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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