Identification of Unknown Bacteria Using Laboratory Tests Report

Aim

This laboratory work aimed to investigate the feasibility of identifying unknown microorganisms through laboratory tests. Since the tests had already been conducted, the practical aim was to evaluate the results and speculate which microorganisms might be in the Petri dishes.

Activities

MacConkey’s Agar

Materials:

• The powder, 51.5 g
• DI Water, 1L
• Heating plate, autoclave, water bath
• Petri dish, one piece

Methods:

1. After dissolving the powder in DI water and mixing thoroughly, the solution was heated until dissolved and then sterilized in an autoclave (121°C, 15 minutes).
2. The microorganisms (Unknown #9) were aseptically distributed into prepared Petri dishes and incubated for 18-24 hours at 35°C.

Gram Stain

Materials:

• Glass
• Crystal violet
• Acetone/ethanol (50:50)
• Gram’s iodine solution
• DI Water
• 1% basic fuchsin solution

Methods:

1. Microorganisms (Unknown #9) were aseptically transferred onto pre-cleaned glass and fixed with ethyl alcohol.
2. The bacterial smear was coated with pigment (crystal violet), and then iodine was added to this.
3. After the smear is washed with the help of alcohol, it is then stained using fuchsin. After drying, the results are evaluated.

Catalase Test

Materials:

• 3% H₂O₂
• Glass slide
• Sterile loop

Methods:

1. Unknown colony #9 was transferred into a test tube using a sterile loop.
2. 3% of H₂O₂ was added to the sample.

Esculin ID Membrane

Materials:

• Surface of the slant
• Autoclave
• Sterile loop

Methods:

1. Several swabs (Unknown #9) from a previously prepared colony were aseptically transferred to the surface of the slant.
2. An autoclave incubation was performed for 48 hours (35-37°C).

Nutrient Agar

Materials:

• Nutrient Agar (28 g powder, 1L DI)
• Autoclave
• Sterile loop

Methods:

1. Unknown #9 bacterial colony was aseptically transferred to the Nutrient Agar surface previously transferred to a Petri dish.
2. The culture was incubated in an autoclave for 48 hours (35-37°C).

Mannitol Salt Agar (MSA)

Materials:

• Mannitol Salt Agar (100 g powder, DI water 1L)
• Autoclave
• Sterile loop

Methods:

1. Utilizing sterile technique, a sample of bacterial colony Unknown #9 was inoculated onto the surface of a MSA plate.
2. For 48 hours, the culture was incubated in an autoclave (35-37°C).

Results

As can be seen, MacConkey’s Agar (Figure 1) did not give positive results. The Gram stain (Figure 2) showed purple staining, and the catalase test (Figure 3) revealed the presence of gas bubbles.

The Esculin ID Membrane (Figure 4) did not show any blackening, indicating a negative result. Nutrient Agar (Figure 5) exhibits the presence of large, lush, orange-yellow colonies. Finally, according to the results of the MSA test (Figure 6), the result was negative.

Discussion

The results of the identification tests performed showed that Unknown #9 was a Gram-positive bacterium that was incapable of breaking down lactose. Additionally, the colony was represented by aerobic bacteria capable of forming bubbles. Since no blackening was visible in the Esculin ID Membrane test, the colony did not contain enterococci. Mannitol also could not be fermented by this bacterium (negative control).

In other words, this bacterium was shown to be Gram-positive and capable of oxidative phosphorylation. It can be assumed that this colony belongs to the genus Staphylococcus, which is consistent with the described characteristics (LT, 2020). Since the test results were not obtained directly but were already prepared, their quality cannot be guaranteed. Additionally, during the processing procedure, some traits, such as the description of the Nutrient Agar test results, were subjective and could lead to errors.

Conclusion

This work aimed to qualitatively identify an unknown bacterial colony using six laboratory tests. Findings indicate that the organism is characterized by a thick peptidoglycan cell wall (Gram-positive) and generates energy through the process of oxidative phosphorylation. Based on these findings, it can be assumed that it belongs to the genus Staphylococcus. In other words, the study’s aim was achieved, which may indicate the successful completion of the work. Such strategies are vital to the clinical industry, where pathogen identification is the primary goal of patients’ laboratory tests, leading to the use of damage minimization protocols for point-of-care treatment.

Reference

LT. (2020). Lab 15: Isolation and identification of Staphylococci. LibreTexts Biology.