

1. to identify the microorganisms, present on capped and uncapped

toothbrushes that are stored inside and outside the bathroom

2. to evaluate the bacterial contamination of the toothbrushes particularly

in between tufts of bristles after 30 days of usage by the respondents

Significance of the Study

This study could be benefited by all citizens that they may be informed

about the importance of the implementation of the need of proper storing of

their toothbrushes. This research may not be new to the field of investigating

the cause of bacterial contamination on toothbrushes but this study could be

their bases in identifying the needs of many in dealing with dental health

problems and further knowledge on how one can get specific illnesses from

their toothbrushes.

Further, this study could be a great help to the medical students that

they may be aware about the issues involving the causative agents of

illnesses particularly on the formation of plaques and the damage of teeth,

how one can obtain a disease and detrimental effects out from improper
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storing of toothbrushes and for them to gain knowledge about how to identify

isolates of bacteria from toothbrushes grown on various mediums.

In addition, this study could also be beneficial to medical field as it will

give them an assessment on how to addresses issues regarding the

predisposition of people to various health problems even just only on their

households.

This study could also be a reference or an additional source of information

to academe for studying issues regarding bacterial contamination on

toothbrushes

Lastly, it is helpful for us researchers for it will help us answer our

questions regarding the causes of growth of pathogenic and non-pathogenic

bacteria on toothbrushes and how the number of times of its usage can

contribute to higher colonization.

Scope and Limitation

This study was limited to its main focus which is the identifiaction and

comparison of the changes in microbial contamination on toothbrushes that

are stored inside and outside the bathroom, toothbrushes that are capped and

uncapped and bacterial population after one month of usage of the

toothbrushes. This research primarily will focus on the isolation and

identification of bacteria grow on various media from the streak of deposits

between bristle tufts. In regards with this, researchers will employ

experimental methods and thus the data collection strategy is by means of

laboratory procedures for inoculation of cultures and observations under

microscope.
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In addition, only toothbrushes 1-month duration will be examined to know

their microbial contamination. In line with this, further studies have to be

collected to assess the microbial contamination of toothbrush head used for

half month, 2 months, 2 and one half months, and 3 months to know the

pathogenicity of the microorganisms and to evaluate the occurrence of any

infections in the subjects following the use of contaminated toothbrushes.

And due to the aim of this study, which is to provide accuracy of the results

and of the risk of possible predisposition to isolated pathogens from the

specimens, the researchers will seek help to a registered Medical

Technologists who are currently working at the microbiology section of a

hospital since proper equipment such as incubators, appropriate agars and

proper training on inoculation of microorganisms are highly needed for the

growth and isolation of bacteria.

Definition of Terms

The following terms are operationally defined for the purpose of getting a

clear understanding of the Study.

Aerosol- is a collection of pathogen-laden particles in air and particles may

deposit onto or be inhaled by a susceptible person.

Agar- a gelatinous substance obtained from various kinds of red seaweed

and used in biological culture media

Bathroom- used to refer to any room in a residence that contains a toilet,

regardless of the inclusion of a bath or shower.

Capped toothbrush- any toothbrush with cover or lid on its head.

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Contamination- non-intended or accidental introduction of infectious material

like bacteria, yeast, mold, fungi, virus, prions, protozoa or their toxins and by-

products on certain surfaces such as the toothbrushes.

Culture- is a method of multiplying microbial organisms by letting them

reproduce in predetermined culture medium under controlled laboratory

conditions.

Dental- is of relating to teeth which the bacteria can inhabit and destroys.

Disinfectants- are chemical agents designed to inactivate or destroy

microorganisms on inert surfaces.

Normal biota- are the microbes typically found in healthy people. These

microbes are present at various places in the body and may be pathogenic

(capable of causing disease) but are not in the process of doing so.

Resistant- occurs when microorganisms such as bacteria, viruses, fungi and

parasites change in ways that render the sanitary solutions and medications

used to cure the infections they cause ineffective.

Toothbrush- is made up of bristles and tufts used for cleaning the teeth by

removing food debris and plaques. This will be the primary source of the

streaked specimen used for the inoculation of culture.

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CHAPTER II

MATERIALS AND METHODS

This section presents the sample collection, materials in the study and the

steps in the analysis.

General Flow Chart of the Procedures

SCHOOL’S CONSENT INFORMED CONSENT TO TOOTHBRUSHES

THE RESPONDETS DISTRIBUTION

STERILE PLASTIC TOOTHBRUSHES

RECOLLECTION USAGE FOR 30 DAYS
CONTAINER

MICROBIOLOGY LAB

TOOTHBRUSHES’ SWAB

DIRECT PLATING ON
ENRICHMENT/NURTIENT DIFFERENTIAL AND
BROTH SELECTIVE MEDIA

COLONY MORPHOLOGY

GRAM STAININGS COLONY COUNT

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BIOCHEMICAL TESTS

Sample Collection

The researchers will conduct their research with the help of selected

individuals aged 18-25 years old. A total of 8 toothbrushes will be given to the

respondents and will be collected, 4 toothbrushes for each group based on

place of storage indicated by this study. The 8 toothbrushes will be divided

into two groups in which 4 toothbrushes will be kept inside the bathroom after

usage and another 4 toothbrushes will be kept outside the bathroom. Out of 4

toothbrushes stored each from inside and outside the bathroom, 2 will be kept

with cap and the other 2 will be kept with no cap in each group indicated. All

of the 24 toothbrushes will be collected back after 30 days of usage and will

place inside a plastic sterile container after drying the toothbrush naturally and

the bristles of the toothbrushes will be trimmed using scissors and sample

inoculation will be taken from the streak of deposits between the bristles tufts

taken using sterile swabs. These will be sent immediately to the microbiology

laboratory at the Madonna Medical Center Incorporated in Kidapawan City for

microbiological analysis of the bristles.

Materials
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Materials to be used in this study include toothbrushes as the main

source of streaked sample from deposits between tufts.

Furthermore, different agar media and agar plates will be used for

inoculation of the sample and for the growing of the bacteria This study will

also use nutrient broth which will give the required nutrients that will support

the growth of the bacteria such as the thioglycollate broth to find out various

bacteria based on their oxygen requirements.

In addition, scissors will be used to cut the bristles of the toothbrush and

allowing the researcher to acquired samples between tufts and sterile swabs

for streaking the deposits between tufts for inoculation.

And plastic sterile container for maintaining aseptic measures and

adhering to strict protocol of exposure to pathogens.

Lastly, compound microscope will be used for better observation of

microorganisms that will grow into the medium.

Analysis

In gathering the data, the researchers will be using experimental research

design. The researchers will be conducting their study by first requesting a

consent from the school. After the approval of the letter, the researchers will

automatically conduct the procedures regarding to the study. Then an

informed consent will be obtained from the participants. In addition, the

analysis will be performed in the microbiology section of the laboratory of

Madonna Medical Center Incorporated in Kidapawan City. For the analysis,

the procedures to be used will be based on the study of R. Niveda (2019),

Guatam et. Al (2017) and the microbial test scheme of Cheesbrough, (2006)

Culture
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Under aseptic condition the bristles of the toothbrushes will be trimmed

using scissors, streak of deposits will be taken using sterile swabs. After the

samples are collected from the streak of deposits between the bristles from a

total of eight toothbrushes, it will be inoculated at room temperature into the

nutrient broth and will be incubated for 24 hours (R. Niveda, 2019).

Sterilized agar plates will be selected; agar media to allow growth of

microorganism will be prepared by weighing correct proportion of water and

agar powder according to the manufacturer’s instructions and following

aseptic protocol. The prepared agar media will be poured into the agar plates

and kept in the refrigerator to allow cooling of the agar media and to prevent

contamination. The hard deposit swab will be inoculated into the prepared

agar plates with the prepared agar plates with different agar media (R.

Niveda, 2019).

To isolate the microorganisms, various agar media like MacConkey agar,

Chocolate agar and Blood Agar Plate will be used. After inoculation, agar

plates will be kept in incubator at 37 degrees centigrade to allow for the

growth of microorganisms. Standardization will be done to materials,

instruments, methodology and calibration of the microbial analysis. After 24

hours, agar plates will be observed and examined using compound

microscope and colonies will be counted using a colony counter and

multiplied by the correction factor in order to obtain colony forming unit/ ml.

Furthermore, the degree of contamination will be calculated by: total colony

forming unit/ total area sampled. Growths were identified to genus and

species level following standard bacteriological technique (Gautam et. Al,

2017).
 23

Gram staining

On the following day, colonial morphology will be noted for the isolated

colonies. Gram staining will be performed for isolated colonies.

To perform gram staining, thin smear of about 200mm in diameter will be

made on grease free slide which will be also fixed over a burning flame. A

crystal violet solution will be applied to cover the smear for 30 seconds and it

will be then washed with distilled water. Secondly lugol’s iodine will be applied

to the surface for good 30 seconds. Acetone will be used to decolorize the

stain and lastly, the safranin solution will be covered on the surface for a

minute, which will be washed and allowed to dry at room temperature. Then

the stains will be observed under microscope with oil immersion.

Consequently, red stains will be indicating gram- negative bacteria

(Cheesbrough, 2006)

Biochemical tests

The bacterial isolates will be characterized and identified on the basis of

their colonial, morphological study and biochemical characteristics.

Coagulase, Catalase, Motility, Voges Proskauer, Indole production, Citrate

utilization, Oxidase, Methyl red tests will be performed according to the

scheme of Cheesbrough (2006).

Catalase test will be performed for further differentiation of bacteria such

as the staphylococcus (positive) and streptococcus (negative) using a slant

method. Using a sterile technique, each experimental organism will be

inoculated into its appropriately labeled tube by means of a streak inoculation.

It will be Incubated for 24-48 hours at 37˚C. Three or four drops of the 3%

hydrogen peroxide will be allowed to flow over the entire surface of each slant
 24

culture and each culture will be examined for the presence or absence of

bubbling or foaming. Furthermore, the coagulase test will be used to

distinguish between pathogenic and nonpathogenic members of the genus

Staphylococcus and results will be noted such as all pathogenic strains of S.

aureus are coagulase positive whereas the nonpathogenic species (S.

epidermidis if present from the contaminated toothbrush) are coagulase

negative. This study will be using tube coagulase test method.

Both the methyl red and Voges-Proskauer tests were commonly used in

conjunction with the indole and citrate tests, to form a group of tests known as

IMVIC which aid in the differentiation of Enterobacteria.

Indole Test will be performed for the confirmation of the presence of E.

coli. A wire loop will be used to inoculate overnight growth cultures into a test

tube of 5ml peptone water. The inoculation will be incubated at 37 degrees

celcius for 24 hours after which 5 drops of Kovac’s indole reagent will be

added and shaken gently. A positive reaction will be indicated by the

development of a red color formation on the top layer.

Methyl Red- Voges-Proskauer: Organism will be grown in 5ml MR-VP

broth and will be incubated for 48-72 hrs at 35 degrees celcius after

incubation, 1ml of the broth will be transferred into a test tube and 2-3 drops

of methyl red will be added. Formation of red color indicates positive methyl

red test, a yellow color will indicate negative test. To the rest of the broth, 15

drops of 15% alpha – napthal in alcohol will be added. 5 drops of 40% KOH

will also be added followed with shaken gently. The cap of the tube will be

loosened and development of a red color within 1hr will indicate a positive

test. No color change will indicate negative test (Cheesbrough, 2006).

 25

Citrate Test: The isolates will be inoculated into Simmon’s citrate agar in a

Bijou bottle and will be incubated for 24- 72hrs. Development of a deep blue

colour will indicate a positive reaction (Cheesbrough, 2006).

Motility: The isolate will be inoculated into the motility medium by making

a fine stab with a needle to a depth of 1-2cm long in the tube. It will be then

incubated at 35 degrees celcius for 24-48hrs. Sharply undefined line of

inoculation and cloudiness in the media will indicates a positive result

(organism is motile). Sharply defined and restricted growth to the line of

inoculation will indicates a negative result (Cheesbrough, 2000).

 26

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 29

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APPENDICES

Appendix A
Informed Consent

Appendix B
Transmittal letters

Appendix C
List of materials and equipment

Appendix D
Gantt Chart

Appendix E
Budget proposal

Appendix F

CV/Biography Sketch
 30

INFORMED CONSENT STATEMENT

Introduction
This study is entitled “COMPARISON OF BACTERIAL POPULATION
ISOLATED FROM UNCAPPED AND CAPPED TOOTHBRUSHES STORED
INSIDE AND OUTSIDE BATHROOM DURING 15 AND 30 DAYS OF USAGE’’

Pasiuna
Ang pag-aaral po na ito ay tungkol sa “COMPARISON OF BACTERIAL
POPULATION ISOLATED FROM UNCAPPED AND CAPPED
TOOTHBRUSHES STORED INSIDE AND OUTSIDE BATHROOM DURING 15
AND 30 DAYS OF USAGE’’

Purpose
This study is in partial fulfillment of the requirements for the program: Bachelor
of Science in Medical Technology
Layunin
Ang pag-aaral na ito ay may kaugnayan sa requirements ng kasalukuyang kurso
ng mananaliksik: Bachelor of Science in Medical technology

Process
The researcher will provide toothbrush and collect and gather data from the
healthy students of North Valley College Foundation Incorporated. The data gathering
will be facilitated by the researcher of this study

Paraan
Ang mananaliksik ay magbibigay ng toothbrush, kukunin, at kukuha nga datus
,mula sa mga malulusog na estudyante ng North Valley College Foundation
Incorporated; ito ay pangangasiwaan ng mananaliksik nga pag-aaral na ito

Participant Confidentiality
 31

Any data collected for the study like your personal details and opinions will be
strictly held with utmost confidentiality. This will not be used for any purposes other
than this study.

Pagsasaalang-alang sa Kapakanan at Pagkatao ng Pasyente

Kung ano man ang makuhang datus, mga sagot sa tanong at iba pang mga
detalye tungkol sa iyong pagkatao, ito ay sa pangangalaga ng mananaliksik at hindi
ito gagamitin sa ibang layunin maliban sa pag-aaral na ito.

Cancellation of Consent and Authorization

The participation in this study is voluntary. You may inform the researcher if you
wish not to participate on the study.

Pagkansela ng Pagpapayag na Maisali Bilang Partisipante

Ang pagsali bilang partisipante sa pag-aaral na ito ay buluntaryo lamang at
hindi pinipilit ang sinuman na maging bahagi ng pag-aaral. Kung ayaw nyo po na
maging parte ng pananaliksik, mangyari po lamang na ipagbigay alam sa
mananaliksik.

Participant Certification
I have read and had the opportunity to ask questions on the kind of data asked
from me. I am granting my permission to utilize my answers as part of the data to be
used in this research.

Sertipikasyon
Nabasa ko at naintindihan ang layunin ng pag-aaral. Binigyan ako ng
pagkakataon na magtanong tungkol sa mga kinukuhang datus mula sa akin.
Ibinibigay ko ang aking pahintulot na gamitin ang mga naibigay ko na datus para
sapag-aaral na ito.

ACCEPTED AND AGREED

Sumasang-ayon at Tinatanggap

Signature (Lagda): __________________________ Date (Petsa):

_______________

Printed Name (Pangalan):

_________________________________________________
Contact Nos. (Numero ng
Telepono):_________________________________________
Address (Tirahan):
_______________________________________________________
 32

Interviewed by: _______________________________

If illiterate:
A literate witness must sign (if possible, this person should be selected by the
participant and should have no connection to the research team). Participants who are
illiterate should include their thumbprint as well.
I have witnessed the accurate reading of the consent form to the potential
participant, and the individual has had the opportunity to ask questions. I
confirm that the individual has given consent freely.

Sa Mga May Problema sa Pagbabasa:

Kung may problema sa pagbabasa, maari siyang pumili ng isang taong marunong
bumasa at pinagkakatiwalaan nya namaaaring tumulong para maintindihan ang mga
tanong at ang mga kinakailangang datus sa pananaliksik.

Signature of witness: ___________________________

Print Name of Witness: _________________________
Date: ________________________

Thumbprint of the Participant

33