Lecture 34: microbial isolation and concentration

accuracy

closeness of measurements to the standard/ known value

precision

closeness of measurements to each other

Sensitivity

true positivity rate, contaminated food correctly identified as contaminated

Specificity

true negativity rate, non-contaminated food correctly identified as non-contaminated

LOD

the lowest analyte concentration likely to be reliably detected

LOQ

the lowest concentration at which the analyte can be detected and at which some predefined goals for bias and imprecision are met

Qualitative methods of detection

enrichment, allows for multiplication of microorganism, intensifies the signal, presence or absence

Quantitative

the ability of the cells to multiply in nutrient -rich medium, plate count (absolute quantification), plaque assay, real-time PCR

Why is it difficult to detect microorganisms in food and water?

usually low in concentration, bound to matrices, not homogenously distributed, formation of aggregates

Why do we use bacterial enrichment

results in resuscitation and rapid growth, increases the odds of detection, used for qualitative only and improved estimation using MPN technique

absolute quantification

CFU per mL

relative quantification

higher or lower signal

reasons to isolate and concentrate microorganisms

separate pathogens from sample particulates, provision of sample size reduction, removal of matrix-associated assay inhibitors

define isolation

the removal of a select population from a complex mixture

define concentration

a sample preparation process to reduce sample volume while simultaneously recovering the initial bacteria population of interest

describe filtration

membrane filter retains cells to be transferred to culture medium

describe Adsorption-elution

2 step process to adsorb microorganisms to a surface through manipulating the pH or ionic strength via isoelectric point (example lower the pH), then eluting/removing the attached microorganisms (increase pH), sample can then be further concentrated

describe centrifugation: differential/pelleting

centrifugal force to separate sample based on size- pellet may contain the microorganism, or the supernatant may contain it

describe centrifugation: density/gradient

preformed layers of a solution with different densities, highest density towards the bottom, sample placed on the top and following ultracentrifugation each zone represents the separation of particles based on their size and density

describe PEG

polyethylene glycol polymers bind to particles forming larger aggregates, sediments after centrifuged, supernatant is discarded and the particles in the solution are resuspended

Immunoaffinity

affinity resin has an immobilized ligand (ligand can be a target small molecule, protein, or antibody), similar process to adsorption/elution