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Experiment #14 Lab Report
By: Mimi Ton
Due: 12/5/2013
Section #: 006
Unknown #: 19
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Abstract:
The purpose of this experiment was to identify three unknown organisms through
a series of tests and mediums. The identification process involved a variety of tests
that narrowed down the possibilities of the three unknowns. Over several weeks of
experimentation, the results were analyzed and concluded. The results indicate that
the three unknowns appear to be Streptococcus pyogenes, Escherichia coli, and
Yersinia enterocolitica.
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Introduction:
This experiment was conducted to establish an accurate identification process of
bacteria in a mixed culture. The culture contained a mix of three unknown bacteria out of
a possible eight. The process of identifying bacteria is applicable in many fields of
science and healthcare. It is also very applicable in the food industry to limit and prevent
the spread or source of any food borne illnesses. Microbes exist in a wide range of forms
and structures, which can be identified and isolated for further examination (Carson,
n.d.).
To accurately identify the three unknowns, a series of differential and selective
tests were performed. Based on the results, further tests can be performed to distinguish
the differences between similar bacteria. The eight possibilities were further narrowed
down to three categories, four possibilities were among the gram-positive bacteria and
two possibilities were each among the gram-negative coliform and paracolon categories.
The gram-positive category includes Bacillus subtilis, Staphylococcus aureus,
Staphylococcus epidermidis, and Streptococcus pyogenes. Each of these gram-positive
bacteria contains a thick peptidoglycan layer that retains the crystal violet dye.
Staphylococcus, Streptococcus and Bacillus bacteria are distinguished upon their cell
morphology. While Staphylococcus bacteria can be identified as having a cocci shaped in
the form of grape like clusters, Streptococcus bacteria are cocci shaped in the form of
chains or pairs and are a part of the normal human flora (Carson, n.d.) . The identification
process between Staphylococcus and Streptococcus bacteria is vital to differentiate
among dangerous pathogens and for proper medical diagnosis (Carson, n.d.). Bacillus
bacteria are easily identified since they are the only genus between the other two gram-
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positive possibilities that contain an endospore. This can be further analyzed with an
endospore stain that uses malachite green and a safranin counterstain. The spore
characteristic of the Bacillus genus enables the bacteria to endure harsh environmental
conditions (Slonczewski, 20). If each test indicates an accurate result after proper
performance, then the three unknowns should be accurately identified.
Materials and Methods:
In the first two weeks of the experiment, several selections of media were
available to assist in the identification process. In the first week, a TSA and MacConkey
plate were streaked out for observation in the proceeding two weeks. The TSA plate is a
general media that enables the growth of both gram-positive and gram-negative bacteria.
This is to ensure that all unknown colonies are collected on a single media to ensure extra
colonies for gram-stains and further inoculations. The MacConkey agar plate is a
differential and selective media that inhibits the growth of gram-positive bacteria but
enables the growth of gram-negative bacteria. The selective component of the
MacConkey plate comes from the bile salts and crystal violet that select for gram-
negative bacteria (Carson, n.d). This plate is essential to differentiate between gram-
negative coliforms and paracolons. The difference between the two types of gram-
negative bacteria will result in different color appearances (The Microbugz, n.d).
In the second week of experimentation, several other plates were used and
include: the blood agar plate, EMB, SS, MSA, TSI tube, citrate tube and starch plate.
The blood agar plate is useful in the identification process of gram-positive bacteria. It is
a differential media that is essentially a TSA plate prepared with 5% sheep blood for
enrichment ingredients (Carson, n.d) . The blood agar plate will distinguish between
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types of Staphylococcus and Streptococcus bacteria based on their hemolytic activity.
The Eosin-Methylene Blue agar plate is a selective and differential medium. The
selective component comes from the methylene blue and eosin dyes which select for
gram-negative bacteria (Carson n.d.). EMB also contains lactose in which the
differentiation factor involves the result of either a lactose or non-lactose fermenter. The
salmonella-shigella agar plate is another medium that is both selective and differential.
The brilliant green dye and the bile salts are useful in identifying one of the gram-
negative paracolon bacteria, Salmonella enterica. The mannitol salt agar also falls into
the selective and differential medium category (Carson, n.d). The selective component
comes from the 7.5% NaCl concentration, which is useful in the identification of
Staphylococcus (Carson, n.d).
The triple sugar iron agar is a slant that differentiates among bacteria. To
differentiate among bacteria, it consists of the phenol red indicator, sodium thiosulfate,
ferrous sulfate and fermentable sugars (glucose, lactose and sucrose) (Carson, n.d). The
citrate agar is another slant similar to that was used in this experiment. Unlike TSI, citrate
can be used to not only differentiate, it can also be used to select for bacteria. It consists
of the pH indicator bromothymol blue and selects for certain bacteria in this experiment
that is able to use citrate as its sole carbon source such as Klebsiella pneumonia (Carson,
n.d). Each slant has a different process of inoculation; TSI agar is inoculated by the stab
and drag method while the citrate agar is inoculated by only the drag method (Carson,
n.d).
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Gram stain
Gram (-) Rod
1)MacConkey Agar
(Selects gram -)
Ex: 12
Paracolon (Non-
Coliform (Lactose lactose
fermentors= Bright fermentors=
pink colonies) colorless colonies)
2) EMB (to
2) EMB (Lactose confirm)
fermenters test)
(No Lactose
( Lactose fermented)
fermented) Colorless colonies/
Metallic green coloration of medium
sheen/ dark purple
color
3) TSI
(No lactose
fermented)
3) TSI (glu,lac, surcose
fermen. gas and H2S)
*Salmonella*
H2S production
4) SS (no black
centers-if
present= pink 4) SS
colonies)
Colorless colonies w/ black Coloress colonies w/
centers no black centers
5) Citrate *Salmonella enterica* *Yersinia
enterocolitica*
(-) Green (+) Blue
5) Citrate
*E. coli* *Klebsiella
pneumoniae* (+) blue
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Gram stain
Gram (-) Rod
1)MacConkey Agar
(Selects gram -)
Ex: 12
Paracolon (Non-
Coliform (Lactose lactose
fermentors= Bright fermentors=
pink colonies) colorless colonies)
2) EMB (to
2) EMB (Lactose confirm)
fermenters test)
(No Lactose
( Lactose fermented)
fermented) Colorless colonies/
Metallic green coloration of medium
sheen/ dark purple
color
3) TSI
(No lactose
fermented)
3) TSI (glu,lac, surcose
fermen. gas and H2S)
*Salmonella*
H2S production
4) SS (no black
centers-if
present= pink 4) SS
colonies)
Colorless colonies w/ black Coloress colonies w/
centers no black centers
5) Citrate *Salmonella enterica* *Yersinia
enterocolitica*
(-) Green (+) Blue
5) Citrate
*E. coli* *Klebsiella
pneumoniae* (+) blue
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Results:
After the incubation period of each slant and plate, the results were observed and
recorded for further analysis. The TSA plate had multiple colonies that were used for the
gram stain. The gram stain revealed pinkish rods and violet cocci bacteria in the medium.
The next medium for week one, the MacConkey agar revealed two forms of colonies one
each side of the plate. One side of the plate consisted of bright pink colonies while the
other side consisted of colorless colonies.
The remaining plates/slants that were inoculated in the second week of
experimentation were finally observed in the third week. To begin the observation
process of the gram-positive tests, the results of the catalase test were first analyzed. The
results of this test indicated no signs of bubble production. Following, the observation of
the catalase test the MSA plate was observed and the results indicated no change to the
medium since it remained red. The last plate observed upon identifying the gram-positive
bacteria was the blood agar plate. The plate revealed clear halos around individual
colonies.
After analyzing the mediums involved in the identification process of the gram-
positive unknown, the mediums involved with identifying the gram-negative unknowns
were observed. The first plate observed was the EMB plate, this plate revealed two
sections of colonies. One section revealed colorless colonies while the other revealed
dark purple colonies. Following the EMB plate, the SS plate was observed in two
different sections as well. One section revealed pink colonies while the other section
revealed colorless colonies that did not contain any black centers. Two individual TSI
slants were then observed, one that revealed a yellow color change and the other a red
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medium with a tiny spec of a black center. The next slants observed were two citrate
slants. Both slants remained green and resulted in no change to the medium.
Discussion:
The process of accurately identifying three unknowns requires an organized
strategic plan. To organize the eight possible bacteria, each were placed into three
separate categories. Four of the bacteria were organized into the gram-positive category
and the additional four were separated into two additional categories. One category
represented the gram-negative coliform bacteria and the other category represented the
gram-negative paracolon bacteria. This enabled an easier process of elimination after the
results were analyzed.
Upon analyzing the observed results, several possible conclusions were made on
the outcome of each. In regards to the MAC plate, the differences in results indicated the
two gram-negative unknowns. The section that fermented lactose appeared bright pink
and was classified as the unknown coliform bacteria. The non-lactose fermenter resulted
in colorless colonies and is referred to as the paracolon gram-negative bacteria. To
further distinguish the two unknowns, an additional lactose fermentation test was
performed. The EMB test was used and revealed a section with dark purple colonies. This
demonstrated the presence of a coliform gram-negative bacterium because the change in
the medium indicates that the bacteria fermented lactose (Carson, n.d).
Further examinations of selective plates helps to narrow down the unknowns that
were presence on the general medias. Since the catalase test did not indicate the presence
of bubble production, this narrows the gram-positive bacteria down to two possibilities:
Streptococcus pyogenes and Bacillus subtilis. Staphylococcus was the only genus among
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the four gram-positive possibilities to contain the catalase enzyme. After interpreting the
catalase test, the MSA plate was used to further confirm the possibility of Streptococcus
pyogenes as one of the unknowns. Since the medium did not change in color (from red to
yellow) that indicated that the unknown bacterium did not ferment mannitol and was
therefore not Staphylococcus. To conclude the presence of Streptococcus pyogenes, the
hemolytic activity from the blood agar plate was interpreted. The results indicated signs
of beta hemolysis since clear halos were observed around individual colonies. Beta
hemolysis indicates complete lysis of red blood cells, which is also indicated in the size
difference between plates that reveals no hemolytic activity. A beta hemolysis result
would have revealed colonies that were larger in size from colonies with no hemolysis
(Carson, n.d.). The concluded gram-positive unknown bacterium is Streptococcus
pyogenes.
The additional tests were examined and interpreted to identify the last two
unknown gram-negative organisms. After interpreting the MAC agar, the EMB plate was
interpreted. The section that revealed dark purple colonies indicated the coliform bacteria
since a dark purple color change to the colonies indicate that the bacteria fermented
lactose. The section with colorless colonies indicates no change to the medium so
therefore the bacteria did not ferment lactose. The fermentation of lactose is essential in
the separation of the coliform and paracolon gram-negatives. The difference is that
coliform bacteria will ferment lactose while paracolon bacteria do not. After, interpreting
the EMB plate, the TSI slant was interpreted to further isolate and identify the two gram-
negative bacteria. The coliform bacteria revealed a yellow color. The change from red to
yellow reveals that the bacterium is able to ferment glucose, lactose, and or sucrose
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(Carson, n.d). Since this is a characteristic of coliform bacteria, this result further
concludes that the test bacterium is indeed a gram-negative coliform. Since the second
TSI slant remained red, this indicated that the bacterium was unable to ferment the three
sugars. This result concludes the paracolon gram-negative bacteria (Carson, n.d.) . The
last medium to further conclude the identity of the two bacteria was the SS plate. The
plate was divided into two sections, one section revealed colorless colonies with no black
centers. This characteristic reveals that the bacterium does not produce H2S and would
therefore represent Yersinia enterocolitica (Carson, n.d.). A citrate slant was interpreted
to conclude if the bacterium was indeed Yersinia enterocolitica, since the citrate test
results indicated no change in the medium, the paracolon gram-negative bacterium is
concluded as Yersinia enterocolitica. The remaining section of the SS plate revealed pink
colonies that indicated no H2S production. The final bacterium was determined through
citrate slants result as well. Since the second citrate slant indicated no change to the
medium and remained green, the unknown coliform gram-negative bacterium is
concluded as Escherichia coli (“Citrate test, n.d).
Each medium used in the process of identifying the unknowns followed the flow
charts that are present on pages six and seven. The consecutive tests are essential for
narrowing down the possibilities. The general media were inoculated first and the
selective and differential medias followed to conclude the unknown identities. The order
of each performed test complimented the previous test, which assisted in the organization
of the experimentation.
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Literature Cited:
Carson, V.A. (n.d.). Microbiology lab. Tampa, FL: Department of Cell Biology,
Microbiology and Molecular Biology.
Slonczewski, Joan L. , John W. Foster, and Kathy M. ) Gillen. Microbiology, An
Evolving Science. W W Norton & Co Inc, print.
"MacConkey Agar." Welcome to Microbugz. N.p.. Web. 5 Dec 2013.
<http://www.austincc.edu/microbugz/macconkey_agar.php>.
"Citrate Test." . N.p.. Web. 5 Dec 2013.
<http://web.clark.edu/rrausch/biolabs/260/Citrate.pdf>.
