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.