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?
Describe Gram-positive bacteria.
- Thick peptidoglycan layer and no outer membrane
- Stain purple
- The peptidoglycan protects the bacteria from osmotic lysis, but not plasmolysis
- The peptidoglycan provides mechanical strength to the cytoplasmic membrane.
Which type of bacteria are responsible for most infections in the bloodstream?
- Gram-positive bacteria (62% of infections)
- Gram-negative bacteria (22%)
- Some Gram-positive bacteria are significant food-borne pathogens
- Some Gram-positive bacteria can be part of the microbiota.
Define: food borne infection.
- Vegetative form or resting form present in food is eaten (if resting form - then germination results)
- Vegetative form grows in the intestines producing exotoxins and causing illness
Define food borne intoxication.
- Vegetative form or resting form present in food (if resting form - then germination results)
- Vegetative form grows in food producing exotoxin
- Food is consumed, causing illness
What is resting form and vegetative form?
Resting form = dormant cellular form, not dead, but not capable of reproduction
Vegetative form = active cellular form capable of reproduction
What is an exotoxin?
- Secreted extracellular proteins
- Enterotoxins (type of exotoxin) bind to membrane proteins of intestinal cells causing them to secrete large amounts of water (diarrhea)
- Neurotoxins (type of exotoxin) bind to nerve cells; have different effects depending on the toxin
- Vary in heat-sensitivity
Give an example of a resting cell.
Endospores formed by some members of Gram-positive bacteria; some endospore formers are pathogenic
Most prokaryotes form resting cells.
True or False?
False.
Most do not form resting cells.
Most prokaryotes do not form resting cells.
True or False?
True.
Describe endospores.
- Core is dehydrated (10-15% water by weight)
- Undetectable metabolic activity due to low water
- Cells are dormant and long-lived
- Resistant to penetration of chemicals (e.g., do not stain)
- Very heat resistant; can survive for hours at normal cooking temperatures, pasteurization temperatures and boiling water.
What is the purpose of endospore formation?
- Protect genome until favourable nutritional conditions return.
- When this occurs, endospore germinates and resumes growth as a vegetative cell
- If present in a food when packaged, endospores might germinate into vegetative cells and grow during room temperature storage and produce the toxin.
- Many packaged foods are not subsequently cooked.
Describe the heat-sensitivity of exotoxins.
- Some are heat labile (denatured by normal cooking temperatures)
- Some are heat stable (not denatured by normal cooking temperatures)
Gram-negative bacteria form spores.
True or False?
False.
They are not spore formers.
Gram-negative bacteria do not form spores.
True or False?
True.
Salmonella causes […].
Food-borne infection or intoxication
Various foods
Camplyobacter jejuni causes […]
Food-borne infection
Typically poultry
Vibrio cholerae causes […]
Food borne infection
Various foods, water
Vibrio parahaemolyticus causes […]
Food-borne infection
Raw or undercooked shellfish
Escherichia coli causes […]
Food-borne infection
Various foods
Compare food-borne infection and food-borne intoxication.
- Food-borne infection – bacteria grow in the intestine, produce toxins
- Food-borne intoxication – toxins present in the food that is ingested
Describe Bacillus cereus.
- Spore former
- Facultative anaerobe (can grow with or without oxygen)
- Can cause both food-borne infection and intoxication
- Most commonly ingested in rice that had been sitting at room temperature after cooking
- A relatively common cause of gastroenterititis worldwide
Describe Clostridium perfringens
- Forms spores
- Anaerobe
- Can cause food-borne infection
- Most commonly ingested from contaminated meat and poultry
- Also causes gas gangrene if it gets into a deep tissue wound.
Describe Listeria monocytogenes
- Does not form spores
- Facultative anaerobe
- Can cause food-borne infection
- Associated with foods such as deli meats, soft cheese (e.g., Brie) and refrigerated seafood (e.g., smoked salmon)
- Grows at refrigerated temperature
- Serious illness for pregnant people and immunocompromised
Describe Clostridium botulinum
- Forms spores
- Anaerobe
- Can cause food-borne infection
- Most commonly ingested from home canned foods
- Most potent toxin - causes flaccid paralysis
- People voluntarily inject diluted toxin for cosmetic procedures (Botox)
Describe Bacillus anthracis
- Forms spores
- Anaerobe
- Can cause food-borne infection
- Ingested from contaminated meats that are consumed raw or are undercooked
- Can cause cutaneous, respiratory and GI illness
- Very high fatality rates for respiratory and GI illness
Describe Staphylococcus aureus
- Does not form spores
- Facultative anaerobe
- Most dangerous of all the many common staphylococcal bacteria, but can be part of the microbiota of a healthy individual
- Can cause food born intoxication
- Ingested from contaminated ready-to-eat food (most likely, contaminated by the food handler)
- Can also cause toxic shock syndrome
