METHODS OF

DETECTION OF
MICROORGANISMS
 Based on established methods CONVENTIONAL METHODS
 Food samples incorporated into
nutrient media and incubated under
specific time-temperature conditions
 Counting done as visual assessment
 Simple, inexpensive, adaptable
 Food sample (accurately weighed) +
known volume of sterile diluent
 Homogenisation
 Serial dilution
 CONVENTIONAL- QUANTITATIVE METHODS

Plate Count Method:

 Elective
 Selective
 Differential
 CONVENTIONAL- QUANTITATIVE METHODS

Most Probable Number (MPN)

Method:
 Results compared to standard
table (ICMSF, 1986)
 More labour- and material-
intensive
 Less accurate than plate counting,
but more sensitive
 CONVENTIONAL- QUANTITATIVE METHODS

Most Probable Number (MPN)

Method:
 Automated Systems
 TEMPO®, France
 https://youtu.be/TctXVpUXNkY?si=
M-p4Ov19V6fsY7V8
 CONVENTIONAL- QUALITATIVE METHODS

 Used when presence/ absence of

organisms needs to be determined
 Pathogen detection
 Sample weighing>> homogenization>>
enrichment in broth medium>> streaking
on agar medium>> biochemical and
serological tests for confirmation
 Rapid test kits for biochemical and
serological tests
CONVENTIONAL- LIMITATIONS

Time
Required

Difficult Data
Interim Entry
Counts Errors
Limita-
tions

Limited Transfer
Accuracy Errors
 Qualitative or Quantitative RAPID METHODS
 Little in common with each other or
with conventional methods
 Unique results Automated
 May work poorly with certain foods
 Maybe unable to detect certain
specific organisms Semi-
 Training of staff
Automated

Manual
 RAPID- ELECTRICAL METHODS

 Particle Counting- Coulter Counter®

 https://youtu.be/Nr9avoRy3Sk?si=ve4J9Lj3Npsa
_VCI
 Metabolic activity- Malthus System, Rabit
System ®, Bactometer ®, Batrac
 Principle: Bacterial growth and metabolism in a
medium causes change in conductivity
 Incubator
 Monitoring unit to measure conductance/
capacitance at frequent intervals (e.g. 6 mins)
 Computer-based data handling system
 RAPID- ATP BIOLUMINESCENCE

 ATP essential in substrate

utilization and cell material
synthesis
 ATP detected using
bioluminescence assay
 Depends on:
 Type of microorganism
 Whether cells have undergone stress
 Whether cells are in a ATP-free
environment (challenging with food)
 RAPID- ATP BIOLUMINESCENCE
 Physical Methods:
 Filtration
Methods to Separate

 Double- filtration
Microbial ATP

 Ion exchange resins

Physical separation of
microbes from other  Extraction Methods:
sources of ATP  Lysis of somatic cells (food cells)

Specific extraction,  Destruction of released ATP with apyrase

removal and destruction  Instruments:
of non-microbial ATP
 Lumac, Foss Electric, Bio Orbit, Biotrace
 Min. detection threshold: 104 bacteria and 103 yeasts
 Analysis time: Under 1 hour
 https://youtu.be/ExStDMExg-s?si=KY3UTcaXg8yUa0l-
 RAPID- GENETICS BASED TECHNIQUES- PROBES

 Small segments of single stranded nucleic acids

 Detect specific genetic DNA/ RNA sequences
 Binding of probe to microbial DNA/ RNA detected
using specific labels
 Methods: radioisotopes (32P) label, non-isotopic
probes (avidin-biotin link system) label, colorimetric
hybridisation
 https://youtu.be/LRf1jYPTduw?si=weAZw3xgsqzi_ldz
 Gene-Trak® probe kits
RAPID- GENETICS BASED TECHNIQUES- PROBES
 RAPID- GENETICS BASED TECHNIQUES- PROBES

 Method of hybridisation:
 Probe: ss DNA probe
 Target: Ribosomal RNA of target organism
 Identification label: Chemiluminescent acridinium ester
Basic conditions
Light Lumino-
Acridinium ester Hydrogen peroxide
meter
PCR
RT-PCR
 OTHER METHODS
 Microscopy:
 Direct Epifluorescent Filter Technique (DEFT)
 Flow cytometry
 Solid phase cytometry