FDSC 409 Exam 1

Purpose of GMLP

prevent uncontrolled spread of microorganisms, protect experiments from contamination, and protect people from infection

sterilization

complete destruction of all microorganisms, including spores

disinfection

destruction, inhibition of all microorganisms using chemicals

aseptic technique

practices that prevent contamination of cultures and protect the worker

autoclave conditions

121 degrees C for 15-20 minutes for media and glassware, as specified in Lab 1

wire loop use

flame a metal loop until red, cool before transfer, and re-flame afterward; do not flame plastic loops

BSL-1

Minimal potential hazard; organisms not known to consistently cause disease in healthy adults

BSL-2

Moderate hazard to personnel and the environment

Broth versus agar

broth is liquid; agar contains approximately 1.5% solidifying agar

nonselective medium

supports growth of a broad range of microorganisms

selective medium

contains ingredients that inhibit some groups while allowing others to grow

differential medium

allows microorganisms to be distinguished by observable reactions

aseptic workspace

disinfect bench, organize supplies to avoid crossing contaminated over sterile items, and work within the prescribed aseptic area

predictive microbiology

mathematical modeling of microbial growth responses to conditions such as temperatures, pH and water activity

limitation of predictive models

predictions guide decisions but do not replace food-specific experimental validation or challenge tests

lag phase

cells adapt to the environment without population doubling; stressed cells may have a longer lag

exponential phase

cells double at regular intervals, characterized by generation time

stationary phase

population plateaus due to nutrient depletion, inhibitory metabolites or space limits

death phase

viable cells decline; culture-based CFU enumeration can detect the decline

generation time equation

G = t / n , where t is elapsed time and n is number of generations

physiological state in ComBase

scale 0 to 1; 0 indicates very poor state/long lag and 1 indicates very good state/no lag

primary growth model

describes changes in microbial numbers over time

secondary growth model

describes effects of conditions such as pH, temperature or water activity

tertiary model

combines primary and secondary models to predict growth under multiple conditions

why log10 CFU?

microbial concentrations span orders of magnitude; log transformation compresses the range

convert 1,000 CFU/mL to log10

3 log10 CFU/mL

Convert 5 log10 CFU/mL to CFU/mL

100,000 CFU/mL (10⁵)

ComBase milk model pH

6.6

ComBase orange juice model pH

5, the minimum accepted by the model, even though typical juice pH is lower

ComBase model temperature

20 degrees C

ComBase initial level

3 log10 CFU/mL, equivalent to 1,000 CFU/mL

purpose of serial dilution

reduce microbial concentration to obtain countable plates

CFU concentration from spread plate

CFU mL = colonies / (dilution plated * volume plated in mL)

Ex: 35 colonies, 10^-5 dilution, 0.1 mL plated

35 / (10^-5 × 0.1) = 3.5 × 10⁷ CFU/mL

goal of lab 3

identify a food spoilage microorganism by isolation and molecular characterization

lab 3.1 isolation

streak spoiled milk on SPC agar and incubate 24 hours at 32 degrees C

Why colony appearance is insufficient

morphology, phenotype and Gram stain alone cannot reliably identify all bacterial species

DNA extraction approaches

physical methods such as heat or bead-beating, and chemical methods such as detergents or enzymes

DNA extraction used in Lab 3

boiling with nuclease-free water to lyse cells; centrifugation removes debris

PCR purpose

amplify selected DNA sequences through repeated thermal cycles

PCR step 1: denaturation

double-stranded DNA separates in single strands

PCR step 2: annealing

primers bind complementary sequences on template DNA

PCR step 3: extension

DNA polymerase synthesizes new DNA strands

PCR components

template DNA, Taq polymerase, dNTPs, primers and MgCl2-containing buffer

Taq polymerase

heat-stable enzyme that synthesizes DNA

dNTPs

DNA building blocks A, C, G, and T

16S rRNA target

bacterial identification target; often resolves genus and sometimes species

can 16S rRNA primers identify yeast or mold

No. the manual identifies 18S rRNA or ITS target for yeast and mold

gel electrophoresis purpose

separate DNA fragments by size

direction of DNA migration

negatively charged DNA moves toward the positive electrode

how to estimate PCR product size

compare sample band migration with a DNA ladder

Sanger sequencing

determines the nucleotide sequence of a PCR product

NCBI BLAST purpose

compare a sequence with database sequences to help identify the microorganism

qualitative method

determines whether a target organism is present or absent

quantitative method

estimates how much of the target organism is present

limit of detection

lowest number or concentration of microorganisms detectable by a method

enrichment

incubation in growth-supporting medium to increase target numbers and improve detection

sensitivity equation

true positives / (true positives + false negatives)

specificity equation

true negatives / (true negatives + false positives)

low sensitivity consequence

more false-negative results

low specificity consequence

more false-positive results

lab 4 case 6 LOD

10,000 cells/g produce after a 1:10 homogenization and testing 1 µL from 1 mL lysate

Lab 4 Case 7 LOD

100 cells/g produce when 10 mL homogenate is concentrated into 1 mL and 10 µL is tested

indicator organisms

organisms used to evaluate microbiological quality or hygiene

index organisms

organisms whose presence indicates increased likelihood of a related pathogen

water analysis methods

Petrifilm and membrane filtration on M-Endo agar

MPN meaning

most probable number: s statistical estimate based on patterns of positive tubes

MPN positive tube criterion

gas production is required; turbidity without gas is not a positive result in the described coliform test

why not vortex MPN tubes before reading?

vortexing may disrupt or release gas accumulated in Durham tubes

LST role

presumptive coliform detection broth

BGLB role

confirmation of coliforms

EC broth role

detection/confirmation of fecal coliforms under specified incubation conditions

three-tube MPN

three replicate tubes are tested at each dilution

how to interpret MPN

record positive-tube counts at three consecutive dilutions and consult the manual’s three-tube MPN table

petrifilm incubation in water exercise

35 degrees C for 24 ± 2 hours

two environmental monitoring programs

hygiene monitoring and pathogen monitoring

what ATP monitoring detects

ATP from living cells or organic material, not specifically pathogens or microorganisms

ATP detection enzyme

luciferase

ATP readout

light emission reported as relative luminescence units (RLUs)

high ATP reading

indicates ineffective cleaning or residual biological materialwh

when to ATP swab

after cleaning and sanitizing

when to swab for pathogens

middle or near the end of processing, before cleaning and sanitation

Zone 1

food-contact surfaces

Zone 2

non-food-contact surfaces close to food-contact surfaces

zone 3

non-food-contact surfaces within processing areas but farther from food contact surfaces

zone 4

areas outside the processing area

priority areas for hygiene swabbing

zone 1, followed by zone 2

priority areas for hygiene swabbing

zone 1 followed by zone 2

priority areas for pathogen sampling

high-risk, hard-to-clean niches such as drains, cracks, equipment-floor junctions and wheels

listeria spp. as an index

environmental Listeria species can indicate risk of L. monocytogenes contamination

Listeria monocytogenes Gram reaction

Gram-positive

disease causes by L. monocytogenes

Listeriosis

groups at elevated risk

pregnant, elderly, immunocompromised and young individuals

listeria-associated foods

vegetables, dairy foods and ready-to-eat meats

why enrichment precedes selective conditions

allow injured cells to recover before selective ingredients inhibit background microbiota

Lab 5 identification workflow

environmental sampling, enrichment, isolation on selective/differential media, biochemical identification with API strips and molecular traceback

purpose of vector sampling

investigate surrounding sites to identify a persistent contamination source

purpose of API strips

biochemical confirmation of Listeria species

purpose of molecular traceback

compare sequence information to investigate possible contamination relationships