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

countries1where 510% of public health expenditure relates to oral health2. In most developing

countries, if treatment were available, the costs of dental caries in children alone would exceed

the total health care budget for children3. 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 Africa4. 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 Streptococci5 (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?

1Petersen, 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] 2US 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] 3Yee R, Sheiham A. The burden of restorative dental treatment for children in third world countries. International Dental Journal 2002;p.52 4World 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] 5Keys, 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 relationship6 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

mutans7. Teeth have non shedding surfaces, so bacteria can form a biofilm8 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

Bacteriology9summarizes the prevalence of certain colonies of oral bacteria and their

pathogenicity.

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

antibiotic10. 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.

10BBC 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 hygiene12. 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.

12Prevention 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 chemicals13. 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 bacteria14. 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

ulcers15.

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 study16 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.

13Akhtar, 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]. 16Ezoddini-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

characteristics17.

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 bacteria18

(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 viridans19) 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

17Highlands.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]. 18Buxton, 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] 19Parte, 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

influenzae20, 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. 20Cdc.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 resource21 helped in the process. These pictures are my own.

1. Small brownish colonies on CAP (-hemolytic on BAP):

21Barenfanger, 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 4th 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 extracts22.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 Research23supplements 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 103) 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:

22Abdelrahman, 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. 23Bhat, 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 literature24 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.

24Almas, 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

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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.

26Pearson 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.