Microbiology Lab Procedures for Week 14

Microbiology Lab Procedures for Week 14

The Week 14 microbiology handout outlines essential laboratory procedures for two projects: the Pseudomonas project and the Streptomyces project. It details the purification of PCR products necessary for 16S rRNA sequencing, including specific steps and materials required. Additionally, it describes the streak test method for testing antibiotic-producing organisms against various test bacteria. This handout is designed for microbiology students and researchers conducting experiments in microbial genetics and antibiotic production. It provides a clear framework for understanding the methodologies and expected outcomes of the laboratory work.

Key Points

  • Details the purification process of PCR products for 16S rRNA sequencing.
  • Outlines the streak test procedure for antibiotic-producing organisms.
  • Lists materials needed for both Pseudomonas and Streptomyces projects.
  • Explains the significance of observing zones of inhibition in antibiotic testing.
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  • Niesha Dupree
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Week 14. Pseudomonas project Day 8 and Streptomyces project Day 6
14.1 Pseudomonas project Day 8: Purification of PCR product
Day 8 Procedure: Purification of the PCR product
Before the sequence of the amplified 16S rRNA encoding DNA can be determined,
the PCR product must be purified, that is, separated from the other components (e.g.,
primers, Taq enzyme, and salts) present in the PCR tube.
Materials (each group)
• 2 PCR reaction products from the previous lab period
• 4 microcentrifuge tubes
• 1 set of PCR purification kit containing 1 tube of Buffer B3, 2 tubes of Wash
solution, 1 tube of Elution buffer, and 2 spin columns.
Procedures
• Transfer your PCR product to a clean microcentrifuge tube.
• Add 500 µL of Buffer B3 into your PCR product and mix it well.
• Transfer the above mixture to the EZ-10 spin column and let it stand at room
temperature for 2 min. Centrifuge at 10,000 rpm for 2 min.
• Remove the flow-through liquid in the collection tube. Add 600 µL of Wash
solution to the column and centrifuge at 10,000 rpm for 2 min.
• Repeat the above washing step and centrifuge at 10,000 rpm for 2 min.
• Transfer the column into a clean microcentrifuge tube and add 20 µL of Elution
buffer. Incubate at room temperature for 2 min and centrifuge at 10,000 rpm for
2 min.
• Purified DNA will be collected in the microcentrifuge tube. Label with your
designated number on the top and place it in the rack on the front bench.
Note: Purified DNA will be shipped off campus to perform 16S rRNA sequencing. We
will be performing sequencing analysis on your sample next week.
14.2 Streptomyces project Day 6: Streak test organisms
Materials (each group)
• Two SpM plates with your inoculated antibiotic producers from last week
• A set of test organisms: E. coli, P. aeruginosa, S. epidermidis, and M. luteus
(shared by 2 groups)
Procedures
• Use the sterilized loop to make a single, straight-line streak of E. coli, P.
aeruginosa, S. epidermis, and M. luteus perpendicular towards the streak of
Streptomyces griseus or your Streptomyces isolate.
Note: It is important to start the streak from the outer edge of the plate and bring the
test organism as close as possible to edge of your Streptomyces culture without
touching it. Streaking from the outer edge of the plate will help reduce the possibility
of picking up some of the antibiotic producing cells (or their secondary metabolites)
from the center streak and carrying it throughout the streak of the test organisms. Try
your best to ensure all cross-streaks finish at the same level.
• Label the streak with the name of the test organism.
Note: It is important to flame the loop thoroughly between each transfer to avoid
contaminating the individual streak with other test organisms.
• Incubate all plates at 37°C for 24 h.
Next week procedures
• Observe each cross-streaked culture for zone of inhibition produced in
response to antibiotic production by Streptomyces.
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End of Document
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FAQs of Microbiology Lab Procedures for Week 14

What is the purpose of PCR product purification in microbiology?
PCR product purification is essential for removing unwanted components such as primers, enzymes, and salts from the PCR reaction. This step ensures that the DNA is clean and suitable for sequencing, allowing for accurate analysis of the 16S rRNA gene. Purified DNA is crucial for obtaining reliable results in microbial identification and phylogenetic studies.
How is the streak test performed in the Streptomyces project?
The streak test involves using a sterilized loop to create a straight-line streak of test organisms, such as E. coli and P. aeruginosa, on a plate inoculated with Streptomyces. The streak should begin at the outer edge of the plate and extend towards the Streptomyces culture without direct contact. This method helps assess the antibiotic activity of Streptomyces against various bacteria, with results observed after incubation.
What are the expected outcomes of the Streptomyces project?
The expected outcomes include observing zones of inhibition around the Streptomyces culture, indicating effective antibiotic production. These results will help determine the antimicrobial properties of the Streptomyces isolate tested against various pathogens. The findings can contribute to understanding the potential applications of Streptomyces in antibiotic development.
What materials are required for the Pseudomonas project?
Materials for the Pseudomonas project include PCR reaction products, microcentrifuge tubes, a PCR purification kit with Buffer B3, Wash solution, Elution buffer, and spin columns. Each component plays a vital role in the purification process, ensuring that the amplified DNA is ready for subsequent sequencing analysis.
What is the significance of the 16S rRNA gene in microbiology?
The 16S rRNA gene is a highly conserved region found in all bacteria, making it a key target for molecular identification and phylogenetic studies. By sequencing this gene, researchers can determine the evolutionary relationships between different bacterial species. This information is crucial for understanding microbial diversity, ecology, and the role of bacteria in various environments.

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