Running head

Running head: Yogurt Unknown Project 1
Identification of Yogurt Bacterium
Christine J. Abragan
BIOL 402
University of Kansas

Yogurt Unknown Project
Abstract
The experiment was performed to determine the identity of the unknown bacterium
sample Blokus and the CFU/gram of a bacterium in Kroger Strawberry Flavored Yogurt. A serial
dilution of the yogurt was performed to calculate the CFU/gram. The TSA plates were inoculated
and incubated. The CFU/gram for Kroger Strawberry Flavored Yogurt was 2.2 x 10^6. The
unknown, Blokus, was used to inoculate the following tests: acid production from sucrose,
mannose, salicin, mannitol, and galactose and growth at 45 C. The bacterium produced acid from
mannitol and galactose and grew at 45 C. It was concluded that Blokus was ?E. faecalis ? based on
the collected data.

Yogurt Unknown Project
Introduction
The purpose of this experiment was to identify unknown yogurt bacterium sample Blokus
and determine the CFU/gram of a bacterium. To have the yogurt to be known as a yogurt with
live and active cultures, it must have 10^8 CFU/g at the time of manufacture (Leboffe and Pierce
2015). Also, a serial dilution was performed to determine if the yogurt sample from Kroger
Strawberry Flavored Yogurt did contain 10^8 CFU/g, The unknown yogurt bacterium sample
was then tested to determine the identity. It was tested to determine if experienced growth at 45
C and if it produced acid from the following sugars: sucrose, galactose, mannitol, salicin, and
mannose. Performing tests in which multiple sugars are tested for acid production makes it less
difficult in identifying a bacterium because different bacteria produce acid from different types
of sugar. The sample was then gram stained to determine whether it was gram positive and gram
negative bacterium. Moreover, it also indicated whether the bacterium’s shape was rod or cocci
and its cellular arrangement. From this test, it was concluded that the unknown bacterium was
gram positive because it retained the crystal violet dye very well and it was cocci-shaped.
Experimental Procedures
First, a serial dilution was performed on the Kroger Strawberry Flavored Yogurt to
determine if it could be labeled as a product with live and active cultures. One gram of yogurt
was first added to a test tube with 9 mL of deionized water and then mixed. The remaining test
tubes were then labeled 10^-2, 10^-3, 10^-4, 10^-5, and 10^-6 and 900 microliters of deionized
water was pipetted into each one. Using a micropipette, 100 microliters of the mixture was
removed from the initial test tube 10^-1. The 100 microliters was then placed into the test tube
labeled 10^-2. After the test tube was thoroughly mixed, 100 microliters was removed from the

Yogurt Unknown Project
tube labeled 10^-2 using a new pipette and transferred into the tube labeled 10^-3. This process
was repeated until the last tube, 10^-6, had 100 microliters added to it. Six MRS plates were
labeled after dilution was finished. Three of the plates were labeled “aerobic”, while the last
three plates were labeled “anaerobic”. Each set of plates had one labeled as 10^-5, 10^-6, and
10^-7. For both sets, aerobic and anaerobic, the following directions were followed. 100
microliters of sample was taken from the test tube labeled 10^-4 using a micropipette. The 100
microliters was then transferred onto the MRS plate labeled 10^-5. Once the sample was plated,
a metal spreader was dipped into ethanol. Once the spreader cooled, it was used to spread the
inoculum over the entire agar plate. The 10^-5 plate was then set aside without inverting it. A
new micropipette was used to remove 100 microliters of the sample from the test tube labeled
10^-6. The 100 microliters was then transferred onto the MRS plate labeled 10^-7. Once the
sample was plated, a metal spreader was dipped into ethanol. Once the spreader cooled, it was
used to spread the inoculum over the entire agar plate. The 10^-7 plate was then set aside without
inverting it. After the inoculums were soaked in their agar plates, the lids were taped. Anaerobic
plates were placed in an anaerobic jar and incubated aerobically at 35 C for 48 hours. The plates
were removed from the incubators and the colonies were counted for each plate.
The unknown sample, Blokus, underwent gram staining, acid production from different
sugars, and streak plating. The sample was first plated from the broth. Then, using aseptic
technique, an inoculating loop collected bacteria from the unknown sample and placed them on
the TSA plate. The sample was plated using the quadrant technique and the plate was incubated
for 24 hours. After completing incubation, the colonies on the plate were used for various tests to
determine acid fermentation. Five tubes of broth containing galactose, mannose, mannitol,

Yogurt Unknown Project
sucrose, and salicin were used for inoculation. After completing the inoculation process, they
were incubated for 24 hours at 37 C. The MRS broth was incubated for 24 hours at 45 C.
All procedures were followed from the laboratory outlines found in ?Microbiology: Laboratory
Theory and Application ? {Leboffe and Pierce 2015).
Results
The number of colonies on the plates was counted and the CFU/g was calculated after
allowing the colonies of Kroger Strawberry Flavored Yogurt to grow on the TSA plates. Because
the bacteria only grew on the anaerobic plates, those were the only plates counted. In Figure 1,
the plates containing colonies are shown. In the plate containing the dilution factor of 10^-5,
there were approximately 22 colonies. In the plate containing the dilution factor of 10^-6 , there
was 1 colony. The CFU/g for the 10^-5 plate was 2.2 x 10^-4 and the CFU/g for the 10^-6 plate
was 1 x 10^-6. In the last plate with the dilution factor of 10^-7, there were no colonies; thus, the
CFU/g was 0.
The broths that were inoculated with the unknown sample Blokus turned different colors
based on if the bacterium was able to produce acid from the sugar in the broth. The MSA broth
was very turbid, indicating a lot of acid production. The mannitol broth turned yellow, indicating
that there was acid production. The galactose broth also turned yellow. There was no acid
production found in the rest of the broths because they all turned purple The results from the
gram stain revealed that the unknown bacterium was a gram positive cocci. The cocci formed
chains making it seem like they were rods at first glance.

Yogurt Unknown Project
Plate Dilution Factor Number of Colonies Calculated CFU/g Aerobic/Anaerobic
10^-5 22 2.2 x 10^-4 Anaerobic
10^-6 1 1 x 10^-6 Anaerobic
10^-7 0 0 Anaerobic

Test Growth at
45 C Acid from
Galactose Acid from
Mannose Acid
from
Mannitol Acid
from
Sucrose Acid
from
Salicin Gram
Stain
Results growth Positive Negative Positive Negative Negative Positive
Cocci

Original Density ?= = ?22 colonies/ 10^-5 dilution = 2.2 xu m b e r o f C o l o n i e s / D i l u t i o n F a c t o rN
10^6 CFU/g
Discussion
The colony forming unit of the Kroger Strawberry Flavored Yogurt was determined
through a serial dilution to be 2.2 x 10^6 CFU.g. According to the National Yogurt Association,
products containing 10^8 CFU/g could be labeled as containing live and active cultures.
However, this certain sample of Kroger Yogurt does not contain the appropriate number of
CFU.g; thus, it cannot be labeled as containing live and active cultures. It contains too few active
bacterial units. Additionally, the unknown bacterium, Blokus, was tested for growth at 45 C and
acid production from sucrose, mannose, salicin, galactose and mannitol. The sample showed
growth at 45 C and showed acid production in galactose and mannitol. After doing the gram

Yogurt Unknown Project
staining process, the sample was determined to be a gram positive cocci bacterium. From these
results it was concluded that the unknown sample, Blokus, was ?E. faecalis ?. To be more confident
about the result, the unknown could be reinoculated in all broths to see if it produces acid from
just galactose and mannitol.

Yogurt Unknown Project
References
Leboffe MJ. Section 9: Food Microbiology. In: Pierce BE, editor. Microbiology: Laboratory
Theory and Application. 4th ed. Englewood, CO: Morton Publishing; 2015.

Yogurt Unknown Project
Figures, Tables, Images, etc.

Figure 1: The plates that were incubated at 37 C for 24 hours anaerobically. They were
inoculated with Kroger Strawberry Flavored Yogurt

Yogurt Unknown Project

Figure 2: A picture showing the results from a gram staining of Blokus