Lab 9: Molecular Methods 1 Flashcards
What is a selective medium?
Am medium that inhibits the growth of some organisms while allowing others to grow, often by omitting essential nutrients or including toxic compounds
Give an exmaple of how a selective medium works.
Using paper as the only carbon source selects for cellulose-degrading microbes, high salt selects for halophiles
What is a differential medium?
A medium that distinguishes between organisms based on visible changes
What is bile esculin azide agar (BEA) used for?
To differentiate enterococcus and streptococcus
What is the purpose of the API test strip?
To identify bacteria based on biochemical reactions in micro tubes, especially useful for staphylococcus and gram negative bacteria
How are API test strip results scored?
Each group of three tests is scored 1, 2, or 4; add positive values for a total between 0-7 per group. Combine into a 7 digit code
What are molecular methods sometimes preferred over traditional microbiology methods?
They are faster, more specific/sensitive, and safer for identifying pathogens that are difficult to culture or require high biosafety levels
What is the most commonly targeted gene in molecular bacterial identification?
The 16S rRNA gene
What gene is targeted to identify MRSA?
The mecA gene
What diseases are caused by helicobacter pylori in humans?
Gastritis, gastric ulcers, and stomach cancer
What species of helicobacter affect lab mice?
H. Bilis and H. Hepaticus
What symptoms might helicobacter infection cause in susceptible mice?
Diarrhea, rectal prolapse, typhlitis, hepatocellular carcinoma, or colon carcinoma
What is the best method to diagnose helicobacter in mice?
PCR from fecal samples
What gene is targeted by PCR to detect helicobacter?
The 16S rRNA gene, using helicobacter specific primers
What are the steps to extract DNA from fecal samples for PCR?
- Add fecal matter to extraction buffer
- Incubate at 75 degrees for 20 min, 95 degrees for 8 min
- Store or use for PCR
What components are in the PCR master mix for detecting helicobacter?
Nuclease free water, fast genotyping mix, and helicobacter primer mix
What are the thermocycler conditions for helicobacter PCR?
- 95 degrees for 5 min (35 cycles: 95 degrees for 15 sec, 60 degrees for 15 sec, 72 degrees for 20 sec)
- 72 degrees extension for 8 min
What technique is used to visualize PCR results?
Gel electrophoresis
