Midterm 3 Flashcards
Challenges of testing
Bacteria not uniformly distributed
Food matrices interfere
Indigenous microbes (not sterile)
Pathogen of interest is in lower #s
Injury to pathogen (give time to recover before selective tests)
MPN
Dilution of food samples
9 or 15 tubes
3 or 5 replicates of each
Based on stats
Dilute to extinction
Higher estimate than colony counts
MPN scores for
Positivity
Total bacteria
Lactose fermenting
LST MUG
Why does broth have higher counts than plates
Easier to grow in broth than on a plate
More ideal to grow in liquid
LST MUG
Select for enteric
MUG cleaved by GUD ⇒ MU (add light = fluorescence)
Yields E coli count
Immunoassays
Antibody specific to antigen
Need specific antigen to organisms of interest
Inject in animal → make antibodies → use for detection
Agglutination
Lattice of antibodies and antigens
Mix colony w/ antibodies → clumping
Immunoprecipitation
Control and test lanes
Antibodies in the sample well
Complex and wick up the wick
Capture antibody captures if antigen is present
Immunoconcentration
Enriched sample
Add to test tube
Add metal beads w/ antibody attached
Mix + incubate
Use magnet to collect beads
Wash off unbound
Streak out (differential and selective)
ELISA
Enzyme conjugated to antibody or antigen
Wash, then react with the substrate
Shows a visible color change
enzyme-linked immunosorbent assay
have to do enrichment 1st
Need antibody specific to microbe
Sandwich Method
Enrichment 1st
Solid matrix (solid is charged) attached to antibodies
Put the enriched sample in well
Antibody look for antigens + capture it
Wash everything not bound
Add 2nd antibody with enzyme tag (same target, diff epitope)
2nd antibody attaches
Wash again
Add substrate
Colorimetric end product
Nucleic acid based
Pros/Cons
Sensitive, specific, fast
Need specific gene
Matrix interference
cost
Live vs dead
PCR
Denature with heat
Need specific primer (brackets target gene)
Anneal the primer to the strand
Add polymerase + replicate
Need Taq polymerase
Takes a few hours
Run a gel electrophoresis to analyze
TaqMan PCR
Removes running of gel with fluorescent tag
2 primers + 3rd probe bracketed by primers
Positive or negative result (fluorescence or not)
If tag is adjacent to the quenching agent → no fluorescence
Polymerase chews probe and releases components –> components spread out → fluorescence
PFGE
Pulsed field gel
Changes orientation to a zig zag pattern
Separate large lengths of DNA (long, tedious)
Contributions to variation
Insertion
Deletion
Recombination
Phage
Plasmid
Most discriminatory subtyping method
Sequencing
Ultimate method to identify differences btwn 2 isolates
Method consideration
Approval status
time
Cost
# of samples
Equipment
Microbiological criteria
Asses the
- Safety
- Quality
- Adherence to GMPs
- Suitability
of a food/ingredient for a particular purpose or target population
Standard
Microbiological criteria that is part of law, ordinance, or regulation
Guideline
A microbiological criterion used by the food industry and regulatory agency to monitor a manufacturing process
Specification
Microbiological criterion that is used as a purchase requirement between a buyer and vendor
Criteria: include
Identify of food
Identity of contaminant of concern
Method for detection
Sampling plan
Microbiological limits
Index organism
Organism that signals the possible presence of pathogenic or toxigenic organisms within a sample
Index Ex
Listeria genus, coagulase + staph, generic E. coli
Indicator organism
Organisms that reflect the general bacteriological quality or safety of a sample
Indicator ex
APC, coliforms, yeast, molds
Indicator characteristics
Easily and rapidly detectable
Easily distinguishable from other food flora
Consistent association with
pathogen whose presence it is to indicate
Growth/survival curve = that of pathogen
Present when pathogen is present
Absent in food free of pathogen
Possible specificity to intestinal tract
May occur in high # in feces
Coliforms
Gram -
Non spore forming
Rod
Ferment lactose to acid and gas
5 genera of Enterobacteriaceae
Fecal coliforms + safety
Coliforms that produce acid and gas (higher tem)
Primarily E. coli
Identify non bacterial microbes (viruses)
Indicators of quality
Usually specific
Yeast, mold, lactic acid bacteria
APC not as specific
Microbial sampling
Identify production lots within a level of confidence that are inferior or unsafe
Production lot
Quantity of food produced, handled, and stored within a limited time period under uniform conditions
Risk assessment considerations
Target population
Transportation and storage
Preparations
What microbes are found in what foods
Incidence
of positives per x samples
6% per 100 samples (ex)
Presence or absence
Level
Enumerate organisms
# of cells per x samples
Representative sample
Similar as possible to that of the lot from which it was drawn from
Random
Consistent and represent the entire lot
2n sampling plan
n = # of samples
m = max level of microbe
c = max # of samples with UNACCEPTABLE results
+ or - (yes or no)
3n sample plan
n = # of samples
c = max # of samples allowed with marginal results between m and M
Accept all below m
Accept c amount between m and M
Reject all above M
Agglugination Pros
Simple, no special equipment, fast
Agglugination Cons
Sometimes difficult to interpret, nonspecific reactions
Immunoprecipitation Pros
Simple, no extra equipment
Immunoprecipitation Cons
Not quantitative, subjective
Immunoconcentration Pros
Very rapid, enhanced specificity, adapt to almost any existing rapid method
Immunoconcentration Cons
Added cost
ELISA Pros
Sensitive, not radioactive, can be quantitative
ELISA Cons
Hands on time + equipment needed
Rapid methods
Mini biochemical kits
Antibody based
Nucleic acid based
Biochemical kits
Use chromogenic or flourescent substrates
X Gal
MUG
Immuno Assays
Agglugination
Immunoprecipitation
Immunoconentration
ELISA
Modeling
Use mathematical tools to predict behavior of 1+ type of microbe
Useful for assessing risk, predicting efficacy of treatments
Subtyping
Characterization below the species or subspecies level
Significance of subtyping
Recognition of outbreaks
Identification of source
Characterization of clones
Discriminatory power
The ability of a method to discriminate, segregate,
or discern differences between two isolates of the
same species
Fermentation def
Catabolism of carbon sources where organic compounds serve as the ultimate e- acceptor to produce ATP in the absence of O2
Phosphorylation
Movement of e- generated from light interaction with pigment via membrane bound transport system creates NRG for ATP synthesis
Oxidative phosphorylation
Movement of e- generated from metabolism through membrane bound transport system creates NRG for ATP synthesis
Substrate level phosphorylation
Phosphate is transferred from high NRG carbon intermediate generated during catabolism
Occurs during fermentation
Glycolysis steps
Activation of glucose
Splitting of hexose
Energy derivation
Glycolysis outputs/inputs
1 Glucose
- 2 ATP
+ 4 ATP (2 Net)
+ 2 NADH
Purpose of fermentation
Free up NAD+ so glycolysis can continue
End products:
Streptococcus, lactobacillus, bacillus
Lactic acid
End products: Yeast
Ethanol
CO2
Benefits of microbes
Preserve foods
Competitive exclusion
Vitamin production
Flavor
Improve safety
Improve starch digestability
Lower toxin level
Fermenatation products
Antibiotics
Hormones
Enzymes
Ethanol
CO2
H2
Acids
Amino Acids
Vitamins
Gums
Fermenters: Bacteria
Lactobacillus
Lactococcus
Streptococcus
Leuconostoc
Propionibacterium
Fresh Pack
Take vinegar + add spice + veg
Not fermented
Pickle fermentation
Cucumber placed in salted water (brine)
Carbs released
Normal flora on the surface ferment carbs
Pickles initial microbes
Leuconostoc mesenteriodes
Enterococcus faecalis
Pickles primary microbes
Lactobacillus plantarum + brevis
Pickles results
Homolactic (only lactic acid)
No CO2 produced
pH drops coliforms/enterics
If yeast grow → bubbles + CO2
Sauerkraut fermentation
Shred cabbage
Add and distribute salt
Place in tank with no brine
Cover with plastic
Water to keep anaerobic
LAB
lactobacillus
Streptococcus
Leuconostoc
Yogurt Starter Culture
Streptococcus thermophilus
Lactobacillus delbrueckii ssp. bulgaricus
Blue, Roquefort, Gorgonzola cheese microbes
Penicillium roqueforti
Penicillium glaucum
Cheese milk contaminant
Psuedomonas
Heat stable lipases
Cheese Steps
Milk Prep
Heat Treatment
Add starter culture
Add rennet
Cut
Separate curds from whey
Salt, press, ripen
Intrinsic and extrinsic factors of cheese
Different pH (based on cheese)
Salt
Temp control
Competition
Cheese and MC
Rate of acid production affects MC
Meat Starter Culture
Drops pH
Need to add glucose
Break down protein, lipids, carbs
Tiem and temp dependent
Control final pH with glucose
Meat Steps
Mixing
Final Grind
Stuff
Thermal Processing
Drying
Meat Main Pathogen
Staph aureus
Probiotics
Live microbes that provide health benefits when consumed
Prebiotics
Select for replication of beneficial microbes
Non digestible oligosaccharides
Strata
Division of population into smaller groups
APC hazard
Utility
E. coli hazard
Indicator
Salmonella hazard
Severe
Staph hazard
Moderate
LM hazard
Severe
Utility microbes
Yeast, mold, total count
Indicator microbes
Coliforms, E.coli, fecal E. coli
EHEC hazard
severe
Why is spoilage not necessarily a bad thing
Competition for pathogens
Quality decrease, people won’t consume the food and won’t get sick
Key point
Lower specifications for incoming ingredients to achieve shelf life
Why retesting is bad
The probability of getting 2 x positives is very low
subtyping methods
Pulse field
Sequencing
Why we subtype
Match product isolate to patient isolate
Helps with sources for outbreaks
Match source of spoilage microbes
Most specific methods
Nucleic acid based
Modeling
If equipment breaks
Plug info into program
Quick
Modeling cons
Data not generated from real food
Some inaccuracies
Modeling pros
Quick
Good tool for ballpark
Feeding grass vs corn
and E coli
Feed corn to cattle lowers intestinal pH
Grass-fed shed lower #s of E. coli per gram (greater amount of feces) (wash)
Feeding corn caused evolution of EHEC
Action doesn’t cause something to evolve
Trait got selected for
Horizontal gene transfer = more rapid than mutations
Farmer processing in open air
Lower CFU/g than commercial birds
Not very significant difference
Who is responsible for keeping food safe
Gov? Everyone? Producers?
Who is responsible for Kevin’s death
Jack in the box
Beef producer
Cook
Feed lot owner
Manufacturing plant
Lobbyist
Ensure another death doesn’t happen
Limit the # of places you get the meat from
Tight specifications
Animal husbandry
Buy local meat
Manage runoff of manure
Make healthy food available to everyone
Consumer education
Government has to be part of solution
Subsitdes or food vouchers to level out playing field
Subsidize small companies instead of large ones
Food trucks in food deserts
Live vs dead solution
Test at diff times
Test for RNA
