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Methods of Detection of Microorganisms

This document discusses various methods for detecting microorganisms, including conventional and rapid methods. Conventional methods include plating samples on media and incubating to allow microbial growth, followed by counting or additional tests. Rapid methods involve electrical impedance measurements of microbial growth, ATP bioluminescence to detect microbial metabolism, using genetic probes to detect specific DNA or RNA sequences, and polymerase chain reaction (PCR) techniques. Both conventional and rapid methods have advantages and limitations for qualitative or quantitative microbial analysis.

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SYBSC 049 Shrishti K.
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13 views

Methods of Detection of Microorganisms

This document discusses various methods for detecting microorganisms, including conventional and rapid methods. Conventional methods include plating samples on media and incubating to allow microbial growth, followed by counting or additional tests. Rapid methods involve electrical impedance measurements of microbial growth, ATP bioluminescence to detect microbial metabolism, using genetic probes to detect specific DNA or RNA sequences, and polymerase chain reaction (PCR) techniques. Both conventional and rapid methods have advantages and limitations for qualitative or quantitative microbial analysis.

Uploaded by

SYBSC 049 Shrishti K.
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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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

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