Efficacy of Normal Oral Hygiene Practices on the

Anaerobic Bacterial and Fungal Content of the Mouth

2009/2010

Class: F.Sci

Name: Martin Walsh

Student No: 7779119J

 Table of contents

Page Title

3 Abstract

4 Introduction

5 Materials and Methods

6 Results

7 Brief Number Run Down

8 Subject 1

9 Subject 2

10 Discussion

11 Discussion (cont.)

12 Identification

13 Research into Candida Albicans

14 Bibliography

15

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 Abstract

Samples from 4 person’s mouths were taken for microbial

analysis. Samples were taken before and after brushing. Recent
studies indicate that brushing alone is not 100% effective at
microbial removal. The aim of this study is to perform a
quantitative analysis of microbial growth in individuals. These
results were calculated taking the following into account; initial
sample size, use of various oral hygiene products as well as the
presence of these products in the sample after its been taken
(in the case of mouthwash and toothpaste). All cases involved
mechanical brushing with a toothbrush and toothpaste, which
for the purposes of this design are being taken as constant
quality. Growth of bacteria was evident in all samples taken
and after cleaning all demonstrated a marked reduction in size
and frequency of colonies on the agar plates used.

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 Introduction

It can be suggested that the best experiments and often the

most revealing and enlightening are the most simple. From the
results of a very simple experiment it is quite easy to deduce
more facts than would be obtained from a larger more complex
design.

The aim of this project was to analyse the bacterial content of

the human mouth and the effects of brushing on those bacterial
levels. It is generally accepted that oral hygiene is necessary
for tooth protection and to prevent halitosis. It is an accepted
fact that the bacteria that live in the mouth are the main
contributors to problems in both of these areas.

While I looked at the effects of other problems associated with

oral bacteria I will mostly focus on the day to day problems
associated with them. The main cause of halitosis is volatile
sulphur compounds (VSC’s). In this instance volatile means
evaporates easily. These VSC’s are the same kind of material
that cause the bad smell found in rotten eggs and flatulence.
These sulphur compounds are the waste products released by
some oral bacteria. Many of these bacteria are anaerobic,
meaning they thrive in an environment without air. As people
sleep with their mouth closed these bacteria thrive and
multiply. This is the cause of “morning breath”. While feeding
they excrete the waste products that create these VSC’s. Other
than VSC’s these bacteria also release these other products as
waste:

• Cadaverine - the smell we associate with corpses.

• Putrescine - the compound responsible for much of the
foul odour produced by decaying meat.
• Skatole - the characteristic smell of human faecal matter.
• Isovaleric Acid - the smell of sweaty feet.

Studies have shown that normal brushing alone does not kill all
the contaminant of the mouth and more steps should be
employed to help kill the harmful bacteria in the mouth.
Different methods of hygiene revealed different results. Though
all samples contained essentially the same bacteria the size

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 and frequency of colonies differed greatly, this may partially
have been related to the unavoidable difference in size of
samples taken the coefficients of these samples (before: after
cleaning) demonstrated the effective differences of the
methods of cleaning employed.

Materials and Methods

Materials –

Cotton swabs, Spoon, Petri dishes, Antiseptic Wipes.

Method –

1. The concave side of a spoon was dragged over the

subjects tongue.

2. A swab was taken from the concave side of the spoon

3. The Petri dish was inoculated. An environment as sterile

as possible (without inhibiting the sample).

4. The subject then brushed their teeth and steps 1 – 3 were

repeated.

5. For each subject in the experiment the same process was

repeated. Each one using their own method of oral
hygiene.

6. Each sample was labelled and stored in a warm and humid

environment just over room temperature.

7. The dish was sealed as air tight as possible to allow the

predominantly anaerobic bacteria to thrive (wrapped in
cling film)

8. The samples were examined after even growth periods,

once at 7 days and once at 14 days, counting from the
day the sample was taken. In each sample, colonies were
counted individually.

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 Martin Walsh
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 Results

Colonies were counted at even periods, once at 7 days and

once at 14 days a 2 day lag phase in all samples was observed.
The information presented in the line charts is the result of an
observed lag phase, extrapolated exponential phase and what
was estimated to be the plateau phase based on the readings
taken at 7 and 14 days. The results for sample 1 are
demonstrated in [fig 1.1] and for sample 2 in [fig 1.2]

Sample 1 uses mechanical brushing and mouth wash (no

tongue scraping) as you can see there is a noticeable
difference in growth trends and numbers as well as speed of
growth of the anaerobic microbes in the agar plates for this
subject.

For sample 2 a much bigger sample was taken in both

instances before and after therefore the numbers for each are
significantly inflated compared to sample1 however the ratios
are fairly representative of their conclusions (ratio’s presented
under charts).

Though great care was taken in all cases ensuring the highest
standards of hygiene were observed in order to minimise
contamination there was clearly contamination evident
displaying in 4 of the eight samples. Subject 3 and 4’s samples
were deemed unreliable and therefore the quantitative analysis
and information put forward are from subject 1 and 2. It was
also noticed that the best performing, both in rate of growth
and countable colonies was the sample from subject 1,
therefore this sample was used in most of my submitted results
as I demonstrated greatest degree in accuracy counting and to
put forward my quantifiable results.

One reason the contaminated specimens were eliminated was

that they were clearly going to inhibit the reproduction and
survival of any fungi that would potentially grow. This was clear
as the “clean” specimens were clearly still in lag phase when
the contaminated bacterial specimens were in log phase.

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 Brief Numbers Run Down

Coloni
es
Subjec Subjec
t1 t2
Day Before Afte Before Afte
r r
1 0 0 0 0
2 0 0 0 0
3 5 3 16 4
4 9 5 23 12
5 17 11 37 20
6 32 20 47 27
7 42 23 55 30
8 53 26 63 32
9 58 31 78 36
10 60 34 89 38
11 63 37 101 40
12 65 39 111 40
13 68 41 118 41
14 69 44 120 43

These numbers are the brief rundown of what later evolved into
the graphs displayed later in this results section.

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 Subject 1

The results for subject 1 demonstrate a reduction from 69

colonies before brushing to 43 colonies after brushing. The full
regime of subject 1 was approximately 3 minutes of rigorous
brushing followed by the use of mouth wash. This shows a ratio
of 1:0.62 demonstrating a 38% reduction in population. It
should also be noted that the sample after brushing itself would
actually still contain some residual remaining quantity of the
mouthwash used while brushing. This seems to have had little
effect on the speed of growth when compared the sample 2.

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 Subject 2

The results for subject 2 demonstrate a reduction from 120

colonies before brushing to 43 colonies after brushing. The full
regime of subject 2 was approximately 3 minutes of rigorous
brushing followed by the use of the OraBrushtm. This shows a
ratio of 1:0.35 demonstrating a 65% reduction in population. As
there were no extra chemicals involved in the process other
than toothpaste and a similar lack of drag was observed, this
indicates that mouthwash itself was having little or no effect on
the rate of growth of the samples in the dish.

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 Discussion

These results show a noticeable superiority of one practice over

another. While no cleaning regime would completely eliminate
all microbial content from the mouth it clearly shows the
efficacy of the OraBrush over generic mouthwash.

Mouthwash has been shown to be one of the most effective

methods of oral hygiene. According to statistics put forward by
their distributors it is capable of destroying up to 99% of
bacteria in the mouth. This is quite likely as many of the
bacteria in the mouth are gram positive and are therefore quite
easy to kill.

The ratios presented in the graphs show a stark contrast

between the efficacy of mouthwash and the efficacy of the
Orabrushtm. Focusing specifically on the levels of gram negative
Candida Albicans it’s quite clear that mouth wash isn’t nearly
as effective as manually scraping the material out.

From these figures and their implications it is relatively simple

to deduce that mouthwash has very little effect on the yeast
present in the samples. The manual removal action of the
OraBrushtm is as much as 27% more effective than brushing
and mouthwash.

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 Discussion (cont.)

Mouthwash

Aqua, Alcohol, Sorbitol, Propyl Alcohol, Poloxamer 407, Benzoic

Acid Sodium Saccharine, Eucalyptol, Methyl Salicylate, Thymol,
Menthol, Aroma, Sodium Benzoate, CL 42053

The alcohols (alcohol, sorbitol and propyl alcohol) present in the

mouthwash are most likely there to kill bacteria. The eucalyptol
has a mild irritant effect on the mucous membranes of the
mouth which should also open them up for reception of the
alcohols so they can act more effectively to kill bacteria.

Toothpaste

Sodium Fluoride (1450ppm), Sorbitol, Aqua , Hydrated Silica,

Glycerine, PEG-12, Sodium Lauryl Sulphate, Aroma, Cellulose
Gum, Sodium Fluoride, Sodium Saccharine, Limonene,
CL74160, CL77891

Toothpaste while containing far less alcohol than mouthwash

also serves its purpose quite well. Many of the ingredients of
toothpaste are focused on the frothing action that manual
brushing causes. Allowing what little alcohol is present to
saturate the mouth to a much greater degree than it could
otherwise.

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 Identification

The identification of the samples was not simple, as a

conclusive reference source could not be found. Due to the
growing conditions there was only one type of organism
evident in the accepted samples. Even through close
examination and thorough research into what it may be
identification was not successful. However through this
research it was found that most gram positive bacteria were
easily killed even by brushing, from that it was deduced that
the organism was most likely a gram-negative organism, there
was no stain available to perform a gram test, however the
evidence supported this postulate. Since the conditions
maintained for the full period were as anaerobic as possible it
was deduced that the main thriving organism found would be
anaerobic in nature but more than likely would have to be aero
tolerant or even a facultative anaerobe, as the conditions were
not entirely anaerobic. These postulates proved to be fruitful.
As research was focused in this area, far more familiar traits
were found in both the morphology and behaviour of the
samples.

Colonies presented with a white smooth texture, multitudinous,

but separate. As the culture grew some colonies would overlap,
the colonies were slow growing and after a 14 day period had
still not reached any noticeable drop off in population, this
suggested that the organisms were not very fast growing and
in fact were not very aggressive either.

From this it was concluded that the samples were actually

yeast which satisfied my reasoning and fitted the circumstance.
(1) Yeast, is a facultative anaerobe, (2) yeast can be gram-
negative (3) morphological comparison of my samples with
known yeast samples concluded that what I had grown was
indeed yeast (4) there have been many cases of yeast growing
very slowly.

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 It is suspected that the slow growing of the oral yeast is due to
a high proportion of inactive ribosome’s [C. Waldron et al
Biochem. J. 1977], its relevance to these samples were merely
conjecture, but is suggested as one explanation for the slow
growth rate.

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 Research into Candida Albicans

This area brought research to the Candida genus of yeasts as

they are known endosymbionts of humans and account for the
majority of yeast presences in and on the human body, with the
main constituent of Candida being C. albicans. C. albicans can
be found on the skin and in the gastrointestinal and
genitourinary tracts and is responsible for the majority of all
Candida infections in the body. These infections include the
generic yeast infection but also extend to a condition known as
Candedemia or Fungemia. The latter conditions are not
typically found in most people but are a severe risk for any
immuneocompromised patients, any patients on
immuneosuppressants and oncology patients. C. albicans
causes 70% of all cases of Candedemia.

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 Bibliography

http://www.wikipedia.com/

http://www.encyclopedia.com

http://www.propeller.com/story/2009/11/26/common-oral-
bacteria-derivative-could-enhance-multiple-sclerosis/

http://www.animated-
teeth.com/bad_breath/t3_causes_of_halitosis.htm

Archives of Oral Biology

Volume 48, Issue 2, February 2003, Pages 117-123,
Copyright © 2003 Elsevier Science Ltd.

Articles from Journal of Clinical Microbiology are provided here

courtesy of
American Society for Microbiology (ASM)
Copyright © 1999, American Society for Microbiology

Biochem J. 1977 December 15; 168(3): 409–415.

J Med Microbiol 29 (1989), 51-54; DOI: 10.1099/00222615-29-1-

51
© 1989 Society for General Microbiology

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 Appendix

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