Lab 1

Objectives

 1. To perform the aseptic technique and dilution series. 

2. To practice the streaking and spreading technique on culture media. 

Materials 

Each student or team of students will require the following items: 

• 99.7% ethanol

• Bacterial culture 

• Vials or Test Tube  

• Pipettes (1000 µL ;100 µL)  

• Sterile distilled water  

• Inoculating loop 

• Glass rod 

• Nutrient agar

• Laminar air flow cabinet  

• Growth chamber 

Procedures

1. The sterilization procedure was performed to eliminate all possible contaminants on the working bench surface and in the laminar airflow. Sterilized utensils were used.

2. A ten-fold serial dilution was conducted for the bacterial culture. For a ten-fold dilution on a 1 mL volume, vials/test tubes were filled with 900 µL of water, and 100 µL of the stock microbial solution was serially transferred, with thorough mixing after every dilution step. This step was crucial to obtain a sufficiently diluted bacterial culture that could be easily counted on a spread plate.

3. A bacterial streaking and spreading technique was exercised on Nutrient Agar (NA) using an inoculating loop. The loop was dipped into the diluted bacterial solution and then spread across one quadrant of the NA. Four-streaking and single-streaking techniques (Figure 7) were used to produce single colonies of bacteria.

4. The plates were incubated in the incubator at a set temperature of 25°C.

5. The plates were observed, and single bacterial colonies were counted after 48 hours of incubation.

Results

Discussion

The total number of bacterial colonies counted on the plates gave an idea of the bacterial concentration in the original culture after performing the dilution and streaking procedures. Comparing the growth patterns on the four-streak and single-streak plates highlighted some key differences in how each method worked to isolate colonies. The four-streak technique, which involved making several passes of the inoculating loop across the plate, resulted in more colonies due to the dilution process with each streak. However, this sometimes caused colonies to overlap or cluster, especially in the center, where the bacteria were more concentrated. While this could make it harder to clearly identify individual colonies, it was useful for generating a large number of colonies for further analysis. In contrast, the single-streak technique involved a single, continuous streak of bacteria, leading to more spaced-out colonies. This method produced fewer colonies, but it was better for isolating individual colonies, allowing for clearer observation of their characteristics. The single-streak technique also made it easier to identify distinct colony morphology and ensured each colony was isolated. Ultimately, both methods had their advantages. The four-streak technique was effective for producing more colonies, while the single-streak method was better for isolating pure colonies. The choice of technique depends on the goal of the experiment—whether it’s counting colonies or obtaining pure cultures for further study.

Conclusion

To wrap up, both the four-streak and single-streak techniques were effective for isolating bacterial colonies, but each had its own strengths depending on the goals of the experiment. The four-streak method worked well for generating a larger number of colonies, which is helpful when you need to count many colonies for analysis. However, it sometimes led to overlapping colonies, which could make it harder to identify them individually. On the other hand, the single-streak technique gave better separation between colonies, making it easier to observe their unique characteristics and ensuring a pure culture. While it produced fewer colonies, it was great for isolating specific strains for further study. In the end, the choice of method depends on whether the experiment requires a high colony count or the isolation of pure bacterial cultures.