LECTURE 2:
PART B - TECHNIQUES ON
MICROBIOLOGICAL
ANALYSIS OF FOOD
MCB106, 2024-2025
Lynn E. Rallos
Criteria for choice of methods
1. A___________
ccuracy
2. S___________
ensitivity
3. Ease of i_______________
mplication
4. E_____________/
quipment
o_______________
perating cost
5. S____________
peed
Common Test Methods
• Direct examination rare
• Culture-based methods some companies are
using this
• Immunological assays
• Rapid tests
Consult Chapter 10 of Moss and Adams
Traditional methods
Divided into three general categories, based on the test
function performed.
1. Presence or absence of microorganisms-
“_____________?"(e.g.,
is something there pathogen detection, absence
of objectionable organisms, sterility testing),
2. Enumeration of microorganisms-“_______________?"
How much is there
(e.g., bioburden testing)
3. Identification of microorganisms “_______________?
What is there
"
Time consuming
Traditional methods
Direct Examination
Culturing
Enumeration
Isolation of presumptive colonies for further
identification.
• If necessary, the food sample must be
homogenized, concentrated, and/or enriched prior
to culturing.
Traditional methods
The ______________of
pre-enrichment bacteria in a
food sample can be performed by a:
- non-selective
- or selective broth culture
- or by the selective agar overlay
technique to resuscitate the injured cells
Traditional methods
Another manner in which the detection levels of
viable cells can be increased is by concentration
of food sample via ____________
filtration or
centrifugation prior to plating.
Traditional methods
The pre-treated food sample can then be plated on:
- non-selective
- selective are types of media; standard media used for different types test; conditioned
- differential media
• ________________or
Non-selective media standard methods agar, for
aerobic plate count, can be used to detect and count
the amount of bacteria in the sample.
Traditional methods
• Bacterial cells can become __________ injured or viable but
____________________(VNC)
nonculturable due to the sublethal
stressors, such as: alive but cannot be cultured
- Heat
- cold because when they are activated to grow; they might not be growing in
a culture medium but they can grow in food. Thus, they are posing
- acid threat.
- osmotic shock during the food processing steps.
These bacterial cells still pose a threat in the food industry and therefore,
methods to improve the detection levels of these injured cells have been
developed.
Commonly used media for
food analysis
lactose fermentors
Traditional methods
• __________________-
Selective medium selectively inhibits the growth of
specific microorganisms (due to component such as
(antibiotic, bacteriocin, a growth nutrient) but
allows the growth of the target. Mannitol - carbon source.
eg: PEMBA medium
-polymyxin pyruvate egg yolk mannitol
bromothymol blue agar - isolation and
enumeration of Bacillus cereus
-can detect <<<B. cereus cells and spores
in the presence of large numbers of other
food contaminants.
Traditional methods
The third type is a _______________
differential medium which contains an
indicator, such a chromogenic or fluorogenic substrate,
which differentiates bacteria by various chemical
reactions carried out during growth.
* direct identification of microorgamisms without further subculturing or biochemical
tests
* based on bacteria producing specific and exact enzymes for available substrates
* as the enzyme acts with the substrate (fluorogenic or chromogenic) the bacterial
growth will fluoresce or change color, respectively.
Traditional methods
color change in colonies
Fluorogenic medium for detecting E. coli Chromogenic medium for detecting
and coliforms Salmonella sp. in food
traditional
Comparison of sensitivity of methods
plating technique done in a diff way, using a tool where the plate is rotated and
the culture(?) is dropped
water analysis
Advantages of
Culture-Dependent Methods
time consuming and laborious BUT
• the isolation and purification of microorganisms
you can identify -
allows for further subtyping analysis and fornecessary
outbreaks
for
storage in culture collections.
• subtyping = phenotypic characteristics based
on:
- biotyping biochemical test
- serotyping
- phage typing
Biotyping
• biochemical growth requirements
• environmental conditions (pH, temperature, antibiotic
resistance)
• morphological and physiological
(colony and cell morphology, cell wall composition by microscopy
(gram staining) and membrane composition such as by fatty acid
analysis)
Lipopolysaccharide - LPS; lipid A, O antigen.
Serological and Phase
g typing Antigenic
*flagella
*pili
*fimbriae
capsules - hide the cell from microphages or harmful microorganisms; lapot
Concentrate more on the surface structure differences
of bacteria.
•specific
Phages are
phage-host
not only useful in subtyping bacteria, but
also in detecting pathogens directly from foods
• Limited since microorganisms are capable of altering
their phenotypic characteristics due to environmental
changes or genetic mutations.
• Therefore, identification by _________________
genotypic
characteristics has been developed to avoid these
problems that can occur with phenotypic methods.
real-PCR - sequence specific
Rapid Microbiological Methods
Based on how the technology works
-Growth-based
1. Methods that measure the growth of microorganisms Technologies
2. Methods that determine the viability of microorganisms
Viability-based Technologies
3. Methods that detect the presence or absence of cellular
components or artifacts -cellular-component or Artifactbased Technologies
4. Nucleic acid methods -nucleic-acid-based Technologies
5. Traditional methods combined with computer-aided imaging
6. Combination methods.
1. method - directly or indirectly quantitates target group of species. not all method directly says that such is present.
2. methods like staining, originally you don't know what is viable or not. direct microscopic count. e.g. APC
3. similar to fatty-acid, you can determine particular kinds of fatty acids because they are ass with cell components.
4. detection via PCR, sequencing, DNA RNA, usually DNA. e.g., canned foods - standard plate counts and
endospore.
5. fluorescence microscopy - in food. use dyes that can only dye viable cells
6. enrichment, selective differential media then molecular identification. always refer to standard method - BAM.
Growth-based Technologies
Based on the measurement of
_____________
biochemical or ____________
physiological
parameters that reflect the growth of
the microorganisms
ex:
*ATP bioluminescence
*Adenylate kinase
*Measurement of change in head space pressure
*colorimetric detection of carbon dioxide production
*Impedance (Electrochemical) Methods
*Conductivity
ATP is found in all living organisms and is an excellent marker for
viability and cellular contamination.
bioluminescence kit - used to measure
for Aerobic and facultatively aerobic cells bioluminescence.
luciferase enzyme - originally from firefly -
is placed to the sample. ATP comes from
the microorganism present. When
luminometers - measures the luciferase acts on the substrate, this will
cause bioluminescence light that can be
captured by the kit and convert to light
units
Light emission: rapid loss
of energy of the
oxyluciferine molecule
from an excited state to
a stable one
the more intense the light produce, the more reaction, the more ATP available, indirect indication of the relative amount of cells
in your sample. It does not tell directly but can do mathematics to convert light readings to relative amounts of light grams per
unit
*insert pic of ATP Bioluminescence in phone
Viability-based Technologies
Varying methods are used to
determine if the cell is _________
viable , and
if viable cells are detected, they can be
enumerated.
Ex.
*DEFT (Direct Epifluorescent Filter Technique)
*Flow Cytometry (Fluorescence)
*Solid-Phase Cytometry
*Microcalorimetry
*insert pic from phone about DEFT
fluorescent light or UV
Polymicrobic biofilm stained with
4,6-diamidino-2-phenyl indole
(DAPI) and examined by
epifluorescence microscopy.
anti-virus; choose antibodies of that specific organisms and idikit sa DAPI.
Cellular-component based Technologies
These technologies look for a specific
_____________________
cellular component or artifact within the cell for
detection or identification
Examples:
• Fatty Acid Profiles (Fatty Acid Methyl Esters [FAMEs])
• Mass spectrometry
• Fourier Transform Infrared Spectroscopy (FTIR)
• Raman spectroscopy
• Enzyme linked immunosorbent assay (ELISA)
• Bacterial endotoxin-limulus amebocyte lysate testing (LAL).
• Endospore detection
Gram stains
antibiotics active only for GN or/and GP
Immunoassays
• Based on the specific reaction between
____________
antigen and ____________.
antibody
*Historically, they were used mainly in clinical diagnostics, but are becoming
popular in rapid detection of microorganisms from food samples.
Immunoassays
• Based on antigen - antibody interactions.
• Binding strength determines the sensitivity and
specificity.
• Involve the use of ___________
polyclonal and
____________
monoclonal antibodies.
• Rapid and sensitive analysis of range of
pathogens and toxins
• Potential for onsite analysis
the more specific the anitbody is, the stronger is the binding strength which determines the sensitivity and specifity.
Poly and monoclonal are ambidextrous; 1 antibody only which increases the specificity
Immunoassays
Immunoassays used in food sample analysis:
- enzyme linked immunosorbent assay (ELISA)
- immunodiffusion tests
- immunofluorescent microscopy - specific antigen for the target
microorganism
- immunomagnetic separation
- immunoprecipitation
used in food or other aspects
1 2 3 4
can have antigenic.
Currently, a couple of commercial
immunoassay-based detection systems
are available to detect toxins or
microbes in the food system
e.g.
ImmunocardSTAT! E. coli
O157:H7detection kit.
like a pregnancy kit.
*insert a pic from phone about ELISA
Lateral Flow Immunoassay
On sight detection by dipstick and
immunochromatographic strips
• Time:2-10 minutes after addition of samples
• Advantage: low cost
reliable easy to operate
• Disadvantage: Requires labeling of antigens or antibodies, enrichment
Cell-colored
latex bead
or colloidal
gold particles
Immobilized
capture Ab
Nucleic acid methods -Nucleic-acid-based
Technologies
___________________is
Polymerase chain reaction the amplification of a
nucleic acid target sequence.
• The targeted sequences can be a specific gene or
repetitive areas in the sequence .
• For foodborne bacterial pathogens, commonly
targeted DNA areas are specific DNA sections for:
- virulence factors
- toxins
- cellular metabolites
- multicopy ribosomal RNA (rDNA)
The PCR-based
techniques have also
been developed for
screening of
genetically modified
organisms and their * Post-PCR detection methods vary
from gel electophoresis and usage of
derived materials in specific nucleic acid probes. In some
cases, probes simplufy the rdetection
foods . of the PCR product, in the similar way
as gel electrophoresis.
• some PCR use DNA primers
and fluorescent probes specific
to the target organism in the
same reaction mix.
•Detection of a fluorescent
signal for the targeted
amplified sequences indicates
the presence of the pathogen in
the sample being tested.
Oligonucleotide DNA microarray
FISH: Fluorescence in situ hybridization
Fluorescent in situ hybridization (FISH) with
oligonucleotide probes directed at rRNA is
the most common method among molecular
techniques not based on PCR.
*whole cell detection
*targets ribosomal RNAs
*16s rRNA of small ribosomal unit
*23s rRNA of large ribosomal unit
*less influenced by the food matrix
*FISH also enables the detection of
foodborne parasites like Giardia and
Cryptosporidium
probes are specific
Traditional methods combined
with computer-aided imaging
• Involves using a classical method for most of the
processing of a sample, and then using imaging software
to detect the growth earlier than methods requiring visual
growth detection
• Detection of growth using human vision typically
requires growth of 105 or 106 cells. Computer-aided
imaging can detect much lower levels of cellular growth,
e.g. less than 100 cells.
pre-enrichment, target using selective media
Computer – Aided imaging
Using advanced image-analysis
software that can significantly reduce the incubation and enumeration
time required
Images are collected using a charge-coupled
device camera
The collected images are digitized on a computer,
using image processing software that has programming capabilities
The digitized picture is processed
to detect colonies present, and the separated colonies are counted
- limit of detection.
- specific to species
run many samples at a time
- number of steps; automated; 1 time training/push button
- less worker direct contact or the researcher
- that's why they choose traditional
methods
- get the sane results even if different time.
- safety;
Recent Advancements
Semi-Automatic and Automatic Systems
API system
• System of miniaturized microtubes with 20 small wells
to carry out 23 standard biochemical tests.
* catabolic reactions between microorganisms and components of the substrate are used
*results-after 18-24h at 35-37dC
*advantage: availability of an extensive database
*disadvantage: Time consuming
BD Phoenix (BD Diagnostics)
• Turbidimetry and colorimetry
methods .
• Simultaneous analysis of 99 panels
can be performed.
• Identification of Gram positive and
gram negative bacteria.
• Time: 8–12 h
* AST results for all the tested antibiotics, except
trimethoprim-sulfamethoxazole and erythromycin,
can be obtained 24 h earlier compared with
standard methods. just like vitek 2 gp and gn
Vitek 2 Compact (bioMerieux)
• Turbidimetrically monitors
bacterial growth during
incubation.
• Gram positive and negative
bacteria
• Simultaneous analysis of 30 or
60 pure cultures
• Time: 1–10 h
min of 5hrs
*insert pic on phone about sensititre - competition of Vitek 2
PALM PCR
PORTABLE PCR SYSTEM FROM
AHRAM BIOSYSTEMS, INC.
• Battery-powered: more than 4
hours.
• Ultra-fast amplification: Turbo Fast
mode ( ≤ 500 bp) 18-21 min/30
cycles; Standard Fast mode ( ≤ 1
kbp) 24-30 min/30 cycles
• For food borne pathogens:
E. coli O157:H7 : 19 minutes
Salmonella spp. : 24 minutes
Droplet Digital PCR
not very used(ha? HAHAHAHAHA), very sensitive; 1nanolitter only
*insert pic of LAMP - loop mediated isothermal amplification
Immuno-magnetic Separation
• Presumptive test, Time:Approx.1 h.
• Affiliation: Department
of Biosystems and Agricultural Engineering, Michigan State University, East Lansing,USA.
