Lab 8 Flashcards
Why has lettuce become important from a microbiological perspective? [4]
- Increased demand for fresh produce
- Increased demand for pre-washed produce (e.g., salad kits)
- Increase in packaging/processing from farm to market
- Shipping (i.e., not only local produce)
What are sources of potentially pathogenic bacteria?
- Contaminated food and water
- Unsanitary conditions
What is the classic method for identification of food borne pathogens? [4]
- Homogenization of solids
- Enrichment
- Selection
- Biochemical characterization
What is Hektoen agar?
- A selective and differential media
What is one advantage and one disadvantage of using the classic method of identification of food borne pathogens (like Salmonella)?
- One advantage of using the classic method of identification of foodborne pathogens is that it is considered the “gold standard” and is well known for its cost-effectiveness, sensitivity, ability to confirm cell viability, and ease of standardization.
- However, one disadvantage of using the classic method is that it can be time-consuming and unable to examine novel organisms. Additionally, traditional typing methods cannot provide sufficient information to differentiate among strains.
List four newer methods for identification of food-borne pathogens.
- Enzyme-linked immunosorbent assay (ELISA)
- Lateral flow devices (LFD)
- Fluorescent In-situ hybridization (FISH)
- Polymerase chain reaction (PCR) (also q-PCR - a variation that quantitates the PCR product as the amplification is in progress)
Other methods exist, but will not be discussed in MICB 353.
What can ELISA be used to detect?
- Bacterial toxins
- Bacterial proteins
- Virus particles
- Cannot detect the actual bacteria
Describe sample preparation for ELISA.
The sample would be homogenized in a phosphate buffer.
What does ELISA entail?
- Two antibodies that bind to different structures of the target of interest (e.g., toxin)
- One antibody is conjugated to an enzyme (E).
- The enzyme cleaves a colourless substrate (S) to a coloured product (P)
- The amount of the product can be measured (qualitative and quantitative assay)
- Time to completion = 4 - 6 hours (not including sample preparation)
What can lateral flow devices be used to detect?
- Pathogens (virus, bacteria)
- Other contaminants (e.g., toxins)
- In water supplies and foodstuffs.
What does a lateral flow device entail?
- Typically contain a control line to confirm the test is working properly, along with one or more target/test lines.
- Can be used in a dipstick or in a housed cassette format.
- Time to completion = 5-10 minutes
- Qualitative
Describe lateral flow device technology.
- Uses immunoassay tech using nitrocellulose membrane, coloured nanoparticles (or labels), and typically antibodies, to produce results.
- When a sample is added, it will flow along the test device passing through the conjugate pad into the nitrocellulose membrane and then the absorbent pad.
- The conjugate pad, which stores the conjugated labels and antibodies, will receive the sample.
- If the target is present, the immobilized conjugated antibodies and labels will bind to the target and continue to migrate along the test.
- As the sample moves along the device the binding reagents situated on the nitrocellulose membrane will bind to the target at the test line. A coloured line will form.
- Very easy to use. Qualitative tests read visually.
What does fluorescent in-situ hybridization detect?
- Detects and locates a specific sequence on a chromosome
- Allows for the direct quantification of pathogens without the need to culture the bacteria first.
Describe how fluorescent in-situ hybridization works.
- Uses a piece of DNA (probe) that is complementary to a sequence in the chromosome. A fluorescent dye is attached to the probe.
- Would have DNA probes – e.g., recognizes the sequence of portion of a gene found in a pathogen like Salmonella.
- Can visualize the test with a fluorescent microscope.
- Time to completion: about 2 – 3 days (would include preparation of the sample).
What is PCR?
- A laboratory technique used to amplify DNA sequences
What does PCR involve?
- Using short DNA sequences called primers to flank (select) the portion of the genome to be amplified
- The temperature is repeatedly raised and lowered to help a DNA replication enzyme copy to target the DNA sequence.
- This technique can produce a billion copies of the target sequence in just a few hours.
- The presence of the PCR product is detected by gel electrophoresis.
- Time to completion = 5 - 8 hours (includes sample preparation)
Describe gel electrophoresis in PCR.
- DNA samples are loaded into wells at one end of a gel, and an electric current is applied to pull them through the gel.
- DNA fragments are negatively charged, so they move towards the positive electrode.
- When a gel is stained with a DNA-binding dye, the DNA fragments can be seen as bands, each representing a group of same-sized DNA fragments.
Think of one advantage and one disadvantage of these methods to identify potential food pathogens.
How could someone interested in food safety (not necessarily microbiological) and/or food quality use these tests?
