Yearbook on Journal of the Japan Society of Nursing Research 2014

Visualization of Oral Bacteria Using a Fluorescent Dye and Its Application for theAssessment of Oral Care
Miki Yamamoto1), Hiroshi Murabayashi1), Yukio Omori1), Shigeko Yasunami2)
Key Words :oral careoral bacteria

I. Introduction

ABSTRACTThe purpose of this study was to investigate the number of oral bacteria and its changes by visualizing thepresence of bacteria using a fluorescent dye to stain bacterial DNA, 4’, 6-diamidino-2-phenylindole (DAPI),and examine the usefulness of this method for oral-care assessment. In 10 healthy adults and 10 elderly
persons, from whom informed consent was obtained, we investigated diurnal changes in the number of oral
bacteria, oral conditions, and their lifestyle. In addition, we measured the number of oral bacteria before
and after oral care training in 11 students belonging to Nursing College A. Bacteria were collected by wiping
3 points in the oral cavity (on the tongue, inside of the cheek, and surfaces of upper and lower teeth) withclean swabs. DAPI facilitated visualization of the presence of bacteria in a sample in a short period andcounting, suggesting its usefulness for oral-care assessment.

bacterial counts with a fluorescent dye can be applied for theassessment of oral care, we investigated diurnal changes in
the number of bacteria in the oral cavity and those before andafter oral care using the following two methods.

Oral care is important to prevent aspiration pneumonia
in elderly persons, persons with consciousness disorder,and severe-status patients under respiratory control1)2).
Fungus, Klebsiella pneumoniae, Staphylococcus aureus, andPseudomonas aeruginosa cause geriatric pneumonia3). Thesebacteria cause chronic respiratory infectious diseases as periodontogenic bacteria by insufficient oral care. In patients whooral ingestion is impossible, aspiration of these bacteria iseasy to cause pneumonia related to the reduction of the oral
self-cleaning function4). As oral indigenous bacteria which do
not usually show pathogenicity, cause respiratory infectious
diseases in infection-prone persons such as the elderly andsevere-status patients, it is necessary to decrease the numberof oral bacteria through oral care.
Therefore, bacteriological examination of the oral cavity
is essential to evaluate the efficacy of oral care. However, asbacterial medium culture and identification of the bacteria iscomplex and take for a long time, there have been few studies and evidence involving elderly persons or chronic-phasepatients regarding oral care. To clarify these issues, the presence and change of number of oral bacteria must be evaluatedusing a simple method.
The fluorescent dye, 4’, 6-diamidino-2-phenylindole (DAPI)5)
which has been used for nuclear staining of cells and microorganisms, facilitates simple microscopic examination andmeasurement of the presence, number, and morphology of oral
bacteria in a short period. In addition, we established conditions for the visualization of bacteria using DAPI, and examined
simply and promptly of the presence, number, and morphologyof bacteria in samples6).
We investigated the number of oral bacteria and its changes
by simply visualizing the presence of bacteria using a fluorescent dye DAPI for staining of bacterial DNA, and examined
whether this method can be applied for the assessment oforal care.

1. Diurnal changes in the number of bacteria in the oral cavity
a. Subjects

The subjects consisted of 10 healthy adults and 10 elderly
persons (total: 20).
b. Sample collection

After wiping 3 points (on the tongue, inside of the cheek,
and surfaces of upper and lower teeth) in each subject’s oralcavity with clean swabs, respectively, they were stored in abottle containing a fixative (4% paraformaldehyde). The subjects were instructed to collect samples themselves. Their procedures were confirmed through an author’s demonstration
based on a leaflet regarding the collection method so that
there were no marked differences in the method.
Sampling was performed at night (before bedtime), in themorning (after waking up), and at noon (before lunch) to
examine diurnal changes in the number of oral bacteria ineach subject. To investigate the association of this parameterwith the oral state or lifestyle, a survey regarding the presenceor absence of dental disease/denture and tooth brushing was
conducted in the subjects.
c. Calculation of the number of bacteria and visualization

Samples were filtered using a syringe filter (proprietary
name: Mini Sarto, pore size: 5.0 µm, Sartorius Stedim Japan).
After large residues and exfoliating mucosal cells were
removed, 10 µL of each sample was collected, dropped ona slide glass, and dried at room temperature for 10 minutes.
These operations were performed on a clean bench.
After washing, each sample was stained with 10 µg/mL
DAPI solution (phosphate-buffered saline, pH 7.4, SigmaAldrich Japan) for 30 minutes, washed in distilled water,
covered with a covering glass, and photographed under a fluorescence microscope (filter No. 2, Zeiss Axioplan 2, Zeiss). In
addition, the photographed images were input to a computer,and bacteria were counted. The number of all bacteria in unit
area (0.013 mm2) was measured. The total number of bacteria

II. Methods

In this study, to clarify whether the visualization of oral

1) The Japanese Red Cross Hokkaido College of Nursing2) Kameda College of Health Sciences

Corresponding author. Tel.: +81 157 66 3336.
E-mail address: mikiy@rchokkaido-cn.ac.jp (M. Yamamoto)

1
This paper was published at “the Japan Society of Nursing Research” Vol.35 No.2 in 2012.

Yearbook on Journal of the Japan Society of Nursing Research 2014

d. Analytical method

in 5 areas (0.065 mm2) per sample was calculated.
d. Analytical method

The number of bacteria was expressed as the mean±
standard deviation. In each subject, the values before andafter oral care were compared.

The data were expressed as the mean±standard deviation(S.D.). For statistical analysis, the Mann-Whitney U test wasused. A P-value of <0.05 was regarded as significant.

e. Ethical consideratione. Ethical consideration

The purpose and method of this study, free will-based
participation, and the absence of identification of individuals were explained to the subjects. Informed consent was
obtained from all subjects. The subjects of this experimentwere students, and it was carefully conducted during lessons so that there was no influence on the lessons. Informed
consent was obtained after explaining the following points:
the results would have no effect on academic evaluation, andthere would be no disadvantage even if they did not participate in this study.

The purpose and method of this study, free will-based
participation, and the absence of identification of individuals were explained to the subjects. Informed consent was
obtained from all subjects. Prior to this study, its protocol
was approved by the Ethics Review Board of The Japanese
Red Cross Hokkaido College of Nursing.
2. Number of bacteria in the oral cavity before and after oral
careIII. Resultsa. Subjects

1. Diurnal changes in the number of bacteria in the oral cavity

The subjects were 11 first-year students attending technicaltraining for daily life support in Nursing College A.

There was no dropout case. The data from all subjects wereanalyzed.

b. Collection of samples

a. Number of bacteria in the oral cavity

Oral bacteria were collected before and after oral caretraining (gargling and brushing) by students. After wiping 3
points (on the tongue, inside of the cheek, and surfaces ofupper and lower teeth) in the oral cavity with clean swabs,
respectively, they were stored in a bottle containing a fixative (4% paraformaldehyde). The students’ procedures wereconfirmed through an instructor’s demonstration and training
by themselves.

(1) Subjects’ age and oral state

The subjects consisted of 10 aged 20 to 39 years and 10
aged 70 to 79 years. There were 14 females and 6 males.
Concerning the presence or absence of dentures, they were
present in 1 of the former and 5 of the latter. Concerning thepresence or absence of dental disease, it was present in 6 of
the former and 2 of the latter.
The frequency of tooth brushing was once a day or twice
a day in 8 of the former and 9 of the latter. It was 3 times ormore in 2 and 1, respectively.
Oral discomfort was absent in 6 of the former and 4 of thec. Calculation of the number of bacteria and visualization

Each sample was treated as described above (1. Diurnalchanges in the number of bacteria in the oral cavity).

Table 1. Subjects

Subject No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

Age group
20s
20s
20s
20s
20s
30s
20s
20s
30s
20s
70s
70s
70s
70s
70s
70s
70s
70s
70s
70s

GenderFemaleFemaleFemaleFemaleFemale
Male
Male
Male
Male
Male
FemaleFemaleFemaleFemaleFemaleFemaleFemaleFemaleMale
FemaleDentures
AbsentAbsentAbsentAbsentAbsentAbsentAbsentPresent
AbsentAbsentPresent
Present
Present
AbsentAbsentPresent
AbsentPresent
AbsentAbsentDental disease
AbsentAbsentAbsentPresent
Present
Present
AbsentPresent
Present
Present
AbsentAbsentAbsentPresent
Present
AbsentAbsentAbsentAbsentAbsentTooth brushing (times/day)
2
2
2
1
3
1
2
1
2
3
2
1
4
2
1
1
2
2
2
2

*Oral discomfort: 1. Food retention between teeth/dentures, 2. Dirty tongue, 3. Halitosis, 4. Dry mouth, 5. Others

2This paper was published at “the Japan Society of Nursing Research” Vol.35 No.2 in 2012.

Oral discomfort*
Absent4, 5AbsentAbsent2
AbsentAbsentAbsent2, 3, 41
1
1
Absent3
3
4
4
AbsentAbsentAbsentYearbook on Journal of the Japan Society of Nursing Research 2014

latter. Subjects with oral discomfort, aged 20 to 39 years,
complained of a dirty tongue, halitosis, dry mouth, andothers. Those aged 70 to 79 years complained of food retention between their teeth/dentures, halitosis, and dry mouth(Table 1).

a fluorescence microscope. The bacteria measured approximately 2 to 3 µm, being globular or in chains. Bacteria wereobserved singly or in colonies. Figure 1 shows fluorescencemicroscopy photographs of diurnal changes in oral bacteria
in subjects aged 20 to 39 and 70 to 79 years, respectively(Subject Nos. 7 and 14). Thus, oral bacteria were fluorescently
visualized on DAPI staining in all subjects.

(2) Visualization of oral bacteria using a fluorescence microscope

Oral bacteria were stained with DAPI, and examined using

Subject No. 7: Before bedtime

Subject No. 14: Before bedtime

Subject No. 7: Before breakfast

Subject No. 14: Before breakfast

Subject No. 7: Before lunch

Subject No. 14: Before lunch

age: 20-30s

age: 70s

Figure 1. Visualization of oral bacteria using a fluorescence microscope

3This paper was published at “the Japan Society of Nursing Research” Vol.35 No.2 in 2012.

Yearbook on Journal of the Japan Society of Nursing Research 2014

aged 70 to 79 years, it was 1.7 to 1.9 times higher than at night
and at noon; the increase was less marked than in the young
subjects. The elderly subjects tended to show a high numberof oral bacteria all day.

(3) Diurnal changes in the number of oral bacteria

The diurnal changes in the number of oral bacteria in eachsubject are shown in Figs. 2 and 3. In most subjects aged 20
to 39 and 70 to 79 years, the number of oral bacteria was
the highest in the morning, followed by those at night and atnoon. In 1 of the young subjects and 1 of the elderly subjects,
the changes were reversed in comparison with the other subjects. In 1 of the former, the number of oral bacteria was thehighest at noon.
We examined diurnal changes in the number of oral bacteria with respect to age. In most subjects aged 20 to 39 years,
the mean number of oral bacteria in the morning was 2 to 3
(maximum: 5) times higher than at night and at noon. In those

The mean number of oral bacteria with respect to age
is presented in Fig. 4. In subjects aged 20 to 39 years, themean number of oral bacteria at night, in the morning, and atnoon was 67.145.4, 138.3±95.7, and 62.5±57.8, respectively.
In those aged 70 to 79 years, the values were 113.6±69.5,
184.5±115.0, and 122.2±86.8, respectively. At all points,
the number of oral bacteria in the latter was higher than in
200

Subject

450

No.1

400

No.2

350

No.3

300

Number of bacteria䠋0.065䟚2

Number of bacteria䠋0.065䟚2500(4) Number of oral bacteria with respect to age

No.4

250

No.5

200

No.6

150
100

No.7

50

No.8

0

No.9

Noon
᫨

ᮅ
Morning

ኪ
Night

No.13

300

Number of bacteria䠋0.065䟚2

Number of bacteria䠋0.065䟚2

No.12

350

No.14

250

No.15

200

No.16

150
100

No.17

50

No.18

0

No.19

Noon
᫨

Morning
ᮅ

ኪ
Night

60

77.4
56.7

40

27.7

20
0
ኪ
Night

ᮅ
Morning

40
20

Night
ኪ

MorningᮅNoon᫨䠆P<0.05

350
300

266.4

250

䠆

200

168.8

150
100

Dentures
Present䠆Dentures Absent

110.6

102.6

133.8

58.4

50

Morning
ᮅ

Noon
᫨

270.0

䠆
P<0.05Present

250

Absent

106.7
71.6

62.5

300

Number of bacteria䠋0.065䟚2

Number of bacteria䠋0.065䟚2

80

67.1

60

Figure 5. Mean number of oral bacteria with respect to thepresence or absence of dentures

Present

140

100

80

ኪ
Night

151.9

120

100

0

Figure 3. Diurnal changes in the number of oral bacteria(age: 70s)

160

122.2

113.6

120

400

No.11

400

140

138.3

Figure 4. Mean number of oral bacteria with respect to age

Subject

450

䠆

䠆

160

0

Figure 2. Diurnal changes in the number of oral bacteria(age: 20-39)500

20-30s
70s

184.5

180

᫨
Noon

䚷䚷䚷䚷䚷䚷䚷䚷㻞㻜㻙㻟㻜㼟200

Absent
177.0

163.1

150
100

124.5 121.6

97.8

50
0
ኪ
Night

ᮅ
Morning㻣㻜㼟Figure 6. Mean number of oral bacteria with respect to the presence or absence of dental disease4
This paper was published at “the Japan Society of Nursing Research” Vol.35 No.2 in 2012.

᫨Noon

Yearbook on Journal of the Japan Society of Nursing Research 2014250

Present
193.8

200

Number of bacteria䠋0.065䟚2

Number of bacteria䠋0.065䟚2

250

Absent

150
101.3

89.8

100

53.5

52.0

50

68.5

208.5
200

168.5

150
110.2

Morning
ᮅ

Night
ኪ

171.8

89.2

100
50

ኪ
Night

Noon
᫨

Absent

118.8

0

0

Present㼍㼓㼑㻚㻞㻜䡚㻟㻜㼟

MorningᮅNoon᫨㼍㼓㼑㻚㻣㻜㼟

Figure 7. Mean number of oral bacteria with respect to the presence or absence of oral discomfort

㻝㻠㻜㻌㻌

Subject B: Before oral care㻮㼑㼒㼛㼞㼑㻌㼏㼍㼞㼑㻌㻝㻞㻜㻌㻌㻭㼒㼠㼑㼞㻌㼏㼍㼞㼑㻌㻺㼡㼙㼎㼑㼞㻌㼛㼒㻌㼎㼍㼏㼠㼑㼞㼕㼍㻛㻜㻚㻜㻢㻡㼙㼙㻞㻌

㻝㻜㻜㻌㻌

㻤㻜㻌㻌

㻢㻜㻌㻌

㻠㻜㻌㻌

㻞㻜㻌㻌

㻜㻌㻌
㻭㻌

㻮㻌

㻯㻌

㻰㻌

㻱㻌

㻲㻌

㻳㻌

㻴㻌

㻵㻌

㻶㻌

㻷㻌

㼟㼡㼎㼖㼑㼏㼠㻌

Figure 8. Number of oral bacteria before and after oral care

Subject B: Aer oral carethe former. It was significantly higher at night and at noon(p<0.05).
(5) Number of oral bacteria with respect to the oral state

The mean number of oral bacteria with respect to the presence or absence of dentures in subjects aged 70 to 79 years
is shown in Fig. 5. In those with dentures, it was 58.4±24.6 atnight, 102.6±38.0 in the morning, and 110.6±119.2 at noon.
In those without dentures, the values were 168.8±51.5,
266.4±107.4, and 133.8±48.8, respectively. At all points, thenumber of oral bacteria in those without dentures was higher
than in those with dentures. It was significantly higher atnight and at noon (p<0.05).
The mean number of oral bacteria with respect to the presence or absence of dental disease in each age group is shownin Fig. 6. Dental diseases were present in 6 subjects aged 20
to 39 years, but absent in 4. They were present in 2 subjects
aged 70 to 79 years, but absent in 8. There were no significant
differences. However, in the two age groups, the number oforal bacteria in subjects with dental diseases was higher thanin those without dental diseases.
The mean number of oral bacteria with respect to the presence or absence of oral discomfort in each age group is shownin Fig. 7. Oral discomfort was present in 4 subjects aged 20
to 39 years, but absent in 6. It was present in 6 subjects aged

Figure 9. Oral bacteria on fluorescence microscopy before
and after oral care

70 to 79 years, but absent in 4. There were no significant differences. However, among subjects aged 20 to 39 years, thenumber of oral bacteria at night and in the morning in thosewith oral discomfort was higher than in those without oral
discomfort. Among those aged 70 to 79 years, the number oforal bacteria in those without oral discomfort was higher thanin those with oral discomfort.

5This paper was published at “the Japan Society of Nursing Research” Vol.35 No.2 in 2012.

Yearbook on Journal of the Japan Society of Nursing Research 2014

requiring nursing indicated that bacteria that cause aspiration
pneumonia, opportunistic infection, or endocarditis were present on dentures9). In this survey, the number of oral bacteria
was estimated to be higher in subjects with dentures. However, the healthy subjects of this study washed their dentures
after meals, and many subjects removed them before sleep.
This may have contributed to the finding that the numberof oral bacteria on waking-up in subjects with dentures was
significantly lower than in those without dentures.
Concerning changes in the number of oral bacteria withrespect to the presence or absence of dental disease, thenumber of oral bacteria in both young and elderly subjects
with dental diseases was slightly higher than in those without
dental diseases. As dental diseases such as caries and periodontal disease are primarily caused by periodontal diseaseassociated bacteria10), this may have resulted in the findingthat the number of bacteria was higher in subjects with dental
diseases.
Concerning changes in the number of oral bacteria withrespect to the presence or absence of oral discomfort, thenumber of oral bacteria in young subjects with oral discomfort was slightly higher than in those without oral discomfort.
Halitosis, a dirty tongue, and dry mouth reflect oral uncleanliness or a reduction in the oral self-cleaning function by saliva;
the number of oral bacteria may have been high. However,
the oral state was not confirmed, and its association with thenumber of bacteria was unclear.
Fluorescent staining of bacteria with DAPI based on thepresence of oral bacteria, their count, and diurnal changes inyoung and elderly subjects facilitates simple, prompt measurement of the presence of bacteria and their relative count.
This method may be applicable for bacteriological assessmentof the oral state.

2. Number of oral bacteria before and after oral care

All required samples were collected, and the data from all
subjects were analyzed.
The subjects were 11 female students, aged 20 to 39 years,
belonging to Nursing College A. The number of bacteria before
oral care ranged from 15 to 119, with a mean of 59.5±9.1.
After oral care, it ranged from 23 to 84, with a mean of
51.2±6.1. After oral care, the number of bacteria decreasedin 7 students, and increased in 4 (Fig. 8).
Representative photographs of oral bacteria on fluorescence microscopy before and after oral care are presented inFig. 9. Thus, the number of bacteria before and after oral careand its increase/decrease were visualized.
IV. Discussion

Oral care influences oral cleanliness, oral/swallowing functions, and activities of daily living (ADL). Thus, although theoral care is important, it is difficult to standardize its techniques and procedures and its standardization is not yet
established.
In addition, neither the necessity nor assessment of oral
care has been standardized. Although there are several
parameters for oral assessment, most are based on subjective
assessment. Concerning subjective assessment, a previous
study reported the results of assessment related to differenceson occupation/specialty of nurses and dental hygienists andtheir reliability7). However, it is difficult to apply them in clinical practice. As bacterial culture for research as a method of
objective assessment requires many hours and is costly, it is
impossible to use it for the assessment of oral care in clinicalpractice.
In this study, we used a fluorescent dye DAPI as a marker
for the visualization of bacteria and measurement and as an
objective evaluation method available for the assessment oforal care.

2. Number of oral bacteria before and after oral care

In the students specializing in nursing, we confirmed anoral care-related increase or decrease in the number of oral
bacteria. Appropriate brushing and gargling cleaned the oralcavity, decreasing the number of oral bacteria.
However, the number of bacteria increased after oral care insome subjects. This was possibly because gargling was insufficient after brushing to promote the exfoliation of biofilms,
such as acquired capsules and plaque, on the tooth surfaces.
This was consistent with the results presented by Sakota etal.11), who performed bacterial culture to evaluate the efficacy
of oral care in students specializing in nursing. Therefore, it
may be possible to bacteriologically evaluate the effects oforal care using DAPI.
In addition, the evaluation method used in this study facilitates observation of fluorescence microscopy photographs, inwhich bacteria are visualized, on a personal computer; therefore, it can also be applied to confirm effective procedures fororal care in nursing education.

1. Diurnal changes in oral bacteriaWith respect to diurnal changes in oral bacteria, we investigated the presence of oral bacteria in healthy subjects aged
20 to 39 and 70 to 79 years. In the present study, oral bacteria were stained with DAPI and it was possible to confirm
the presence of bacteria and count them using a fluorescencemicroscope. Furthermore, the fluorescence of bacterial DNA
was observed in all bacteria in the oral cavity. Their shape andrelative number could be observed and measured. It was also
possible to accurately evaluate diurnal changes in the numberof oral bacteria. The following results were consistent withthose using the bacterial culture method: (1) the number oforal bacteria before meals was the highest, and it decreased
after meals, but gradually increased thereafter; and (2) the
number of oral bacteria increased at night, reaching a maximum at the time of waking-up8).
In addition, the number of oral bacteria in the elderly group
was higher than in the young group. This may be due to theoral self-cleaning function reduced with a decrease in saliva
secretion at an advanced age.
Concerning dentures which are commonly used by elderly
persons, the number of oral bacteria in subjects without dentures was higher than in those with dentures. Dentures may
become a reservoir for bacteria. A study involving individuals

3. Significance of this study and indication for nursing

This study showed that the observation of bacteria withDAPI facilitated the objective assessment of the oral state andefficacy of oral care, as it is simple and makes the visualizationof the presence of bacteria possible.
Medium culture and identification of bacteria are complex, requiring much time. They do not reflect the oral state
6This paper was published at “the Japan Society of Nursing Research” Vol.35 No.2 in 2012.

Yearbook on Journal of the Japan Society of Nursing Research 2014

at sampling. Therefore, it is difficult to apply these proceduresfor clinical care. However, the observation of bacteria withDAPI made it possible to confirm the presence of bacteria anda care-related increase/decrease in the number of bacteria ina short period, suggesting its usefulness for oral-care assessment and the examination of methods/procedures.
Effective oral care and assessment are also important toprevent respiratory infection in elderly persons and severestatus patients. Simple bacteriological evaluation may be significant as an objective oral assessment method.

of The Japanese Red Cross Society of Nursing Science (Junein 2007, Aichi) and the 33rd meeting of the Japan Society of
Nursing Research (July in 2007, Iwate).
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8) Nakagaki, H, Kanbara, M, et al. [ed.]: Dental hygiene for clinicalpractitioners, revised version. Nagasue Shoten Publisher, 2000.
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4. Limitations of this study and future issues

In this study, most oral bacteria were globular or in chains,suggesting the presence of cocci or Streptococcus. However,it was impossible to accurately identify the type of bacteria,
which is a limitation of this study, raising a future issue.
In the future, the usefulness of our method for oral-careassessment should be established by clarifying changes in
the number of bacteria before and after oral care in clinicalpractice.
V. Conclusion

Using a fluorescent dye, DAPI, oral bacteria were investigated. It was possible to visualize the presence of bacteria ina sample in a short period and count them. This method may
be applicable for oral-care assessment.
Acknowledgments

We thank the subjects and relevant persons for their cooperation.
An abstract of this study was presented at the 8th meeting

7
This paper was published at “the Japan Society of Nursing Research” Vol.35 No.2 in 2012.