Solange Sylvester
Instructor: Nasreen Haque
BIO 3302 E178 (45034)
LAB PRACTICAL: Identification of Unknown tube #6
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Introduction
Bacteriology is the study of bacteria. It includes characteristics such as morphology, biochemistry, and
ecology. Microbial tests and techniques are imperative in the identification and classification of
microorganisms. They are important to our survival and health as well as the assessment and identification of
diseases caused by pathogens. This study was done to identify unknown bacteria in test tube #6. To identify
unknown bacteria, one can conduct multiple tests. The aim of this study was to perform various lab tests and
techniques we learned throughout the semester to identify a mix of an unknown gram-positive and gram-
negative bacteria, interpret the results of the tests performed, and discuss the different characteristics of the
identified bacteria.
Materials and Methods
The tests I used to identify the bacteria were, nutrient agar plate, Gram stain, Blood agar plate, Mannitol
salt agar plate, MacConkey agar plate, Phenylethyl alcohol agar plate, Triple sugar iron agar,
Sulfide/Indole/Motility (SIM), Catalase, Urease, and Methyl-Red & Vogues-Proskauer (MR&VP).
The nutrient agar plate is a general medium used to grow a wide range of bacteria. The Gram stain is a
differential test that differentiates gram-positive and gram-negative bacteria. The composition of the cell wall
sets the two groups of bacteria apart. Gram-positive bacteria’s cell wall is made up of a thick layer of
peptidoglycan which when stained with the primary stain, crystal violet, retains a bright purple color. Gram-
negative bacteria’s cell wall is made up of a thin layer of peptidoglycan surrounded by an outer membrane of
lipid-polysaccharide (LPS). When stained using the counterstain, safranin, gram-negative cells retain a pink
color.
The blood agar plate (BA) was used to observe growth characteristics. It is an enriched, differential
medium. Some bacteria will simply grow on this agar while some will partially or completely break down the
blood cells in the agar. The mannitol salt agar (MS) plate is a selective medium, which allows the growth of
halophilic organisms only, while inhibiting the growth of other organisms. It is also a differential medium
because it is used to differentiate closely related organisms, Staphylococcus aureus and Staphylococcus
epidermidis. The MacConkey agar plate (MAC) is a selective medium that inhibits the growth of gram-positive 2
bacteria, but allows the growth of gram-negative bacteria due to crystal violet and bile salts present. It is also
differential because it differentiates non-lactose and lactose fermenters. The Phenylethyl alcohol agar plate
(PEA) is a selective medium that allows the growth of gram-positive bacteria but inhibits the growth of gram-
negative bacteria due to the phenylethyl alcohol present.
Triple sugar iron agar (TSIA) is a differential medium used to differentiate and identify gram-negative
enteric bacteria. It tests bacteria’s ability to produce H2S and ferment specific carbohydrates. The SIM test is
used to detect H2S production from thiosulfate reduction, indole production from hydrolysis of tryptophan, and
determine the motility of the bacteria. The catalase test is used to identify catalase positive bacteria and
differentiate gram-postive cocci. The Urease test is used to identify members of the genus, Proteus, rapid
urease-positive bacteria. Finally, the MRVP test differentiates between organisms that follow the mixed-acid
and butylene glycol pathways by examining carbohydrate metabolism.
Results
Figure 1.
Figure 2A & 2B
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Figure 3. Figure 4.
Figure 5.
Figure 6. Figure 7.
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Figure 8. Figure 9.
Figure 10. Figure 11.
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Figure 12. (Gram-negative) Prepared and used as references.
Figure 12. (Gram-positive) Prepared and used as references.
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Discussion
Figure 1 shows my gram stain. My gram stain only showed a gram-negative, rod shaped bacteria. This
was actually my second gram stain because the first gram stain I performed did not have any cells on it. I
obtained several colonies from a nutrient agar plate I prepared using the unknown test tube for this gram stain.
Though I transferred samples from several different colonies on the nutrient agar plate, I probably only
transferred a gram-negative bacterium. Figure 2A shows my blood agar plate while closed and held up to the
light. Figure 2B shows my blood agar plate opened with greyish/whitish colonies. Upon further examination of
my blood agar plate, I realized majority of the cells in the middle of my plate were gamma hemolytic meaning
no lysis occurred and no change in the surrounding agar. In the corners of my blood agar plate, there were very
small areas of beta-hemolysis meaning complete lysis of red blood cells and break down of hemoglobin causing
a colorless clearing of the agar surrounding the colony. The plates remained in the incubator for more than 24
hours and this could be why there was so little beta-hemolysis seen on the plate, causing one bacteria to grow
more than the other. I now knew I had a beta and a gamma hemolytic bacteria. Figure 3 shows my mannitol salt
agar plate. This plate had no type of growth on it. From this observation, I knew I did not have Staphylococcus
aureus or Staphylococcus epidermidis, which are both halophiles. Figure 4 shows my MacConkey agar plate
with clear/colorless colonies. Growth on this agar showed that I had a non-lactose fermenting, gram-negative
bacterium. I eliminated the possibilities of gram-negative bacterium Enterobacter aerogenes, as well as
Escherichia coli, because both grow in pink colonies due to lactose fermentation. I also eliminated the
possibility of Serratia marcescens, because it grows in red colonies on MS agar. I narrowed down my gram-
negative bacteria to either Pseudomonas aeruginosa or Proteus vulgaris. Figure 5 shows my PEA plate with
greyish/whitish colonies. With this observation, I knew I had a gram-positive bacterium but it was not
Staphylococcus aureus or Staphylococcus epidermidis because of the absence of growth on my MS plate.
Figure 6 is my TSIA tube. I inoculated the TSIA tube using a sample from my gram-positive PEA plate.
I used the TSIA tube to help identify my gram-positive bacterium because the results ranges between the
various gram-positive bacteria we’ve used throughout lab. Using my chart in Figure 12 (Gram-positive), I
already eliminated Staphylococcus aureus and Staphylococcus epidermidis, and all of the other results for gram-
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positive bacteria using the TSIA test differ. The slant and the butt of my tube were both yellow meaning the
unknown organism used glucose and another sugar, and continued to ferment throughout incubation to produce
acid. Enterococcus faecalis and Staphylococcus aureus produce acid throughout the tube. I knew I did not have
Staphylococcus aureus. Figure 7 is my SIM test I performed. I used this test mainly to help identify the motility
of my bacterium from my PEA plate since majority of the gram-positive bacteria we used are negative for
indole production. The stab inoculation showed a non-motile bacterium because it grew in a well-defined line. I
added kovac’s reagent to the surface of the tube to detect indole production. There was no color change when I
added the reagent, which meant the bacterium was indole negative. When thiosulfate located in the medium is
reduced, a black precipitate is formed due to H2S production reacting with the iron salt also in the medium. My
tube did not have a black precipitate. My bacterium was a non-motile, non-indole producing and negative for
H2S. I narrowed down my choices to Enterococcus faecalis, Micrococcus luteus, and Mycobacterium
smegmatis. Looking back at my BA plate, I knew I actually did not have Micrococcus luteus because I didn’t
have any vivid yellow colonies present, which is a characteristic according to my chart in Figure 12 (gram-
positive). Figure 8 is the catalase test I used to narrow down my identification since Enterococcus faecalis is
negative for catalase but Mycobacterium smegmatis is positive. I obtained a sample from the TSIA slant to
perform the catalase test. After adding two drops of H2O2 to the slide containing my sample, there was no
bubbles production meaning my bacterium was negative for catalase. After gathering my observations, I
confirmed my gram-positive bacterium was Enterococcus faecalis.
Figure 9 shows the urease test I performed which I inoculated using a sample from my gram-negative
MAC plate. After incubation, the broth turned a bright fuchsia pink, which means my bacterium is rapid urease-
positive. Since I already narrowed down my choices to Pseudomonas aeruginosa and Proteus vulgaris using the
observations from my MAC plate, I chose the urease test to differentiate. This positive result showed that my
bacterium was probably Proteus vulgaris. Figure 10 is my MR test, and figure 11 is the VP portion. After
incubation I divided the tube into two test tubes. I added methyl red to one of the test tubes to perform the MR
portion of the test. The broth turned red which meant the bacterium produced an acid end product and is a
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mixed-acid fermenter. I added VP reagents A and B to the second test tube and there was no change in color.
After performing these tests, I confirmed my gram-negative bacterium to be Proteus vulgaris.
Enterococcus faecalis is a gram-positive, cocci, non-motile, gamma hemolytic, non-indole, non-H2S
producing, catalase-negative and acid-producing bacterium. The identification is clinically important because
the Enterococcus species is the fifth most common cause of healthcare-associated infections (CDC). It causes a
range of diseases, which includes bloodstream infections, surgical site infections and urinary tract infections
(CDC). Proteus vulgaris is a gram-negative, rod-shaped, beta-hemolytic, rapid urease-positive, and mixed-acid
fermenter. It makes up part of the normal flora of the human gastrointestinal tract (O’Hara et al. 537). The
identification of this organism is clinically important because it ranks third as the cause of uncomplicated
cystitis, pyelonephritis, and prostatitis (O’Hara et al. 537). Identification of microorganisms is important when
distinguishing one bacterium from another. It is important to determine how these organisms affect us.
References
CDC. “Antibiotic Resistance Patient Safety Atlas.” Centers for Disease Control and Prevention, CDC,
www.cdc.gov/hai/pdfs/patient-safety-atlas/AR-Patient-Safety-Atlas-Phenotype-Definitions.pdf.
O'Hara, Caroline Mohr, et al. “Classification, Identification, and Clinical Significance of Proteus, Providencia,
and Morganella.” Advances in Pediatrics., U.S. National Library of Medicine, Oct. 2000,
www.ncbi.nlm.nih.gov/pmc/articles/PMC88947/.
Finazzo, Susan, and Steven Obenauf. Laboratory Manual, Microbiology Fundamentals: A Clinical Approach.
2nd Edition ed., McGraw Hill.
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