1. Isolation and Quantification of
Normal Mouth Flora Relative to
Diet and Soft Drink
Consumption
Jane M. Tonello
Jason M. Dillon
Microbiology 222
Spring 2009

2. The average human mouth contains a multitude of
bacteria. Such bacteria include: Staphylococcus
epidermidis, Streptococcus mitis, Streptococcus salivarius,
Streptococcus mutans, Lactobacillus sp., Spirochetes,
Actinomyces odontolyticus, anaerobes, and numerous
others (Hanks 1,Todar 1). The human esophageal cavity
contains many nutrients and remains very moist, thus it
provides an environment that is vital to common oral
bacterial growth. Many of these common bacteria found in
the human mouth have the ability to ferment
carbohydrates, such as lactose and glucose, into lactic
acid.
Introduction

3. Acid fermenting bacteria have the ability to multiply
greatly at pH’s of between 4.0 and 5.2, demonstrating that
artificially lowering the pH by introducing carbonic acid
from a soft drink may further promote bacterial growth
(Bibby 2). The normal flora of the mouth also helps
prevent transient flora growth and infections from
pathogenic bacteria by utilizing the available resources in
the mouth. This experiment will quantify the oral bacteria
for a number of individuals, and determine if factors such
as vegetarian versus non-vegetarian diet, and the amount
of daily soft drink intake affects oral bacterial growth.
Intro contd.

4. Materials
Materials used in the experiment include: TSY agar media, Petri-
dishes, sterile swabs, 0.6% saline, sterile screw-cap glass tubes,
P-1000 Micropipettor, P-200 Micropipettor, nystatin antifungal,
9.9 mL water blanks, 9.0 mL water blanks
Figure 1.1
Figure 1.1 shows the utensils
utilized in the gathering and
storage of each bacterial sample.
Sterile swabs can be seen wrapped
in sterile paper towels, and the P-
1000 pipettor was used to transfer
1.0ml of sterile saline solution to
each saline blank tube.

5. Methods 250.0mL of TSY agar recipe is made, and TSY
agar plates poured
Six screw-cap vials are filled with 1.0mL of
0.6% saline
Bacterial samples are taken from six subjects
by swabbing mouth with sterile swabs and put
into saline screw cap vial
Six TSY agar plates are streaked from the
inoculated saline solution
After one week of incubation in the room
temperature (20-25o
C) incubator, all plates
are observed and morphologies recorded
Figure 1.2
Figure 1.2 demonstrates the
electronic scale that was used during
the process of TSY Agar media
creation. All ingredients were
weighed out to the hundredths place.

6. Gram stains are performed for isolated colonies and all observations are recorded
500.0mL of TSY agar plates poured
Bacterial samples obtained through previous method
Serial dilutions are made for each sample possible (10-3,
10-4
, 10-5
, and 10-6
) using P-
1000 and P-200 micropipetters
Plates are incubated for one week at room temperature, and then observed for the
quantity of bacteria for each subject
500.0mL of TSY agar plates are poured with Nystatin
More samples are collected and dilutions are made for each sample possible (10-5
, 10-
6
, 10-7
, 10-8
)
Plates are incubated for one week, and then removed for final observations and
recordings of bacterial amount for each subject
Methods contd.

7. Results-Gram Stains
Two streak plates were performed for the very first
bacterial sampling. Gram stains were completed for
each of these two pure cultures, and Gram positive,
tiny cocci were observed for each plate. Also, Gram
stains were performed for two of the first spot
plates that were made, and Gram positive, tiny
clustered cocci were also observed for these plates.
For all four Gram stains, pairing of two to three cocci
were observed.

8. Results
Figures 2.1 and 2.2 show the
spot plates that were
prepared for the second
bacterial samples. The
dilutions were 10^-3, 10^-4,
10^-5, and 10^-6, and
demonstrate that many of
the plates contained bacteria
that was too numerous to
count. As can be seen, there
is a diverse amount and types
of bacteria present.
Figure 2.1
Figure 2.2

9. Figure 2.2
Figure 2.2 displays the
serial dilutions prepared
for the third bacterial
sample. The dilutions were
10^-5 and 10^-7. The P-
200 and P-1000 pipetters
were utilized in the dilution
process, and sterile pipette
tips had to be utilized
between each dilution.
During the actual plate
spreading the 10^-6 and
10^-8 concentrations were
prepared by pipetting only
0.1ml from each of the
initial dilutions.
Results Contd.

10. Results-Bacterial Quantification
Non-
vegetarian
(NV), soda
#/day
10^-5
(4-14-09)
10^-5
(4-20-09)
10^-7
(4-20-09)
10^-8
(4-20-09)
AH (NV, 2
sodas)
20,800,000 NG NG NG
HK (NV, 1
soda)
NG NG 580,000,000 TNTC
KK (NV, 1
soda)
NG 11,700,000 NG 123,000,000,000
MC (NV, 1
soda)
TNTC 400,000 NG NG
As seen in Figure 2.3, some of the spot plates demonstrated no growth
(NG) at all, and many of them demonstrated bacterial growth that was
too numerous to count (TNTC). This table represents the data that
could actually be quantified, and displays that the bacterial
concentration found in a person’s mouth is particular to him or her.
Figure 2.3

11. Vegetarian/Non-Vegetarian Sodas/Day TNTC
CJ NV 4 5
AH NV 2 2
HK NV 1 1
VC V 0 1
MC NV 1 3
Figure 2.4
This figure demonstrates that there is a general trend toward
increased bacterial growth for those who drank more soda pop. There
is a positive correlation that can be seen in subjects AH and CJ with a
higher soda intake and higher bacterial counts. HK and VC had less
plates that were too numerous to count, and they also drank less soda.
MC is the only subject that did not follow the normal trend.
Diet and Soda Related Bacterial Growth Trend

12. Discussion
Six different subjects were chosen, all with different diets, and
relatively different soda consumption, including one subject that did not
drink any soft drinks at all as a control. Since it is nearly impossible to
have all six subjects to have the exact same oral hygiene and eat the
exact same thing every day, bacterial levels varied greatly sample from
sample. In gathering the bacterial samples using sterile swabs for each
subject’s mouth, the same amount of sample can’t be extracted every
time. This leads to further variation in bacteria amount for each subject.
In the initial inoculation of TSY agar plates mold growth became
overwhelming. The mold spores could have been present in the subject’s
mouth, or could have been introduced from the air. Nystatin anti-fungal
was used in the second inoculation to limit mold and promote bacterial
growth.

13. Discussion Cont.
Due to further fungal contamination only three plates could be
inoculated for the second bacterial sampling, and only five plates
for the third. Also, the Gram stains exhibited Gram positive,
paired cocci, which indicates Streptococcus sp., which is very
common in mouth flora. A fourth sample was taken from each
subject, but an autoclave malfunction prohibited TSY Agar plate
production, prematurely ending the experiment. The data,
despite all errors, showed that there was a trend between diet
and soda consumption in comparison with bacteria growth.
Subjects with a non-vegetarian diet and larger soft-drink
consumption tended to have more bacteria growth compared
with those who did not drink soda and had a vegetarian diet.

14. Literature Cited
Bibby,B.G., J.F. Volker, and M. Van Kersteren. Acid Production and
Tooth Decalcification By Oral Bacteria. Journal of Dental
Research. 1942. 5 May 2009.
http://jdr.sagepub.com/cgi/reprint/21/1/61
Hanks, Hershell. Microbial Flora of the Mouth-Experiment 60.
Collin County Community College District. 2009. 15 March
2009. http://209.85.173.132/search?
q=cache:aAXhEayMyjgJ:iws.ccccd.edu/hhanks/Lab
%2520Lectures/MICROBIAL%2520FLORA%2520OF
%2520THE%2520MOUTH,%2520Exp.
%252060.doc+mouth+flora&hl=en&ct=clnk&cd=3&gl=us.
Todar, Kenneth. The Normal Bacterial Flora of Humans. Todar’s
Online Textbook of Bacteriology. 2008. 15 March 2009.
http://www.textbookofbacteriology.net/normalflora.html.

15. Acknowledgments
A special thanks to:
Dr. Anderson
Dikshya Bastakoty (and all other TAs)
And to our anonymous test subjects!