Isolation and Quantification of Normal Mouth Flora

Relative to Diet and Soft Drink Consumption

Jane M. TonelloJason M. Dillon

Microbiology 222Spring 2009

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

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.

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.

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 (20-25oC) 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.

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.

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.

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

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.

Results-Bacterial QuantificationNon-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

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

DiscussionSix 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.

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.

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.

Acknowledgments

A special thanks to:Dr. Anderson

Dikshya Bastakoty (and all other TAs)And to our anonymous test subjects!