12/8/2013

BIOLOGY EXTENDED ESSAY

Comparative study on Miswak (Salvadora persica) and Colgate
toothpaste effectiveness on the normal oral flora

Research Question: To what extent is Miswak more
effective than toothpaste in reducing certain colonies
of oral bacteria, as observed by inoculation of pre and
post usage oral swabs?

Momina Amjad
Candidate Number: 002223-0028

Word Counts
Abstract: 288
Essay: 3,973


Abstract
Salvadora persica or miswak has been used for a long time in Middle East and North Africa
regions by Muslims for maintenance of oral hygiene. I wanted to investigate whether miswak
actually had an antimicrobial effect and if this was greater than or less than that of toothpaste.
Subsequently, I also wanted to determine which specific microorganisms were reduced the
most and the implications of miswak on tooth decay prevention and worldwide healthcare
costs associated with that. Therefore this research question was formulated for the Extended
Essay: To what extent is Miswak more effective than toothpaste in reducing certain colonies
of oral bacteria, as observed by inoculation of pre and post usage oral swabs?
A sample of 10 people was selected for this study, 5 for the miswak trials and 5 for toothpaste
trials. An oral swab was taken by the subjects and then they were asked to brush their teeth
with either miswak or toothpaste for two minutes, after which another swab was taken. These
swabs were later inoculated in Blood agar, Chocolate agar, MacConkey agar and Sabouraud
agar to qualitatively observe (using an ACFOR scale) the reduction of specific colonies between
the pre and post treatment samples. Gram staining was then performed to help identify various
bacterial species.
The results of the experiments indicated that miswak was more effective on cariogenic (tooth
decay causing) species of bacteria such as Streptococcus mutans. Toothpaste, on the other
hand, was more effective on potential pathogens like Staphylococcus aureus. Miswak and
toothpaste were equally effective on gamma hemolytic colonies of Neisseria species. More
research has to be done with quantitative aspects in order to be conclusive about these
findings and integrate miswak as a major means of tooth decay prevention.




Acknowledgements
I would like to thank the Ministry of Health- North Batinah region, Oman, for letting me carry
out my experiment and use all the required resources at the Pathology department in Sohar
Hospital. I would also like to thank Naima Salem Khalfan Al Raisi and Ahmed Abdullah
Mohammed Omer for all of their support and time, during and after their working hours, and
for teaching me all the practical skills in microbiology that were required in this research.

Table of Contents
Introduction .................................................................................................................................... 1
Biology of plaque formation and oral microflora .................................................................... 2
Secondary Research ....................................................................................................................... 5
Primary Research ........................................................................................................................... 7
Methodology .............................................................................................................................. 7
Materials ..................................................................................................................................... 7
Variables ..................................................................................................................................... 8
Notes on some controlled variables ........................................................................................ 9
Justification of the agar media ................................................................................................ 9
Procedure .................................................................................................................................. 10
Results ........................................................................................................................................... 13
a) Processed results ................................................................................................................... 14
b) Gram staining ........................................................................................................................ 15
Discussion of the results .............................................................................................................. 19
a) Explanation ............................................................................................................................ 19
c) Anomalies .............................................................................................................................. 19
Limitations .................................................................................................................................... 21
Conclusion .................................................................................................................................... 23
Bibliography .................................................................................................................................. 26
Appendix 1 .................................................................................................................................... 29
1.1: Streaking ............................................................................................................................. 29
1.2: Aseptic technique ............................................................................................................... 29
Appendix 2 .................................................................................................................................... 30
2.1: Colony Morphology ............................................................................................................ 30
2.2: Gram staining procedure ................................................................................................... 30
Appendix 3 .................................................................................................................................... 32
Appendix 4 .................................................................................................................................... 33

1

Introduction
Oral disease is the fourth most expensive disease to treat in most industrialized
countries
1
where 510% of public health expenditure relates to oral health
2
. In most developing
countries, if treatment were available, the costs of dental caries in children alone would exceed
the total health care budget for children
3
. With this serious economic impact it is important to
find out cost effective alternatives to preventative dental care.
The aim of this study is to identify any antimicrobial effect that Salvadora persica might have. S.
persica grows naturally in environments from India to West Africa
4
. If there is indeed a
significant antimicrobial effect of Miswak, it can be introduced to other communities besides
people from the Middle East North Africa region that have traditionally used it as the primary
means of oral hygiene maintenance. Infective endocarditic (IE) is a rare infection with high
mortality rate in the lining of the heart or heart valves that could destroy these valves. The
most causative organisms in infective endocarditic are viridans Streptococci
5
(a pseudo-
taxonomic grouping) which is the predominant subtype Streptococci species found in the oral
cavity. If cost effective alternatives are available, more people will be motivated to use them
reducing the aforementioned economic impact and chances of acquiring IE. This led to the
development of the following research question for my extended essay in Biology:
Research Question: To what extent is Miswak more effective than toothpaste in reducing
certain colonies of oral bacteria, as observed by inoculation of pre and post usage oral swabs?

1
Petersen, P. et al. C. 2005. The global burden of oral diseases and risks to oral health. Bulletin of the World Health
Organization, 83 (9), pp. 661-669. Available at:http://www.scielosp.org/scielo.php?pid=S0042-
96862005000900011&script=sci_arttext&tlng=p>t[Accessed: 31 Oct 2013]
2
US Department of Health. Health care financing administration. National health expenditures, 1998. Available at:
http://www.nidr.nih.gov/sgr/sgrohweb/toc.htm [Accessed 2 Nov 2013]
3
Yee R, Sheiham A. The burden of restorative dental treatment for children in third world countries. International
Dental Journal 2002;p.52
4
World Agroforestry Centre, 2013. Species Information. [online] Available
at:http://www.worldagroforestrycentre.org/Sea/Products/AFDbases/AF/asp/SpeciesInfo.asp?SpID=1477
[Accessed: 3 Nov 2013]

5
Keys, T. 2012. Infective Endocarditis. [online] Available at:
http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/infectious-disease/infective-
endocarditis/#t0015 [Accessed: 3 Nov 2013].
2

Biology of plaque formation and oral microflora
The oral cavity in human beings is host to a large variety of bacteria. The surface of teeth,
gingival, hard palate, soft palate and all the areas of the tongue contain both gram positive and
gram negative bacteria. Most of these bacteria are not particularly harmful on their own; they
have a commensal relationship
6
with human beings wherein they derive benefit from the food
and the environment provided by our mouth but we are not directly harmed or benefitted by
their presence. Indirectly, these bacteria seem to be both harmful and beneficial to human
beings. The benefit arises from the simple fact that they occupy space in the oral cavity and that
to an extent prevents colonization by the more dangerous pathogenic microbes in our
surroundings and food.
A predominant number of the numerous bacteria that live in the oral cavity is Streptococcus
mutans
7
. Teeth have non shedding surfaces, so bacteria can form a biofilm
8
in which various
colonies stick to each other embedded in an extracellular matrix while being firmly attached to
the tooths surface. This is called plaque and when plaque accumulates over time, it mineralizes
forming calculus or tartar. S. mutans is a gram positive bacterium that consumes sugar and
converts it into lactic acid. Lactic acid erodes the enamel on the tooths surface and sustained
erosion develops dental caries. The table overleaf adapted from Todars Online Textbook of
Bacteriology
9
summarizes the prevalence of certain colonies of oral bacteria and their
pathogenicity.

6
Encyclopedia Britannica. 2013. commensalism (biology). [online] Available at:
http://www.britannica.com/EBchecked/topic/127789/commensalism[Accessed: 3 Nov 2013].
7
Oliver, D. 2003. Microbes and You: Normal Flora. [online] Available at: http://www.scq.ubc.ca/microbes-and-you-
normal-flora/[Accessed: 22 Sep 2013]
8
Encyclopedia Britannica. 2013. biofilm (biology). [online] Available
at: http://www.britannica.com/EBchecked/topic/1368883/biofilm[Accessed: 3 Nov 2013].
9
Todar, K. n.d. The Normal Bacterial Flora of Humans. [online] Available
at: http://textbookofbacteriology.net/normalflora.html [Accessed: 22 Sep 2013]
3


Table 1: Abundant bacteria in the oral flora


To prevent dental caries and gum diseases (gingivitis, periodontitis), most people nowadays
brush their teeth with toothpaste at regular intervals. Maintenance of oral hygiene, however, is
not a novel concept. In the past many people recognized that various plants and organic
material had medicinal properties; for instance Neem or Azadirachta indica has been used in
South Asia for millennia as an
antibiotic
10
. As mentioned in the
introduction, people in the Middle
East/North Africa region and the wider
Muslim world have used Salvadora
persica(Figure 1)
11
(commonly called
Miswak, Sewak or Arak in Arabic) for
protecting their teeth from disease.

10
BBC News. 2006. Neem: India's tree of life. [online] Available at:
http://news.bbc.co.uk/2/hi/south_asia/4916044.stm [Accessed: 3 Nov 2013].
11
Roxburgh, W. 1793. Illustration of Salvadora persica. [image online] Available at: http://www.kew.org/plants-
fungi/Salvadora-persica.htm[Accessed: 3 Nov 2013]
Bacterium Presence
in the Oral
Cavity
Staphylococcus
epidermidis
++
Staphylococcus aureus* +
Streptococcus mitis ++
Streptococcus salivarius ++
Streptococcus mutans* ++
Neisseria sp. +
Neisseria meningitidis* +
Haemophilus influenzae* +
Lactobacillus sp. ++
Key:
++ = nearly 100
percent
+ = common
(about 25
percent)
* = potential
pathogen

Figure 1: Illustration of Salvadora persica
4

This practice is still prevalent today because of the availability, low cost, simplicity, and religious
or traditional associations of chewing sticks. The World Health Organization has also
recommended the use of these sticks as an effective tool for oral hygiene
12
. Miswak sticks act
as natural toothbrushes with toothpaste in it; its chemicals constituents have some
antibacterial properties and the fibers in the stick act as bristles of a toothbrush.

12
Prevention methods and programmes for oral diseases. 1985. [E-book] Geneva: World Health Organization.
[Accessed: 5 Nov 2013].
5

Secondary Research

In a chemical investigation, miswak was reported to contain trimethylamine, salvadorine,
chlorides and fluorides in large amounts as well silica, sulfur, vitamin C and small quantities of
tannins among other chemicals
13
. The fluoride content is known to be beneficial because it
helps prevent tooth decay by repairing the enamel layer. This makes the tooth more resistant
to acid attacks from plaque bacteria
14
. The silica helps to clean the teeth by acting as an
abrasive. Chlorides are known disinfectants and tannins are astringents (chemical compounds
that tend to shrink or constrict body tissues) which are sometimes useful in healing oral
ulcers
15
.

In addition to these chemicals naturally present in miswak, it is also the mechanical cleaning by
the chewing stick that helps prevent dental decay. A clinical study
16
was conducted regarding
the efficacy of miswak. 380 participants were examined dentally in this study. Miswak was
distributed to the case group and required trainings were given to both groups. After the
interval one year, the examinations were repeated. In this study, Decayed/Missing/Filled Teeth
(DMFT) was used as a dependent variable to investigate the effectiveness of miswak compared
to that of simple tooth brushing. Overleaf are the final summarized results of the study as taken
directly by the journal.


13
Akhtar, M. and Ajmal, M. 1981. Significance of chewing sticks (miswaks) in oral hygiene from a pharmacological
viewpoint. Journal of the Pakistan Medical Association, 31 (4), pp. 89-95.
14
Web MD. n.d. Dental Health and Fluoride Treatment. [online] Available at: http://www.webmd.com/oral-
health/guide/fluoride-treatment [Accessed: 5 Nov 2013].
15
Botanical-online.com. n.d. Remedies for mouth ulcer. [online] Available at: http://www.botanical-
online.com/remediesmouthulcers.htm [Accessed: 30 Nov 2013].
16
Ezoddini-Ardakani, F. 2010. Efficacy of Miswak (Salvadora persica) in preventing dental caries. Health, 2 (5), pp.
499--503.
6

Table 2: DMFT in miswak and toothbrush controls (Ezoddini-Ardakani, F. 2010)

Before the study commenced, the case group had more incidence of DMFT than control group.
Initially, incidence of DMFT increased in both groups, but the increase was far bigger in the
control group (12.34% to 20.35%) compared to the case group (13.94% to 14.78%). After
continued and regular use for a year, however, the incidence of DMFT in the case group was
more than nine times less than that of the control group. These results indicate the increased
efficacy of miswak compared to tooth brushing over a prolonged period of time.


7

Primary Research
Methodology
Initially a different approach was used for this study which was purely in vitro (lab based). This
was attempted using miswak samples on agars inoculated with S. mutans and S. aureus and
consequently measuring their inhibition zones. The results were poor because the miswak had
not been extracted properly. Moreover, miswak works when its bristles interact with the
tooths surface; this method had a major limitation in that it only used the extract itself without
considering the mechanical action of the stick. Therefore this approach was rejected and the
pre and post usage swabs method was adopted instead. With the swabs it was possible to
monitor the experiment in vivo(in a real life situation); subjects oral flora before and after the
treatment was inoculated and the differences were observed on the agar plates and in the
gram stain results. The following few pages describe the details of this method.
Materials
Inoculation Materials Justification
20 charcoal swabs

Charcoal enables preservation
2Tayyebah miswak sticks cut in 5 cm pieces

2 sticks were used to limit
variation as much as possible.
130 g tube of Colgate Cavity Protection fluoride
toothpaste
-

20 MacConkey agar plates (MAC)
20 Blood agar plates (BAP)
20 Chocolate agar plates (CAP)
20 Sabouraud agar plates

5 for pre-treatment & 5 for post-
treatment of miswak; repeated
with toothpaste. Choice of agar
explained on page 9.
1 Bunsen burner
Sterile gloves
Face mask
Sterile inoculating loops
Aseptic technique- Appendix 1.2.




8

Gram staining materials (explained on appendix 2.2)
Microscope slides
Sterile inoculating loops/needles
Gloves
Normal saline
Laboratory heater
Crystal violet
Iodine
De-colorizer
Safranine
Optical microscope
Variables
Independent:
Usage of Miswak/ toothpaste
Dependent:
The extent of antimicrobial effect on oral flora
Controlled:
5 cm length of miswak
Brand of miswak
Toothpaste
Swabs
Method and 2 minutes duration of application
Method of swab taking
Agar brand
Aseptic technique
Streaking method of inoculation
Time in the incubator- 24 hrs.
Temperature at 25C
O2 and CO2 concentrations depending on the medium
9

Notes on some controlled variables
Streaking and aseptic technique are explained in Appendix 1.1 and 1.2 respectively. Although a
temperature higher than 25C would have encouraged better grown, it was not used due to the
possibility of spreading pathogens; which would have been unethical. Method of application,
swab taking and O2 and CO2 concentrations were kept constant for ensuring a fair test and are
explained in the procedure.

Justification of the agar media
In order to promote the growth of the bacteria in normal flora and make it easier to identify
them, selective and differential media were used instead of simple nutrient agar. Selective
media support the growth on one group of microbes and inhibit the growth of another whereas
differential media distinguish microorganisms from one another based on growth
characteristics
17
.
Blood agar plate (BAP) was used in this experiment because it is an enriched differential
medium. It consists of 5% mammalian blood which acts as a nutrient for fastidious bacteria
18

(bacteria that will grow only if special nutrients are provided). Blood agar differentiates related
bacteria based on their hemolytic activity (ability to breakdown erythrocytes). Thus it is helpful
in identifying viridans streptococci species (S. mutans, S. salivarius, S. mitis are all viridans
19
) as
they cause alpha hemolysis; partial breakdown of erythrocytes resulting in a dark and greenish
colony. Staphylococcus aureus causes beta hemolysis; complete breakdown of erythrocytes
around and under the colonies. Gamma hemolysis is represented by no breakdown of the
surrounding medium typically caused by Neisseria species. Chocolate agar (CAP) is a type of
blood agar that has had lysis of the blood cells because they are heated, which gives it the

17
Highlands.edu. n.d. Use of selective and differential media. [online] Available at:
http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivedifferential.htm [Accessed: 22
Sep 2013].
18
Buxton, R. 2005. Blood Agar Plates and Hemolysis Protocols. [online] Available
at: http://www.microbelibrary.org/component/resource/laboratory-test/2885-blood-agar-plates-and-hemolysis-
protocols[Accessed: 22 Sep 2013]
19
Parte, A. 2013. Genus Streptococcus. [online] Available at: http://www.bacterio.net/s/streptococcus.html
[Accessed: 5 Nov 2013].
10

brown color. It is enriched to promote the growth of certain bacteria such as Haemophilus
influenzae
20
, however it is not a differential medium.
MacConkey agar (MAC) is also a selective and differential medium. Bile salts and crystal violet in
it inhibit gram positive bacteria growth, selecting only gram negative bacteria. It further
differentiates the gram negative bacteria among those that ferment lactose (pinkish coloration
of the medium) and those that do not (no coloration of the medium).
Sabouraud plates were used to detect and grow fungal species if they were present in any test
subjects. It is selective because it has a low pH that will kill most bacteria only allowing fungi to
grow.
Procedure
Day 1
1. Set a group of five people as test subjects.
2. Ask them to take oral swabs after rinsing their mouth. NB: no toothpaste, mouthwash or
chewing gum should have been used in the last 5 hours as this might interfere with the
results of what is supposed to be an untreated normal flora.
3. The technique should involve taking a swab from the surface of teeth, gums, upper
tongue, lower tongue, hard palate and inner surface of cheeks. This should take 30
seconds.
4. The swab should be immediately put back into its charcoal container for preservation,
with subject identification written on it.
5. Give the subjects a piece of miswak stick each and ask them to clean all the surfaces
mentioned in Step 3 as thoroughly as possible. The time is controlled here as 2 minutes
for thorough cleaning.
6. As soon as the miswak is done, ask the subjects to take their oral swab in the same
manner described in Step 3.
7. Immediately put them back in charcoal containers. The five pre and post miswak oral
swabs are now ready for inoculation.

20
Cdc.gov. 2012. Identification and Characterization of Haemophilus influenzae. [online] Available at:
http://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-hi.html [Accessed: 8 Nov 2013].
11

Day 2 (morning)
8. Wear a lab coat and sterile gloves throughout the inoculation. This is crucial as a safety
measure and it is also important to prevent contamination of the samples.
9. Follow aseptic technique.
10. Take out all the required mediums from the storage.
11. Inoculate the first sample on BAP using the streaking technique in Appendix 1.1.
12. Inoculate the sample on CAP and MAC and Sabouraud in the same way.
13. Repeat steps 11 and 12 with all the remaining nine samples.
14. After the inoculation is done,
Leave BAP and CAP plates in the same aerobic incubator with 7% CO2 at 25C,
Leave MAC and Sabouraud plates in the aerobic incubators with no added CO2 at 25C
15. The cultures have to be in the incubators for 24 hours to ensure proper growth,
especially given the relatively lower temperature.
16. Discard the swabs, gloves and all of the disposable inoculating needles used. Turn off
the Bunsen burner and clear the working area.
Day 2 (evening)
17. Similar procedure to day1 (steps 1-7); this time replacing miswak with toothpaste. The 5
new subjects have to bring their own toothbrushes. A pea sized amount of toothpaste
has to be squeezed on them, roughly estimating an equal volume. The swab taking and
cleaning surfaces are the same as day 1.
Day 3
18. Follow the aseptic technique.
19. Inoculate the toothpaste samples on the media in the same way miswak samples were
inoculated the previous day (steps 11-13).
20. Incubate the plates in the way described in step 14 and leave the cultures for 24 hours.
21. Repeat step 16.
22. By this time, 24 hours of incubation period has passed for the miswak samples.
23. Wear new gloves. Collect all the plates from the incubators and record the presence of
microorganisms; making comparisons between the before and after samples on the
12

basis of differences in morphology (form, shape, color, explained in Appendix 2.1) and
//-hemolysis.
24. Distribute the hemolyses on BAP using an ACFOR scale that stands for: Abundant,
Common, Frequent, Occasional and Rare. This scale can help in qualitatively
distinguishing between the relative abundances of a colony and will form the basis for
the graphical results.
25. Gram stain all the visibly different colonies in CAP individually for identification.
Explained in Appendix 2.2. Apply the ACFOR after gram staining, this will be used for
graphical presentation of the bacterial species themselves.
Day 4
26. After 24 hours have passed repeat steps 23-25 from the previous day.
27. Clear the working area. Take all the used agar plates from Days 2-4 and put them
properly in the hospital container that takes care of used media.

13

Results

Below are a few of the pre and post miswak/toothpaste results as photographs of the cultures.
The annotations demonstrate the process of identifying morphology characteristics (appendix
2.1) and/or hemolytic activity.


Some other photographs of the results are grouped together in a collage in appendix 3.

14

Raw data tables of results can be found in appendix 4. The results presented here are processed
bar charts of the data. These charts are not representative of the raw data in its entirety. For
example the colonies termed moderate, gray, semi-opaque and wrinkled were rare and
negligible for most subjects except toothpaste trial 4 and 5 whereas the translucent colonies
were not identified in the gram stains and were rare too, so these were omitted from the
charts. Other data not represented is the colorization of MAC which showed whether the
bacteria were lactose fermenting or not; this was irrelevant to all trials except toothpaste 3.
Processed results

The units on the left were derived from the notes made during
the experiment and are true as a rough estimation


Chart 1: Pre and post Miswak results on BAP

0
20
40
60
80
100
120
Pre Post Pre Post Pre Post Pre Post Pre Post
1 2 3 4 5
A
C
F
O
R
Miswak trials on BAP
hemolysis hemolysis hemolysis
A Abundant =100
C Common = 80
F Frequent =60
O Occasional=40
R Rare=20
15


Chart 2: Pre and post toothpaste results on BAP
Gram staining
The procedure is outlined in appendix 2.2. Gram staining allows the identification of the
bacterial species. This was incredibly important for the analysis of the results. Microbiologists in
the lab and this resource
21
helped in the process. These pictures are my own.
1. Small brownish colonies on CAP (-hemolytic on BAP):





21
Barenfanger, J. and Drake, C. 2001. Interpretation of Gram Stains for the Nonmicrobiologist. [e-book] Springfield:
Laboratory Medicine. p. 2. http://labmed.ascpjournals.org/content/32/7/368.full.pdf [Accessed: 11 Nov 2013].
0
20
40
60
80
100
120
Pre Post Pre Post Pre Post Pre Post Pre Post
1 2 3 4 5
A
C
F
O
R
Toothpaste trials on BAP
hemolysis hemolysis hemolysis
The purple color indicates that it
is a gram positive bacterium and
there are two round cells
conjoined together called
diplococci.
Gram positive diplococci indicate
streptococci, specifically viridans
streptococci group

16

2. Moderate, opaque, white colonies on CAP (-hemolytic on BAP):









3. Yellow opaque colonies on CAP(-hemolytic on BAP):


Gram positive cocci in
clusters indicate
Staphylococci. A coagulase
test can be performed to
confirm if these are
Staphylococcus aureus,
however for the purpose
of this study they will be
assumed as S. aureus
Pink color indicates gram
negative bacterium. They
are round and in pairs, i.e.,
diplococci.
Gram negative diplococci
belong to Neisseria species.
An oxidase test was also
performed which supported
this but extra testing has to
be done to find out the
exact species, but they are
most likely nonpathogenic
species of Neisseria.
17

4. Gray, semi-opaque, wrinkled colonies on CAP (-hemolytic on BAP):








Following the gram stains, it is possibly to graph the results with respect to the actual species.
Note that the 4
th
species, H. influenzae, is omitted from the graphs due to it being negligible in
majority of the results.

Chart 3: Pre and post Miswak results on CAP after gram stain identification.

0
20
40
60
80
100
120
Pre Post Pre Post Pre Post Pre Post Pre Post
1 2 3 4 5
A
C
F
O
R
Miswak trials on CAP after gram stain
viridans Streptococci S. aureus Neisseria spp.
Gram negative small rods
called coccobacilli. Most
likely Haemophilus
influenzae because they
were rare and secluded
and existed mostly on
CAP, however, further
testing is needed.
18



Chart 4: Pre and post toothpaste results on CAP after gram stain identification.

0
20
40
60
80
100
120
Pre Post Pre Post Pre Post Pre Post Pre Post
1 2 3 4 5
A
C
F
O
R
Toothpaste trials on CAP after gram stain
viridans Streptococci S. aureus Neisseria spp.
19

Discussion of the results

Explanation
The results of CAP and BAP graphs seem repeated because they correspond to the same
species. However they may not be exactly repeated in all cases due to differences in streaking
technique. In general, the results of both miswak and toothpaste show a reduction of different
types of bacteria between the pre and post samples. Miswak seems to be more slightly more
effective on the -hemolytic viridans Streptococci; 2 trials showed marked reduction in miswak
compared to only 1 in toothpaste notwithstanding the anomaly subject 3. On the other hand,
toothpaste seems more effective on the -hemolytic S. aureus, again, notwithstanding subject
3. Toothpaste and miswak were equally effective on the -hemolytic colonies of Neisseria spp.,
this can be observed on the four charts and the raw data tables in appendix 4.
The coloration of the MacConkey agar in most instances was a slight red/pink tinge, signifying a
small amount of lactose fermenting bacteria. For majority of the samples this remained
unchanged, but for toothpaste subject 3 there was a visible increase in the colorization of the
sample. This coincides with the dramatic increase in abundance of S. aureus in the post
treatment sample (Chart 2 & 4) as S. aureus is a lactose fermenting bacteria.
Anomalies
In miswak subject 3, theres virtually no change detected in the pre and post cultures (Charts 1
and 3).This subject, unlike the other, habitually uses miswak. It is possible his oral flora has
already adjusted at that level due to frequent miswak use.
Toothpaste subject 3 has the most unusual results. Although -hemolytic and -hemolytic
colonies were reduced, there was a sharp increase in the -hemolytic colonies from pre to post
toothpaste (Chart 2). Gram staining established that these are most likely S.aureus, which is a
pathogen and should normally be present only in small amounts (Table 1). The dramatic
increase of S. aureus can be seen in figure 4 below, where previously rare moderate to large
white colonies became very abundant in the after sample. This infestation of S. aureus can also
account for the uncharacteristic decrease of -hemolytic and -hemolytic colonies in Chart 2
20

because there was a lack of nutrition for other microorganisms. In figure 5, MAC is shown to be
colorized pink because S. aureus is lactose fermenting. There are two possible explanations for
this anomaly; either there was an infection in the subjects mouth that spread out after she
brushed her teeth or that her toothbrush was really old and was infected with bacteria itself.

Figure 4 Toothpaste, subject 3- Blood agar plate

Figure 5: Toothpaste, subject 3- MacConkey agar





Before After
Before After
21

Limitations

The principal flaw of the methodology is the lack of quantitative data. A spectrophotometer or
its modified version called a flow cytometer could be used to optically count all the cells but
this equipment was not present at the pathology lab. Although a counting chamber for
microscopic counting was available, the microbiologists were reluctant to set it up as it would
have been too time consuming for all the cultures. A colony forming unit where cells can
simply be counted on a culture with dilutions would have been another possible method of
garnering quantitative data, however this was not researched beforehand and all the data
acquired for this experiment was qualitative.
The other main problem is the sample size. Only 10 subjects were used in this study, 5 for
miswak and 5 for toothpaste. This is too small a sample to say anything conclusive about these
results; the statistics are too limited to claim that similar reduction of colonies would happen in
50 or 100 subjects. However, reliable secondary data was utilized to compensate for this and
numerous and varied sources of data were included.
Additionally, the experimental design also needs improvements. The toothbrush should have
been controlled, it not being controlled possibly led to the anomaly subject 3. The experiment
itself had no control. The effect of normal saline on oral flora should have been investigated to
ensure that the colonies were being reduced by the antimicrobial action of miswak or
toothpaste only, and not anything else in the media or the surroundings. There were also
problems with inoculations on different media as the same swab was used to inoculate all 4
media; each time reducing the concentration of bacteria.
Some bacteria such as Lactobacillus species exist abundantly in the oral cavity but were not
found on any of the media or gram stains. This might be due to the media preferring some
species over others based on the nutrients. Simple nutrient agar could be used as a control to
see how the species would grow differently and which other species would or would not grow.
22

Furthermore, there were limitations to the aseptic technique. Occasionally a contaminant
would be visible on the culture. This didnt alter the readings as the contaminants were always
secluded so they werent factored in the results. That being said, working in a laminar flow
cabinet would have provided a more sterile environment.

23

Conclusion

The results of the experiment indicate that miswak is more effective on -hemolytic viridans
streptococci. This group of streptococci contains cariogenic bacteria Streptococcus mutans.
These results agree with prior research done which states that S. mutans was the most
susceptible organism to miswak extracts
22
.The clinical trials in mentioned in the Secondary
Research that monitored the DMFT variable in miswak and toothbrush users also support the
experimental results of this research.
Another research published in the Journal of Advanced Oral Research
23
supplements my
findings. The study was conducted clinically using participants saliva and it measured the effect
of miswak extract (Group 1), toothbrush (Group 2), and the control normal saline (Group 3) on
S. mutans in the saliva. The authors used the colony forming unit method to analyze their
results which are adapted below:

Table 3:Evaluation of Streptococcus mutans CFU (colony forming unit) (at 10
3
) count in three different groups
The highest difference in the before and after counts was noted in the miswak group, which
was more than twice the difference in toothpaste counts. Furthermore, this research also
specified the difference in S. mutans on the basis of the participants gender. It was discovered
that miswak is more effective on females compared to males, which can be seen in the chart
overleaf adapted from the journal:

22
Abdelrahman, H., Skaug, N. and Francis, G. 2002. In vitro antimicrobial effects of crude miswak extracts on oral
pathogens. Saudi Dental Journal, 14 (1), pp. 26--32.
23
Bhat, P., Kumar, A. and Sarkar, S. 2012. Assessment of immediate antimicrobial effect of miswak extract and
toothbrush on cariogenic bacteria--A clinical study. Journal of Advanced Oral Research, 3 (1).
Groups S. Mutans count before S. Mutans count after Difference
Group 1 28.701.77 11.902.18 16.802.15
Group 2 26.102.08 18.601.84 7.502.37
Group 3 26.401.65 21.101.85 5.302.11
24


Chart 5:Comparison of the difference (Post-Pre) of Streptococcus mutans CFU count (at 103) in males and females
This research confirms my findings quantitatively and the specification of gender is a relevant
extension for further research carried in this topic.
Toothpaste reduces the pathogenic -hemolytic S. aureus better than miswak and this is also
supported by existing scientific literature
24
which suggested no inhibition of S. aureus by
miswak extracts. Miswak and toothpaste were equally effective on the -hemolytic colonies but
these results are not as significant medically or economically; -hemolytic bacteria dont
contribute to tooth decay or gum diseases.
The result of toothpaste trial three also revealed a major disadvantage to using toothpaste; the
toothbrush has to be renewed often to avoid the risk of contamination and subsequent
infection in the oral cavity. Miswak contains antibiotics within the stick itself; therefore it
cannot be contaminated in the same way.


24
Almas, K. and Al-Bagieh, N. 1999. The antimicrobial effects of bark and pulp extracts of miswak, Salvadora
persica. Biomedical Letters, 60 p. 71.
25

The implications of this research are large because it demonstrates how miswak can perhaps
control tooth decay in a more cost effective way than toothpaste. This would reduce healthcare
costs mentioned in the introduction and alleviate oral health standards in lower socioeconomic
class and poorer countries. However, this research is limited; the experiment has to be
repeated a number of times minimizing the aforementioned limitations and further
investigation should be done to provide quantitative data for these results to be truly reliable.

26

Bibliography
Abdelrahman, H., Skaug, N. and Francis, G. 2002. In vitro antimicrobial effects of crude miswak
extracts on oral pathogens. Saudi Dental Journal, 14 (1), pp. 26--32.
Akhtar, M. and Ajmal, M. 1981. Significance of chewing sticks (miswaks) in oral hygiene from a
pharmacological viewpoint. Journal of the Pakistan Medical Association, 31 (4), pp. 89-95.
Almas, K. and Al-Bagieh, N. 1999. The antimicrobial effects of bark and pulp extracts of miswak,
Salvadora persica. Biomedical Letters, 60 p. 71.
Barenfanger, J. and Drake, C. 2001. Interpretation of Gram Stains for the Nonmicrobiologist. [e-
book] Springfield: Laboratory Medicine. p. 2.
http://labmed.ascpjournals.org/content/32/7/368.full.pdf [Accessed: 11 Nov 2013].
BBC News. 2006. Neem: India's tree of life. [online] Available at:
http://news.bbc.co.uk/2/hi/south_asia/4916044.stm [Accessed: 3 Nov 2013].
Bhat, P., Kumar, A. and Sarkar, S. 2012. Assessment of immediate antimicrobial effect of
miswak extract and toothbrush on cariogenic bacteria--A clinical study. Journal of
Advanced Oral Research, 3 (1).
Botanical-online.com. n.d. Remedies for mouth ulcer. [online] Available at:
http://www.botanical-online.com/remediesmouthulcers.htm [Accessed: 30 Nov 2013].
Buxton, R. 2005. Blood Agar Plates and Hemolysis Protocols. [online] Available at:
http://www.microbelibrary.org/component/resource/laboratory-test/2885-blood-agar-
plates-and-hemolysis-protocols [Accessed: 22 Sep 2013].
Cdc.gov. 2012. Identification and Characterization of Haemophilus influenzae. [online] Available
at: http://www.cdc.gov/meningitis/lab-manual/chpt09-id-characterization-hi.html
[Accessed: 8 Nov 2013].


27

Encyclopedia Britannica. 2013. commensalism (biology). [online] Available at:
http://www.britannica.com/EBchecked/topic/127789/commensalism [Accessed: 3 Nov
2013].
Encyclopedia Britannica. 2013. biofilm (biology). [online] Available at:
http://www.britannica.com/EBchecked/topic/1368883/biofilm [Accessed: 3 Nov 2013].
Encyclopedia Britannica. 2013. phytochemistry. [online] Available at:
http://www.britannica.com/EBchecked/topic/458909/phytochemistry [Accessed: 22 Sep
2013].
Ezoddini-Ardakani, F. 2010. Efficacy of Miswak (salvadora persica) in preventing dental caries.
Health, 2 (5), pp. 499--503.
Highlands.edu. n.d. Use of selective and differential media. [online] Available at:
http://www.highlands.edu/academics/divisions/scipe/biology/labs/rome/selectivediffere
ntial.htm [Accessed: 5 Nov 2013].
Keys, T. 2012. Infective Endocarditis. [online] Available at:
http://www.clevelandclinicmeded.com/medicalpubs/diseasemanagement/infectious-
disease/infective-endocarditis/#t0015 [Accessed: 3 Nov 2013].
Oliver, D. 2003. Microbes and You: Normal Flora. [online] Available at:
http://www.scq.ubc.ca/microbes-and-you-normal-flora/ [Accessed: 22 Sep 2013].
Parte, A. 2013. Genus Streptococcus. [online] Available at:
http://www.bacterio.net/s/streptococcus.html [Accessed: 5 Nov 2013].
Petersen, P., Bourgeois, D., Ogawa, H., Estupinan-Day, S. and Ndiaye, C. 2005. The global
burden of oral diseases and risks to oral health. Bulletin of the World Health Organization,
83 (9), pp. 661-669. Available from: doi: 10.1590/S0042-96862005000900011 [Accessed: 3
Nov 2013].

28

Prevention methods and programmes for oral diseases. 1985. [e-book] Geneva: World Health
Organistaion. Available through: Google Scholar [Accessed: 5 Nov 2013].
Roxburgh, W. 1793. William Roxburgh, from Plants of the Coast of Coromandel. [image online]
Available at: http://www.botanicus.org/page/280108 [Accessed: 3 Nov 2013].
Roxburgh, W. 1793. Illustration of Salvadora persica. [image online] Available at:
http://www.kew.org/plants-fungi/Salvadora-persica.htm [Accessed: 3 Nov 2013].
Todar, K. n.d. The Normal Bacterial Flora of Humans. [online] Available at:
http://textbookofbacteriology.net/normalflora.html [Accessed: 22 Sep 2013].
Tonzetich, J. 1977. Production and Origin of Oral Malodor: A Review of Mechanisms and
Methods of Analysis*. Journal of periodontology, 48 (1), pp. 13--20.
US Department of Health. Health care financing administration. National health expenditures,
1998. Available at: http://www.nidr.nih.gov/sgr/sgrohweb/toc.htm [Accessed 2 Nov 2013]
Web MD. n.d. Dental Health and Fluoride Treatment. [online] Available at:
http://www.webmd.com/oral-health/guide/fluoride-treatment [Accessed: 5 Nov 2013].
Worldagroforestrycentre.org. 2013. Species Information. [online] Available at:
http://www.worldagroforestrycentre.org/Sea/Products/AFDbases/AF/asp/SpeciesInfo.asp
?SpID=1477#Addinfo [Accessed: 3 Nov 2013].
Yee R, Sheiham A. The burden of restorative dental treatment for children in third world
countries. International Dental Journal 2002;p.52



29

Appendix 1
1.1: Streaking
25

1.2: Aseptic technique
Work near a Bunsen burner throughout; it sterilizes the area to an extent and keeps circulating
the air around it. Gloves and lab coat are compulsory; a face mask may also be worn. Make sure
the apparatus in use is brand new; only open the packaging when it is ready to be used. Check
the agar plates for any contamination before inoculating. If contamination is visible, discard the
plate and start with a new one. Start with a new inoculating loop with each subject and never
let the loop touch anything else in the working space. Refrain from talking during inoculation
and try to breathe nasally. Never physically touch the culture at any point. This aseptic
technique must be followed both for the accuracy of the experiment(s) and for protection from
potential pathogens for the person performing the experiment(s).


25
Abedon, S. 1998. Using a loop to streak a plate. [image online] Available at: http://www.mansfield.ohio-
state.edu/~sabedon/biol4035.htm [Accessed: 5 Nov 2013].
30

Appendix 2
2.1: Colony Morphology
26

2.2: Gram staining procedure
1. If a pure sample is required, re-inoculate a specific colony on a new BAP
2. After incubation is over (3-4 hours are enough), put a droplet of normal saline on a
sterile microscope slide.
3. Using a new inoculating needle, gently remove a tiny piece of a colony from the pure
culture and smear it with the normal saline already on the slide.
4. Let it dry for 10 to 15 minutes on the heater.
5. Take it near the sink and pour crystal violet on the slide.
6. Wait for a minute, then rinse the slide.
7. Pour iodine, leave for a minute, rinse.
8. Pour de-colorizer on the slide, leave for a minute, rinse again.

26
Pearson Education. 2006. Colony Morphology. [image online] Available at:
http://www.studyblue.com/notes/note/n/microbiology-ch6-notes/deck/4047388 [Accessed: 5 Nov 2013].
31

9. Pour Safranine on the slide, leave for a minute and rinse for the last time and leave it for
drying on the heater.
10. After the slide has completely dried up, put a droplet of oil on it. The slide is now ready
to be viewed under an optical microscope.

32

Appendix 3
Some of my own photographs of the cultures arranged randomly. There are also some pure
cultures in these collages that were required for gram staining.

33

Appendix 4
Raw data tables detailing the ACFOR results.



Table 4: Pre and post miswak inoculation findings
Subject
1 2 3 4 5
Pre Post Pre Post Pre Post Pre Post Pre Post
Blood agar plate
hemolysis C O A C C C A A C A
hemolysis F O F F O O O O F R
hemolysis C F C O C C C O C -/-
Chocolate agar
Yellow, opaque,
smooth and
circular colony
C F C R C C C O C -/-
Small brownish,
opaque and
smooth colony
A C A O C C A A C A
Moderate,
white, opaque
and smooth
colony
F O F F O O O O F R
Translucent
colonies
R -/- R O R R O R O R
Moderate, gray,
semi opaque
and wrinkled
colony
-/- -/- -/- -/- -/- -/- F O R R
MacConkey agar
Any colorization
of the medium?
-/- -/- +/- -/- +/- +/- +/- +/- +/- +/-

A Abundant
C Common
F Frequent
O Occasional
R Rare
+/+ Strong positive
+/- Positive
-/- Negative
Key
34

Table 5: Pre and post toothpaste inoculation findings
Subject
1 2 3 4 5
Pre Post Pre Post Pre Post Pre Post Pre Post
Blood agar plate
hemolysis A A A A C R C A C C
hemolysis F R O R O A F R F O
hemolysis C F F R O R C -/- C O
Chocolate agar
Yellow, opaque,
smooth and
circular colony
C C F R C R F -/- C O
Small brownish,
opaque and
smooth colony
A C A A A R C A C A
Moderate,
white, opaque
and smooth
colony
F R O O R A F O F O
Translucent
colonies
R -/- R R -/- -/- C R C O
Moderate, gray,
semi opaque
and wrinkled
colony
R R -/- -/- -/- -/- C -/- C R
MacConkey agar
Any colorization
of the medium?
+/- -/- +/- +/- -/- +/+ -/- +/- +/- +/-

NB: None of the subjects showed any fungal growth in the Sabouraud agar.