efficacy of normal oral hygiene practices on the anaerobic bacterial and fungal content of the mouth

8/7/2019 Efficacy of Normal Oral Hygiene Practices on the Anaerobic Bacterial and Fungal Content of the Mouth

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Efficacy of Normal Oral Hygiene Practices on theAnaerobic Bacterial and Fungal Content of the Mouth

2009/2010

Class: F.Sci

Name: Martin Walsh

Student No: 7779119J


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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

Martin Walsh


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Abstract

Samples from 4 persons 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 maincontributors to problems in both of these areas.

While I looked at the effects of other problems associated withoral bacteria I will mostly focus on the day to day problemsassociated with them. The main cause of halitosis is volatilesulphur compounds (VSCs). In this instance volatile meansevaporates easily. These VSCs are the same kind of materialthat cause the bad smell found in rotten eggs and flatulence.

These sulphur compounds are the waste products released bysome oral bacteria. Many of these bacteria are anaerobic,meaning they thrive in an environment without air. As peoplesleep with their mouth closed these bacteria thrive andmultiply. This is the cause of morning breath. While feedingthey excrete the waste products that create these VSCs. Otherthan VSCs these bacteria also release these other products aswaste:

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. Thoughall 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 thepredominantly 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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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 ofgrowth 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 (ratios 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 4s 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 whenthe contaminated bacterial specimens were in log phase.

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Brief Numbers Run Down

Colonies

Subjec

t1

Subjec

t2

Day Before Afte

r

Before Afte

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

742 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 its quite clear that mouth wash isnt 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 yeastpresent 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, BenzoicAcid 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 oftoothpaste 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 ribosomes [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 ofCandedemia.

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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 herecourtesy of

American Society for Microbiology (ASM)Copyright 1999, American Society for Microbiology

Biochem J. 1977 December 15; 168(3): 409415.

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

51

1989 Society for General Microbiology

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Appendix

Martin Walsh

efficacy of normal oral hygiene practices on the anaerobic bacterial and fungal content of the mouth

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