Free Characterization And Identification Of Two Unknown Bacteria Using Biochemical Tests Report Sample

ABSTRACT

Bacteria can be beneficial to other organisms or can be pathogenic. Their diversity and versatility have caused scientists to study them deeply. A classification procedure using morphological and biochemical tests have since been implemented in microbiological studies in order to characterize isolated microbes. In this experiment, two unknown bacteria were characterized using Gram staining and a number of metabolic assays. Using the tests results, the unknown bacteria were successfully classified as Pseudomonas aeruginosa (Unknown A) and Staphylococcus aureus. Both bacteria are commonly found within humans and are occasionally potentially dangerous especially for people with compromised immune system. Additional testing can be performed to further confirm the identities of the unknown bacteria.

INTRODUCTION

Bacteria are prokaryotic organisms that occur in massive numbers throughout the natural world. Some people normally associate bacteria as "bad germs" that cause diseases, and while there are indeed a number of pathogenic species many bacteria are actually non-harmful to humans and other organisms. In fact, many bacteria possess characteristics that humans utilize for scientific and technological improvement. As such, it becomes necessary to determine which bacterial species are beneficial, harmless, or pathogenic. Identification tests have since been developed to characterize bacteria and classify them into various groups in order to better understand them through further study.
Traditional microbiological techniques are routinely used in laboratories for the characterization of bacteria. Staining is performed to visualize the morphology of the bacterial cell and to generally divide microbes into large subgroups, such as in Gram staining which classifies bacteria based on the peptidoglycan content of their cell walls. However, since many bacteria share similar morphological features, staining alone is not reliable proof or a bacterium's identity. As such, metabolic tests are also utilized. Since bacteria have different metabolic properties (i.e. they produce varying proteins), they have varying responses to these tests. The overall profile of the bacterium based on these assays can up to some extent help determine the identity of the microbe.
In this experiment, two unknown bacteria were characterized using Gram staining and four metabolic tests (indole test, urease test, gelatinase test, and lactose fermentation test). Their identities (species) were determined through the assay results.

METHODS

Gram-staining and cellular morphology
Gram staining was performed through the following steps. First, a smear of bacteria was heat-fixed on a glass slide. Next, the primary stain was added and rinsed with water. Iodine was added as a mordant and rinsed. Destaining was done using ethanol. Finally, safranin was used as a counterstain and rinsed. The Gram-stained sample was then viewed under a light microscope to observe cellular morphology and to classify as Gram-positive or Gram-negative.

Indole production test

Bacteria were inoculated onto tryptophan broth with Kovac's reagent. The culture was incubated overnight at 37°C and observed. This assay tests the bacterium's ability to produce the enzyme tryptophanase, which converts the amino acid tryptophan into indole and pyruvic acid. Kovac's reagent reacts with indole to produce a red product.

Lactose fermentation test

Bacteria were inoculated onto lactose broth with phenol red as pH indicator. The culture was then incubated at 37°C overnight and observed. This test determines the ability of the bacteria to ferment the sugar lactose as a carbon source.

Gelatinase test

A bacterial sample was stab-inoculated onto a nutrient gelatin slant and incubated overnight at 37°C. The culture was then refrigerated for a few minutes and observed for liquefaction of the solid media. This test determines if the bacterium produces gelatinase, which hydrolyzes the protein gelatin.

Urease test

Bacteria were inoculated onto urea broth with phenol red as pH indicator. The culture was incubated overnight at 37°C and observed. This test determines the bacterium's ability to produce urease, which splits off ammonia from urea.

RESULTS

Unknown A
Gram Stain Results
The unknown bacterium A was observed to be pinkish in color, which indicates that it is Gram-negative. Bacterial cells were rod-shaped and occurred in singles.

Metabolic Test Results

Only urease test and lactose fermentation test were performed on the bacterium Unknown A. Both tests were negative, as indicated by the yellow color of the cultures. Based on these tests, Unknown A does not produce urease nor does it utilize lactose as a carbon source.

Unknown B

Gram Stain Results
The unknown bacterium B was purple in color, which means it is Gram-positive. The bacterial cells were coccus in shape and were arranged in grape-like clusters.

Metabolic Test Results

Unknown B tested positive for urease, gelatinase, and lactose tests and tested negative for indole production. The urease and lactose tests showed a yellow to red color change in the culture, which indicates that bacterium B produces urease and is able to ferment lactose. The gelatinase test showed liquefaction of the medium, which determines that the bacterium is able to hydrolyze gelatin.

CONCLUSIONS

Unknown A
Based on the tests the bacterium was observed to be Gram-negative, rod-shaped, arranged in singles and does not hydrolyze urea nor ferment lactose. The bacterium was subsequently identified as Pseudomonas aeruginosa.
P. aeruginosa is a Gram-negative, rod-shaped, motile bacterium with a single polar flagellum (Madigan et al. 27, 490). It is an opportunistic pathogen that attacks a host with a compromised immune system (CDC; Madigan 315; Tortora 591). It has the ability to enclose itself in a complex matrix called a biofilm, which increases its pathogenicity by evading the immune system and preventing the penetration of antibiotics (Madigan 133; Tortora 57, 593). P. aeruginosa contributes to most nosocomial infections and are often present in the respiratory tract of cystic fibrosis patients (CDCm “Pseudomonas aeruginosa”; Tortora 593).
Additional tests that may be done to confirm the identity of the unknown bacterium as P. aeruginosa include motility test and flagellar staining. Since this bacterium is monoflagellated, expected results would be positive test result for motility and an observed single flagellum. Amplification of the 16S rRNA with subsequent sequencing can also confirm the identity of the bacterium.

Unknown B

The bacterium was observed to be Gram-positive, coccus in shape and arranged in grapelike clusters, produced gelatinase & urease, ferments lactose, and does not produce tryptophanase. The bacterium was subsequently identified as Staphylococcus aureus.
S. aureus is a Gram-positive, coccoid bacterium that arranges in an irregular, grapelike cluster (Madigan et al.). S. aureus is a common commensal and occasional parasite that causes skin infections such as boils and pimples. S. aureus is also common in nosocomial infections and have developed strains that are drug-resistant such as MRSA, VISA, and VRSA (CDC, “Staphylococcus aureus”).
Additional tests to confirm the identity of S. aureus include microbial growth on mannitol salt agar, which would result in the growth of yellow colonies. Amplification of the 16S rRNA with subsequent sequencing can also confirm the identity of the bacterium.

Works Cited

CDC. "Pseudomonas aeruginosa in Healthcare Settings." 7 May 2014. Web. 11 April 2015.
CDC. "Staphylococcus aureus in Healthcare Settings." 17 Jan 2011. Web. 11 April 2015.
Madigan, Michael, et al. Brock Biology of Microorganisms. 13th Ed. San Francisco, CA: Pearson Education, Inc., 2012.
Tortora, Gerard, Funke, Berdell, and Christine Case. Microbiology: An Introduction. 10th Ed. San Francisco, CA: Pearson Education, Inc., 2010.