Jessica Romano

Introduction
In the late 1800s, the science known as bacteriology was greatly influenced by a German
physician, Robert Koch. Derived from his work, he advanced the ways in which the medical
field can determine the cause of a disease. He explained that there were 4 major guidelines to
acknowledge while studying infectious diseases and were later known as Kochs postulates. In
the study of medicine, these guidelines continue to hold the enduring standard for determining
the connection of microorganisms and diseases.
Although during his education bacteriology was not common, he still continued to study
medicine at the University of Gottingen, where he acquired skills that concentrated on scientific
research, pathology, and microscopy. These skills lead him to create a medical practice that
included a laboratory which focused on microbiology research. With his new staining methods
that he developed to visualize bacteria along with growth culture techniques, he utilized his
guidelines to find the bacterial causes for anthrax and tuberculosis. Kochs postulates at first was
an attempt to develop a definitive cause of diseases and a way to argue that microorganisms can
in fact be the cause of some infectious diseases. The first postulate states the same pathogen must
be present in every case of the disease in order to be a considered a probable cause. The second
postulates explains that the pathogen that was present in every case must be able to be isolated
from the diseases host and grown on its own in a pure culture. From the pure culture that the
pathogen was grown on, the third postulates states that the pathogen will cause the disease when
inoculated into a healthy susceptible animal. Finally, the fourth postulates states that the
pathogen must then be isolated from the inoculated animal and must be shown to be the same

pathogen present in the original animal. Being that the fourth postulate was not considered a
requirement, if the first three standards are met, you can conclude that the pathogen is in fact the
cause of the disease you are studying. Kochs Postulates have been used widely when studying
the causes of human diseases and have greatly influenced the world of medicine today.
A common food-borne pathogen known as Staphylococcus aureus, is considered to be an
important cause of infectious disease that comes from animals and can be transmitted to humans
depending on how close they come in contact with each other. In order to treat and prevent this
disease a developing strategy has been used known as Antibacterial photosensitization-based
treatment and research shows that this treatment is cherished when dealing with microbial
infections. Staphylococcus aureus CECT 239 was obtained from the Spanish Type Culture
Collection (CECT). Bacterial cells were grown overnight in sterile tryptic soy broth at 37C.
Stock inoculum suspensions were prepared in PBS and adjusted to optical densities of 0.4 at 600
nm. This experiment is a clear example of how models of human disease are used in modern
medical research.
Procedure
Students were required to experiment with yogurt and milk to learn about microbial
growth and what pathogens can cause disease. The main goal of this project is to be able to use
Kochs Postulates to identify disease by using aseptic techniques, isolation plating for pure
cultures, microscopy and simple staining to visualize the bacterial isolates. This project was
spread out into 3 weeks of experiments; on the first experimental day various different brands of
yogurt and milk were given to the students on MRS agar plates. By the end of the first day, the
students should have isolated bacteria from the yogurt and milk samples along with separating
signs and symptoms from a diseased individual, in this case the yogurt from the healthy

individual being the milk sample given to each student. In order to complete the first day of
experiments, the students would used the following materials:

4 mini-Lactobacillus- MRS agar plates

Loop for isolation and incinerator
Milk and yogurt samples

Once the students obtained all the necessary materials, they followed step-by-step procedures
provided from their lab instructors. Bacterial infections will not all have the same growth
requirements, in other words some may require oxygen and some may be able to grow without
oxygen. Being that it was the first day of the experiment the students were unable to determine
the preferred growth method for the bacteria they isolated from the milk and yogurt samples.
Therefore this study aimed to test two conditions of growth; he first being Plus Oxygen, with
oxygen and the second being Minus Oxygen, without oxygen. The students took the 4 mini-MRS
Agar plates and labeled two of them for milk and two of them for yogurt. But they couldnt
forget to distinguish which plates would grow with oxygen and which plates did not have oxygen
for their growth method. They then sterilized the loop they were provided for an aseptic
technique to isolate the bacteria sample they took from the milk and yogurt samples. They took
one small drop of milk in the sterile loop and spread the sample across the MRS-Milk plates
attempting to isolate the bacteria into individual colonies. The students repeated the same steps
for each of their yogurt plates and all four plates are placed in the 37-degree incubator. The
second part of this day involved simple staining techniques to be able to see the bacteria under a
microscope. Students were required to prepare a heat fixed slide of the milk and yogurt samples
and observed the slides at 1000x magnification.
During week 2 of the procedure, the students had to observe the plates made from the
previous week and determine if they had a pure culture of bacteria. The bacteria from the yogurt

that was grown, was then inoculated into a healthy individual to recreate the disease. The
materials needed for this day 2 sterile tubes of LB broth that acted as the healthy individual, a
sterile loop to inoculate the bacteria, an incinerator to sterilize the loop before each inoculation
and 3 tubes of scalded milk. The students then selected their 2 different colony types that were
grown on their yogurt agar plates and they were required to use an aseptic technique to transfer
one colony to the first tube of LB and repeat this technique for the second colony you chose from
the yogurt plate. One both broth tubes have been inoculated with the bacteria, the students
prepared smears on clean slides from each LB tube. The students had to make sure the smear was
thick enough, dried, fixed and stained with a simple stain. When the students observed the slides,
the needed to take note of the purity of the sample and the morphology and arrangement of the
bacteria. In order for the students to prove that there was a bacterial species present in their
yogurt sample, the students needed to complete the 3rd postulate so they re-made the disease by
inoculating a loopful of the first colony LB tube into the first sample of scalded milk and
repeated the aseptic technique for the second colony LB tube. The third tube of milk the students
had remained as the constant and acted as the healthy individual for the experiment. At the end of
this day the students placed both LB tubes in the 37-degree incubator for the next week.
The final week of this experiment the students completed the observations for postulates
2 and 3. The students prepared simple stain slides of the bacteria that was grown in the 2 LB
tubes from the week before. The students noted observations of all 3 milk tubes such as the pH of
the sample, the color, the odor, and the texture of the sample.

Results

During the first week of the experiment, I used the yogurt sample that was labeled #1, it
was a brand called Brown Cow. The yogurt appeared to me as soft, mushy and thick. It was a
creamy white color and that had an odor that was a mixture of sour and sweet and a pH level of
4. The milk sample was the same for every student in the class; I observed that the milk was a
creamy white liquid that had a sour smell and a pH level of 5. After I prepared my simple
staining slides and observed them underneath the microscope, the milk sample appeared to have
bacteria that were Bacillus, rod shaped. The bacteria present in the yogurt when observed under
the microscope had an arrangement of streptococcus.
After observing the plates I made the week prior, it appeared to be that there were no
isolated colonies that formed on either of my yogurt plates. Being that I had to pick a little dot
that was semi-isolated from the first plate and the second plate I pulled a sample from where the
plate has the least growth. These two colonies that I pulled from the yogurt plate where then
inoculated into 2 separate tubes of LB. Those tubes were now seen as infected and were used to
inoculate into 2 tubes of milk to re-create the disease and prove that the bacterial species that was
grown is the cause of the initial illness. Both milk MRS agar plates had no growth but the plate
that was grown with oxygen contained 2 contaminations, resulting from poor sterilization of the
inoculating loop. Although there were no isolated colonies on either of the yogurt plates, growth
was still present and able to test. After simple staining two slides for each LB tube, colony 1 was
grown with no oxygen and had no bacteria present while colony 2 was grown with oxygen but
only one bacteria was present having the morphology of Bacillus.

The last week focused on completing observations for postulates 2 and 3.

Macroscopic Observations
Tube Number
Color
Odor
Texture
pH

Milk tube #1 with

oxygen
White, creamy
Sour
Liquidy, semi-sold
5.5

Milk tube #2 w/o

oxygen
White, creamy
Sweetish Sour
Liquidy, semi-solid
5.5

Control tube
White, creamy
Sour
Solid
5.5

The second part of the observations where of the simple staining from sample from each
LB tube. LB #1 that had oxygen, the bacteria was seen in a staphylobacillus arrangement and LB
#2 was without oxygen, the bacteria appeared to be in a staphylococcus arrangement.
Conclusion
Being that the MRS agar plates that I prepared for this experiment in the first week
exhibited no isolated colonies, it was difficult to find a sample to pull in order to continue testing
Kochs postulates. I pulled from where the plate had the least growth and a dot that was semiisolated, which I used in order to complete the third postulate. The 3rd postulate is the remaking
of the disease from the isolated bacterial species found and allows us to prove that the species
found is the cause of the initial illness. Complications were seen from my MRS milk agar plates,
there was simply no growth of bacteria at all. However the Plus oxygen milk plate contained 2
contaminations and as I said before could have resulted from poor sterilization techniques used
while I was using the loop. To explain why there was no growth, I could have held the loop in
the incinerator too long killing off any bacteria that were present from either milk sample. Also
being that one of the plates did not have oxygen, if there were any bacteria present in the milk
sample that needed oxygen to grow, that would also explain why there was no growth because
the bacteria did not have the correct growth methods. Although there was growth seen on both
MRS yogurt agar plates, like I stated before there were still no isolated colonies present. From

the LB tubes, only colony 2 contained one bacteria present concluding that the colonies that I
pulled from each of the plates were in fact not bacteria at all. To improve my experiment, I
should be more patient when sterilizing the inoculating loop, making sure all contaminants are
killed and making sure not to keep the loop in the incinerator for too long to avoid killing any
bacteria present in my loopfulls.

Sources

http://www.journalofdairyscience.org/article/S0022-0302%2812%2900266-4/fulltext
http://cmgm.stanford.edu/micro/MI209/postulates.pdf
http://www.journalofdairyscience.org/article/S0022-0302(14)00788-7/abstract